Innovative Approaches in Medicine and Health Sciences II
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INNOVATIVE APPROACHES IN MEDICINE AND HEALTH SCIENCES II Editor Nizami DURAN Lyon 2025
INNOVATIVE APPROACHES IN MEDICINE AND HEALTH SCIENCES II Editor Nizami DURAN Lyon 2025
Innovative Approaches in Medicine and Health Sciences II Editor • Prof. Dr. Nizami DURAN • Orcid: 0000-0002-2766-3491 Cover Design • Motion Graphics Book Layout • Motion Graphics First Published • October 2025, Lyon e-ISBN: 978-2-38236-920-3 DOI: 10.5281/zenodo.17382037 copyright © 2025 by Livre de Lyon All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without prior written permission from the Publisher. The author or authors of the relevant section are responsible for any copyright infringement that may occur due to the images and graphics used in the book. The editor or publisher does not assume responsibility in this regard. Publisher • Livre de Lyon Address • 37 rue marietton, 69009, Lyon France website • http://www.livredelyon.com e-mail • [email protected]
I PREFACE In today’s rapidly advancing world of science, one of the foremost missions of medicine and health sciences is not only to find solutions for the treatment of diseases but also to focus on preventive, personalized, and holistic approaches. Interdisciplinary collaborations and innovative thinking have become the key elements that define the direction and objectives of modern medicine. This book, “Innovative Approaches in Medicine and Health Sciences II,” is a comprehensive scientific compilation that examines innovative perspectives in the field of health sciences from multiple dimensions. The chapters of this volume present the most current approaches extending from basic sciences to clinical practice and include discussions on novel methodologies in health research. Among the highlighted topics are the concepts of health literacy and genetic literacy, which have gained significant importance both nationally and globally. These sections address the accessibility of individuals to health services, the awareness of genetic knowledge, and the role of scientific information in public dissemination. Such discussions offer valuable insights and serve as guiding resources for both medical education and public health. The book also includes sections that review gene transfer methods, immunological mechanisms, and cellular analysis techniques in the light of current scientific knowledge. Particularly, the understanding of immune responses in acetaminopheninduced hepatotoxicity and the evaluation of stereological analysis methods in quantitative health research are of great significance, providing a foundation for future studies. These chapters emphasize the importance of translational medicine, bridging the laboratory and clinical applications. In other sections, the protective and therapeutic roles of natural products and bioactive compounds on human health are discussed. The potential of plant-based agents used in psoriasis treatment and the anti-aging effects of bioactive compounds derived
from seaweed are evaluated in the context of innovative biomedical research. Furthermore, the chapters focusing on clinical applications cover topics such as adrenal diseases, splenic artery aneurysm, and surgical approaches, highlighting the relevance of clinical practice in contemporary medicine. In conclusion, this book serves as an important scientific guide for both young researchers and clinicians working in the field of health sciences. Prepared with the contributions of esteemed academicians from diverse disciplines, this work presents a clear vision for the future of health sciences. Each chapter, written in the universal language of science, aims not only to provide knowledge but also to inspire readers to develop new ideas and perspectives. I would like to express my sincere gratitude to all those who contributed to this work and wish the readers an inspiring and fruitful reading experience. Editor Prof. Dr. Nizami Duran Hatay Mustafa Kemal University, Faculty of Medicine Hatay-2025
III CONTENTS PREFACE I CHAPTER I. HEALTH LITERACY OF ACADEMICIANS AND ITS AFFECTING FACTORS 1 GülşenULAŞKARAAHMETOĞLU&Nesibe SümeyyeKÜTAHYALIOĞLU CHAPTER II. GENETIC LITERACY: CONCEPTUAL FRAMEWORK, IMPORTANCE, AND INTERDISCIPLINARY REFLECTIONS 15 SelihaSeçilBAYRAM CHAPTER III. FUNDAMENTALS AND METHODS OF GENE TRANSFER 23 MuratÖZTÜRK&MustafaSANDIKÇI CHAPTER IV. IMMUNOLOGICAL MECHANISMS AND INNATE IMMUNE CELL–TARGETED THERAPIES IN ACETAMINOPHEN-INDUCED HEPATOTOXICITY 45 ZeynepKARAKOY&ZekaiHALICI&ElifÇADIRCI CHAPTER V. QUANTITATIVE ANALYSIS METHODS IN HEALTHCARE: STEREOLOGY AND CAVALIERI’S PRINCIPLE 59 MuhammetLütfiSELÇUK&FatmaKAYIKÇIHEKİM CHAPTER VI. NATURAL PRODUCTS USED IN THE TREATMENT OF PSORIASIS 67 MehmetBERKÖZ CHAPTER VII. THE ANTI-AGEING ROLE OF BIOACTIVE COMPOUNDS DERIVED FROM SEAWEED 87 MehmetBERKÖZ
IV SCIENTIFIC APPROACHES IN LANDSCAPE ARCHITECTURE CHAPTER VIII. CONTEMPORARY APPROACHES TO ADRENAL DISEASE AND SURGERY 111 TimuçinSİPAL&ÇağlarSARIOĞLU CHAPTER IX. SPLENIC ARTERY ANEURYSM 141 MehmetBurakÇİLDAĞ&TuğbaÖZTÜRK CHAPTER X. REINVENTING THE HERITAGE: WHEN TRADITION BECOMES INNOVATION IN MODERN HEALTH 147 SinemTUNÇERÇAĞLAYAN CHAPTER XI. OBESITY AND GUT MICROBIOTA 165 BüşraÇALIŞIR CHAPTER XII. BIOCHEMICAL AND MOLECULAR MECHANISM OF APOPTOSIS AND PHARMACOLOGICAL IMPORTANCE OF CASPASES 175 SongülAYDEMİR&İsmetYILMAZ
HEALTH LITERACY OF ACADEMICIANS AND ITS AFFECTING FACTORS 7 had higher scores (17.25 ± 3.16) compare to males (5.48 ± 3.32). However, there was no significant difference in the health literacy score for either marital status or region where the participants have lived the longest (p> 0.05) (Please see Table 1 for detailed information). To understand whether health literacy scores of the participants change with their academic title, length of work-experience, occupational satisfaction, and income-related satisfaction, a Kruskal Wallis H-test was conducted. There was no statistically significant difference found in the end (p>0.05) (Please see Table 1 for detailed information). Likewise, to test whether health literacy scores vary with general health situation, chronic disease, and regular medicine intake, a Kruskal Wallis H-test was conducted. There was no statistically significant difference found in the end (p > 0.05). While there was no significant difference in health literacy scores with smoking, alcohol consumption, and regular exercise (p > 0.05), there was a significant difference in having proper diet and daily sleeping hours. The academics who have a proper diet (16.47 ± 3.29) had more health literacy scores compare to have not (15.41 ± 3.41). Similarly, there was a significant difference in the health literacy scores between different sleeping hours, X2 (2) = 8,824, p = .012, with a mean rank health literacy score of 16.54 ± 3.04 for people who sleep 7-8 hours, 15.37 ± 3.62 for people who sleep 5-6 hours, and 14.52 ± 4.66 for people who sleep 9+ hours (Please see Table 2 for detailed information).
8 INNOVATIVE APPROACHES IN MEDICINE AND HEALTH SCIENCES II Table 1. Distribution of Adult Health Literacy Score by Demographic Characteristics (N=301) Socio-demographic Characteristics Adult Health Literacy Score N (%) ± SD U, X2 p Age 22-29 61(20) 15.54 ± 3.49 X2 = 18,651 2>1, 3>4, 2>4 0.001 30-39 138(46) 16.71 ± 3.07 40-49 77(26) 16.37 ± 2.96 50+ 25(8) 13.60 ± 4.47 Gender Female 110(36) 17.25 ± 3.16 U = 6991,00 0.001 Male 191(64) 15.48 ± 3.32 Education Bachelor degree 35(12) 14.51 ± 4.34 X2 = 7,819 2>1, 3>1 0.02 Master’s degree 112(37) 16.44 ± 3.29 Ph.D. degree 154(51) 16.27 ± 3.08 Marital Status Married 211(70) 16.26 ± 3.54 U = 8327,50 0.089 Single 90(30) 15.83 ± 2.91 Region Marmara 39(13) 16.05 ± 2.95 X2 = 8,797 0.117 Aegean 33(11) 16.03 ± 2.39 Mediterranean 20(7) 14.70 ± 3.84 Balck Sea 115(38) 16.32 ± 3.79 Central Anatolia 72(24) 16.58 ± 3.09 Eastern/Southeastern Anatolia 22(7) 15.27 ± 3.13 Academic Title Research Assistant 71(24) 16.35 ± 2.83 X2 = 6,761 0.239 Lecturer 10(3) 14.10 ± 4.81 Instructor 77(26) 16.35 ± 3.86 Assistant Professor 108(36) 16.36 ± 2.89 Associate Professor 23(7) 15.47 ± 3.88 Professor 12(4) 14.33 ± 3.86 Length of employment 0-5 91(30) 15.86 ± 3.13 X2 = 8,605 0.072 6-10 70(23) 16.61 ± 3.44 11-15 49(17) 16.75 ± 2.89 16-20 44(14) 16.27 ± 3.20 21+ 47(16) 15.14 ± 4.09 Job Satisfaction Neutral 18(6) 16.0 ± 2.99 X2 = 4,408 0.110Satisfied 156(52) 16.40 ± 3.61 Very Satisfied 127(42) 15.81 ±3.08 Income Satisfaction Yes 190(63) 16.03 ± 3.64 X2 = 3,180 0.204No 25(8) 15.36 ± 3.22 Neutral 86(29) 16.57 ± 2.66
HEALTH LITERACY OF ACADEMICIANS AND ITS AFFECTING FACTORS 9 Table 2. Distribution of Adult Health Literacy Score by General Health Characteristics (N=301) General Health Characteristics Adult health Literacy Score N(%) ± SD U, X2 p General Health Poor 9(3) 14.55 ± 3.46 X2 = 3,812 0.282 Fair 77(26) 16.10 ± 3.27 Good 187(62) 16.28 ± 3.48 Very Good 28(9) 15.71 ± 2.78 Chronic Disease Yes 54(18) 16.48 ± 3.74 U = 6283,50 0.503 No 247(82) 16.05 ± 3.28 Regular Medicine Yes 51(17) 16.49 ± 3.35 U = 6111,00 0.639 No 250(83) 16.06 ± 3.37 Smoking Yes 79(26) 15.73 ± 3.27 U = 7825,00 0,153 No 222(74) 16. 27 ± 3.39 Alcohol Yes 31(10) 16.25 ± 2.59 U = 4181,00 0.993 No 270(90) 16.11 ± 3.45 Proper Diet Yes 203(67) 16.47 ± 3.29 U = 7954,50 0,005 No 98(33) 15.41 ± 3.41 Regular Exercise (#of days in a week) 0130(43) 15.98 ± 3.42 X2 = 8,583 0.072 1 40(13) 15.57 ± 3.21 264(21) 16.39 ± 3.29 3 41(14) 17.29 ± 2.63 4 26(9) 15.26 ± 4.14 Sleeping Hours 569(23) 15.37 ± 3.62 X2 = 8,824 2>1, 2>3 0.012 7-8 211(70) 16.54 ± 3.04 9+ 21(7) 14.52 ± 4.66 4. Discussion and Conclusion Socio-demographic factors affect the health literacy level of academics. In our study, we attempted to determine the health literacy levels of academics. The results showed that health literacy scores of the one who is younger than 30 and older than 50 were found lower. Since younger ones do not have so many health problems, this can cause them not to be so much interested in such issues. Older people might have difficulties due to deteriorating mental and physical abilities. Also, although diseases increase with aging, after the age of 50+, patient relatives generally take care of health problems in Turkey, this might
10 INNOVATIVE APPROACHES IN MEDICINE AND HEALTH SCIENCES II cause the older ones to have lower health literacy as well. The ones who are in the middle can have higher scores due to getting married, having kids, and taking care of their relatives’ health. Similar results were found by Yılmazel. (22) Additionally, in the current study, while a statistical significant existed among health literacy mean scores of the academics by sex and education, there is not a significant difference in marital status and a longest-lived region (8,16). The current study couldn’t find any significant difference in the health literacy scores of academics regarding the length of work experience, the satisfaction of occupation, and income. However, in the study of Yılmazel (22) which was conducted with primary school teachers, ten years or less working experience was significantly related to higher health literacy scores. Also, while some studies showed income level was significantly associated with health literacy (8, 20) others found no significant relation and supported our findings (16,18). According to the general health situation, chronic disease, and regular medicine intake, we could not find any significant difference among health literacy scores of academics. Although Barutcu and Duzen (16) found a significant difference in health literacy mean scores by having a chronic disease, the finding from the studies of Yılmazel (22) and Üçpunar (20) could not find any significant difference between health literacy levels and having chronic diseases and supported our results. Finally, we could not find any significant difference among health literacy scores of academics and smoking, alcohol consumption, and doing regular exercises. It was supported by the studies of Moeini and colleagues (23) Yılmazel (22) and Üçpunar (20). However, a statistically significant difference was found between health literacy scores having proper diet and the daily sleeping hours. In this regard, the ones who sleep 7-8 hours have higher scores. The role of sleep in maintaining good health is essential. Thus, the ones who use sleeping hours wisely behave more carefully about other health issues. We were not able to compare our findings with other studies. Although sleeping is one of the fundamental concept regarding health literacy, according to our knowledge, it was not controlled in the literature. This initial study showed the health literacy level and affecting factors of the academics. The results indiczted that middle age, female, higher educated academics had higher health literacy scores. Also, sleeping hours and having proper diet were the only factors that affected the academics’ health literacy level. The academics play the primary role in preparing and integrating student curriculum, and teaching and guiding students. They should emphasize the
HEALTH LITERACY OF ACADEMICIANS AND ITS AFFECTING FACTORS 11 importance of health literacy. Therefore, this study will contribute to putting health literacy on the agenda in academics in Turkey. Since college students are the future of a country, more health literate academics in the college will lead to more health literate students, and it will provide more healthy communities and developed countries. Limitation The study may include a few limitations. For example, this study used a crosssectional design, which limits the ability to make inferences (e.g., cause-effect) about the results. Also, it limits the prediction of how scores on the variables may change over time. Additionally, we did not collect data about the way of getting information about health literacy, which is important to consider for future studies. Finally, since the finding of the study does not represent the general population, there are potential risks to threat the external validity of the study. Recommendations for Future Research Future studies should look at deeply about the role of sleep in health literacy and the association between sleep disorders and health literacy. There are very few studies that looked at the relationship between sleep disorder outcomes and health literacy (24). Also, future research should be focused on intervention that give a role to academics. Funding This work did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Compliance with Ethical Standards Written ethical permission was obtained from Kastamonu University Scientific Research and Publication Ethic’s Council, with a number of 2017/3. Moreover, volunteer subjects who participated in the research were informed about the research and signed an inform consent form. Conflict of Interest The authors have no conflicts of interest to disclose. REFERENCES 1) Yılmaz M, Tiraki Z. What is health literacy? How to measure? DEUHFED. 2016);9(4):142-147. 2) Sand-Jecklin K, Murray B, Summers B, Watson J. Educating nursing students about health literacy: From the classroom to the patient bedside. Online Journal Issues Nursing, 2010; 15(3).
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15 CHAPTER II GENETIC LITERACY: CONCEPTUAL FRAMEWORK, IMPORTANCE, AND INTERDISCIPLINARY REFLECTIONS Seliha Seçil BAYRAM (Asst. Prof.), Karabük University, Health Services Vocational School, Medical Services and Techniques Department, Karabük, Türkiye E-mail: [email protected] ORCID:0000-0002-6061-6160 1. Introduction Genetic literacy, with its growing importance in modern society, plays a critical role in enhancing individuals’ abilities to understand, interpret, and use genetic information and technologies. Evaluating the current body of knowledge in the field of genetic literacy can provide valuable insights for identifying research gaps and determining future research directions. This information can contribute to the development of genetics education curricula, more effective use of resources, and help policymakers make informed decisions. Genetic literacy refers to individuals’ ability to understand and use genetic information and technologies. This concept reflects the public’s level of knowledge regarding topics such as genetic tests and the interpretation of their results (Milo Rasouly et al., 2020). The importance of genetic literacy can be explained by the fact that, as genetic information and technologies rapidly advance, it is becoming increasingly necessary for society to be informed about these subjects (Bowling et al., 2008). Genetic literacy helps individuals make informed decisions about genetic tests and their results. Low genetic literacy can make it difficult to understand the limitations of genetic tests and may reduce satisfaction with the informed consent process (Milo Rasouly et al., 2020). This situation can negatively impact the clinical effectiveness of genetic tests. Genetic literacy also influences individuals’ attitudes toward genetic research
16 INNOVATIVE APPROACHES IN MEDICINE AND HEALTH SCIENCES II and practices. People with high genetic literacy tend to view DNA research more positively and are more open to genetic testing. In conclusion, genetic literacy is critically important for society’s ability to understand, evaluate, and use genetic information and technologies. Therefore, the development and dissemination of genetic education programs will be a significant step toward increasing society’s genetic literacy (Olwi et al., 2016). The significance of this study lies in its ability to reveal the current knowledge structure in the field of genetic literacy and to identify future directions for research. Additionally, by detecting gaps and potential opportunities for collaboration in the field, it can serve as a guide for researchers, educators, and policymakers (Fauzi, 2023; Wu et al., 2023). In an era where genetic technologies are rapidly advancing, this is important for increasing society’s genetic knowledge and addressing ethical issues. 2. Literature The concept of literacy has evolved beyond basic reading and writing skills to encompass domain-specific knowledge and competencies across various fields. This trend is particularly evident in the emergence of genetic literacy, which focuses on understanding and applying genetic information in healthcare services and decision-making processes. Genetic literacy is especially important in today’s healthcare context, where genomic information is playing an increasingly significant role in clinical care. Research has shown that health literacy, including genetic knowledge, is associated with better understanding and communication of genetic information and family health history (Kaphingst et al., 2016). In a study conducted in a medically underserved population, limited health literacy was associated with lower genetic knowledge and awareness of family health history, highlighting the need for targeted educational strategies to improve genetic literacy (Kaphingst et al., 2016). The importance of genetic literacy extends beyond individual health outcomes to broader societal implications. As genomic information becomes more prevalent in healthcare, there is an increasing need to examine how individuals understand, value, and communicate about this information (Kaphingst et al., 2016). Enhancing genetic literacy can contribute to reducing inequalities in health outcomes and ensure that the benefits of genomic medicine are accessible to all populations. The concept of literacy is used in every field to understand the nature of a concept and to gain competence in that field through the attitudes developed based on this concept. This situation is also noteworthy in the field of genetics. Therefore, this study specifically focuses on the concept of genetic literacy.
23 CHAPTER III FUNDAMENTALS AND METHODS OF GENE TRANSFER Murat ÖZTÜRK1 & Mustafa SANDIKÇI2 1(Dr.), Aydın Adnan Menderes University, Faculty of Veterinary Medicine, Department of Histology-Embryology, E-mail: [email protected] ORCID: 0000-0002-4774-6980 2(Prof. Dr.), Aydın Adnan Menderes University, Faculty of Veterinary Medicine, Department of Histology-Embryology, E-mail: [email protected] ORCID: 0000-0002-9126-1016, 1. Introduction Deoxyribonucleic acid (DNA) is the molecule that contains all the genetic information essential for the formation and functionality of cells. The temporal, spatial, and structural instructions for protein synthesis are encoded within the polymeric structure of DNA. The transfer of genetic information on DNA depends on the sequence of nucleotides along the strand. Specific DNA regions carrying this information are called genes, and genes play a fundamental role in determining the biological characteristics of an organism (1). Advances in molecular biology have enabled genes to be regarded not only as hereditary units but also as macromolecules that can be experimentally manipulated. In this context, genes can be identified, isolated, cut, recombined, and even transferred across species (2). The process of transferring a gene (transgene) into a target cell, in which the gene crosses the cell membrane and integrates into the host genome, is defined as gene transfer (transfection). Today, transfection techniques are broadly divided into two major categories: viral and non-viral vectors, with retroviruses and adenoviruses being the most commonly employed viral vectors. Non-viral vectors are further subdivided into two groups: physical
24 INNOVATIVE APPROACHES IN MEDICINE AND HEALTH SCIENCES II transfection methods and chemical transfection methods. Physical methods include microinjection, particle bombardment, and electroporation (3–5), while chemical methods include cationic lipids (liposomes), calcium phosphate, and cationic polymers (6–8). The purpose of this section is to define the gene transfer process and its basic mechanism, and to outline the main gene transfer methods currently in use, thereby providing a scientific framework for the subject. 2. Gene Transfer DNA carries genetic information through nucleotides arranged in a linear sequence, and its information-bearing segments are organized into functional units called genes (9). Genes contain the instructions necessary for the synthesis of proteins. The genetic information encoded within the gene regions of DNA dictates the amino acid sequence of polypeptides that form proteins. The accurate transfer of genetic information into proteins takes place in two steps, termed transcription and translation. The central dogma articulates the canonical pathway in which genetic information encoded in DNA is transcribed into RNA and then translated into proteins (10). Thus, the flow of genetic information encompasses replication, in which DNA produces its own copy; transcription, in which information in DNA is transferred to RNA; and translation, in which RNA conveys this information into proteins. However, in certain cases, such as in retroviruses, the RNA molecule serves as a template for DNA synthesis. Reverse transcription represents a deviation from the canonical flow of genetic information, occurring in the opposite direction from the usual DNA-to-RNA pathway (11). Reverse transcription is catalyzed by the enzyme reverse transcriptase, which was first identified in 1970 by Temin and Baltimore in RNA genome–containing viruses. Viruses that perform reverse transcription are called retroviruses (12). Advances in molecular genetic techniques have made it possible to recombine DNA molecules from different sources in vitro. A DNA molecule obtained by cutting DNA fragments from various sources with restriction enzymes and inserting them into a vector DNA, such as a bacterial plasmid, to allow replication is termed recombinant DNA. The process of constructing a new DNA molecule with novel gene combinations for a specific purpose is referred to as recombinant DNA technology. Using these technologies, the successful integration of a target gene fragment into the host cell’s DNA molecule, following its passage through the cell membrane, is defined as gene transfer (13–15).
FUNDAMENTALS AND METHODS OF GENE TRANSFER 25 Gene transfer comprises two distinct stages. The first stage involves a mechanism that enables the genetic material to be transported from the extracellular environment, across biological membranes, and into the nucleus. This mechanism allows the transferred genetic information to merge with the genome of the target organism. The second stage provides the means for the new genetic information to become incorporated into the host genome as a functional part of it (16). 3. History Of Gene Transfer The phenomenon of genetic material transfer between cells was initially shown in bacteria. The initial mechanism was discovered in 1928 by Frederick Griffith using two different strains of Streptococcus pneumoniae, known as R (Rough) and S (Smooth). Griffith showed that the lethal S bacteria, even when killed by heat, could render the non-lethal R bacteria pathogenic upon contact (17). However, he was unable to identify the substance responsible for this transformation. Later, in 1944, Oswald Avery determined that the material transferred between cells was DNA (18). A second form of gene transfer was discovered in 1946 by Joshua Lederberg and Edward Tatum in Escherichia coli, and this process was named conjugation. Conjugation entails the transfer of the entire genome or a portion of the donor cell’s genetic material to a recipient cell, occurring either through direct cell-tocell contact or via sex pilus (19,20). Subsequently, in 1951, Joshua Lederberg and Norton Zinder identified a new type of gene transfer in Salmonella species, mediated by a bacteriophage. In this process, the phage occasionally packages a portion of the host cell’s DNA and transfers this DNA fragment to another host cell during subsequent infections. This gene transfer mechanism was termed transduction (21,22). This discovery was of fundamental scientific importance because it explained how bacteria of different species could rapidly acquire resistance to the same antibiotic. Furthermore, understanding that phages could transfer genetic material between bacteria laid the groundwork for potential studies on viruses that infect eukaryotic hosts (23). Focusing on how genes are transferred, modified, and regulated, Szybalski was the first researcher to experimentally demonstrate in 1962 that genetic information could be transferred into mammalian cells via DNA and that genetic defects could be corrected through this method (24). Following studies showing that phages could transfer genetic material from one bacterium to another, Howard Temin discovered that, similarly, specific genetic mutations could be inherited as a result of viral infection. In his work
26 INNOVATIVE APPROACHES IN MEDICINE AND HEALTH SCIENCES II on chicken cells infected with Rous Sarcoma Virus (RSV), he demonstrated that virus-specific genetic mutations carrying the information responsible for the formation of new RSV generations were stably inherited by the cells (25). Because RSV is an RNA virus, Temin’s studies revealed that genetic information could be transferred not only from DNA to RNA but also from RNA to DNA. These findings later paved the way for the discovery of RNA-dependent DNA polymerases (reverse transcriptases). Moreover, it became clear that new phenotypic traits acquired through the chromosomal integration of foreign genetic material into the host genome could be stably inherited (25–27). It was found in various studies that viruses possess features that could be highly useful for transferring genes into relevant cells. Successful results from cell transformation studies raised the idea that genetic engineering could potentially be used in the treatment of genetic disorders (28). Furthermore, viruses have been reported to hold potential in somatic cell genetics and gene therapy. However, it has been emphasized that viruses intended for this purpose must be re-engineered by removing their pathogenic genes and replacing them with therapeutic ones (29). In a study conducted in 1968, Rogers and Pfuderer provided the first evidence of virus-mediated gene transfer. In this study, Tobacco Mosaic Virus (TMV) was used as a vector. A polyadenylate (poly A) tail was added to the viral RNA, and the resulting poly A–tailed virus was used to infect Turkish tobacco plants. The study demonstrated that the transfer of the poly A–tailed virus was successful and that it was expressed in the Turkish tobacco plants. This indicated that, in vitro, a nucleotide sequence could be added to viral RNA, and the virus could be used as a vector to deliver the desired genetic information (30). In the early 1970s, Paul Berg and his colleagues created a recombinant DNA fragment by combining the DNA of the E. coli chromosome with that of Simian Virus 40 (SV40), a monkey virus, marking the first step in recombinant DNA (rDNA) technology. Berg and his team first isolated the DNA from E. coli and SV40. They then digested both DNA samples using the endonuclease EcoRI, and the resulting chromosomal and viral DNA fragments were combined in a reaction tube containing DNA ligase, resulting in a hybrid molecule composed of E. coli and SV40 DNA. This experiment demonstrated that DNA fragments from different sources could be cut with the same enzyme and subsequently joined to form a recombinant DNA molecule (31). In the following years, research in genetic engineering and biotechnology accelerated. Despite the development of various gene transfer techniques, one
FUNDAMENTALS AND METHODS OF GENE TRANSFER 27 of the major challenges in gene transfer studies has been delivering the desired DNA molecules into target cells. Due to the limited ability of DNA to enter cells and the potential for enzymatic degradation, DNA transfection is usually performed using a vector (32). Once the gene to be transferred is obtained, it is inserted into a carrier vector. The vector’s principal function is to deliver the inserted gene to the target cell. Upon entry, it carries the genetic material to the nucleus, enabling the gene to integrate into the genomic DNA. Therefore, appropriate vector selection is critical for the success of gene transfection (33). Today, vectors are generally classified into two groups: viral and non-viral. Transduction denotes gene transfer using viral vectors, in contrast to transfection, which involves gene delivery via non-viral vectors. Examples of viral vectors include adenoviruses, adeno-associated viruses, lentiviruses, and retroviruses. Non-viral vectors are further subdivided into physical and chemical transfection methods (34). 4. Viral Vector-Mediated Gene Transfer Gene transfer via viruses is likely the original asexual gene transfer approach for mammals and was among the first gene transfer methods tested as a laboratory technique (35). The ability of viruses to infect target cells as part of their natural life cycles has made them preferred tools in gene transfer studies. Viruses, being obligate intracellular parasites, depend on host cellular processes for their replication. The mechanisms by which they deliver their genetic material to host cells differ depending on whether they are enveloped or non-enveloped. Typically, enveloped viruses bind to specific receptors on the cell surface and are then taken up either by direct fusion with the plasma membrane or via endocytosis followed by fusion with the endosomal membrane. In contrast, non-enveloped viruses transfer their genetic material into the host cell through a process known as penetration (36,37). Despite their high transfection efficiencies, the major disadvantages of gene transfer via viral vectors are their intrinsic immunogenicity and cytotoxicity (38). The introduction of a viral vector can trigger inflammatory responses and insertional mutagenesis, as viral vectors tend to integrate randomly into the host genome. This can result in disruption of tumor suppressor genes, activation of oncogenes, or interruption of genes essential for cellular function (39). Additionally, the risk of transgene integration, viral recombination, challenges
28 INNOVATIVE APPROACHES IN MEDICINE AND HEALTH SCIENCES II in vector production and repeated administration, and limitations on the size of genetic material that can be carried pose general reliability issues and potential hazards to laboratory personnel (40). For these reasons, although viral vectormediated gene transfer is highly efficient and easy to use, considerable effort has been devoted to developing non-viral gene transfer methods. 4.1.Retroviruses Retroviruses are enveloped viruses carrying a single-stranded RNA genome of 7 to 10 kilobases (kb) in length (41). They enter target cells via receptor-mediated endocytosis. Inside the host cell, viral RNA is transformed into double-stranded DNA by reverse transcriptase, after which integrase mediates its integration into the host genome. The integrated form is referred to as a provirus. Access to the host genome occurs when the nuclear membrane disappears during cell division; therefore, most retroviruses only affect dividing cells, which limits the use of retroviral vectors (42). Retroviruses have three distinct transcriptional units: gag, pol, and env (42). Structural proteins are encoded by the gag region, reverse transcriptase by pol, and envelope proteins by env. Additionally, viral RNA carries long terminal repeats (LTRs) at both ends that function as promoters and enhancers, and psi (ψ) regions necessary for genome packaging. In retroviral vectors, the LTR and psi regions are removed from the viral genome, eliminating the virus’s ability to cause infection (43). The efficient integration of retroviruses into animal cell genomes, their ability to provide stable transgene expression, and the ease of manipulating the viral genome to include therapeutic genes have encouraged researchers to widely use retroviruses in gene therapy studies (44). However, because they integrate only into actively dividing cells, in vivo applications often do not achieve the desired efficiency (45). Another limitation is the physical size restriction of retroviruses (<10 kb), which constrains the size of the genetic material that can be delivered (16). Moreover, random integration of the transferred gene into the host genome can lead to insertional mutagenesis (46). In gene therapy studies, insertional mutagenesis has been associated with the development of leukemia (47). Retroviral vectors exhibit high gene transfer efficiency in animal cells and have been widely used in experimental and clinical studies. However, due to the risk of insertional mutagenesis, researchers have shifted toward using alternative viral vectors.
FUNDAMENTALS AND METHODS OF GENE TRANSFER 29 4.2.Adenoviruses Adenoviruses were first isolated and characterized in 1953 by Rowe and colleagues from human adenoids undergoing spontaneous degeneration in tissue culture, and were shown to be agents causing acute respiratory tract infections (48). In 1977, Graham and colleagues developed the HEK293 cell line, which was transformed with human adenovirus type 5 (Ad5) DNA and enabled the production of recombinant adenoviruses. This development marked an important milestone in the use of adenoviral vectors for gene transfer and has since attracted considerable attention in gene therapy and biotechnological applications (49). Adenoviruses, non-enveloped and structurally complex, range from 70 to 90 nm in diameter and carry a double-stranded DNA genome of 36–38 kb. The integration and replication of viral DNA with the host cell genome occurs in the nucleus of the host cell (50). Unlike retroviruses, they can infect not only dividing but also non-dividing cells. Viral DNA does not integrate into the host cell genome, and therefore, the expression of the transferred gene is transient. Consequently, multiple administrations of the viral vector may be required (34). To date, 57 different adenovirus serotypes have been identified in humans, which are also classified into seven subgroups (A to G) based on their hemagglutination properties (51). In gene transfer studies, serotypes belonging to subgroup C, specifically type 2 (Ad2) and type 5 (Ad5), are frequently used as vectors. The main reason is that these serotypes are not associated with severe pathogenicity in humans and are therefore considered safer options for in vivo applications (52). Adenoviral transcription occurs in two stages, designated early (E) and late (L), with the early phase comprising the principal gene regions E1, E2, E3, and E4. Among these, the E1 region is particularly critical for initiating and sustaining viral replication. The E1 gene region is also critical for host cell transformation and the oncogenic potential observed in adenovirus infections. The E2 region regulates the synthesis of DNA-binding proteins and DNA polymerase required for replication. The E3 region controls immune responses and lysis of the host cell infected by the adenovirus. The E4 region plays a regulatory role in the transition between early and late phases. Deletion of these genes has been used to develop new vector systems with higher transgene capacity (50,53). Initially, the E1 and E3 gene regions of adenoviruses are removed. By deleting the E3 region, the vector’s transgene-carrying potential can be increased.
30 INNOVATIVE APPROACHES IN MEDICINE AND HEALTH SCIENCES II The E3 region does not affect viral replication under in vitro conditions. A transgene (therapeutic gene) is inserted into the deleted E1 gene region. Human embryonic kidney cells (HEK293) are used to amplify the prepared E1 adenoviral vectors. Cells infected with the E1 adenoviral vector (HEK293) produce viral DNA containing the transgene (52,54). Currently, adenoviruses are the most commonly used viral vectors in clinical trials. Compared to retroviruses, they are attractive for gene transfer studies due to their higher transgene-carrying capacity, low risk of mutation since they do not integrate into the host cell genome, high transduction efficiency with strong transgene expression, and ability to be produced at high titers in vitro (4). Their disadvantages include transient transgene expression and the potential to induce immune responses (55). 5. Non-Viral Gene Transfer Methods In general, viral vectors provide enhanced transfection efficiency relative to non-viral methods. However, the major limitations of viral vectors include the risks of immunogenic and cytotoxic responses. In addition, the potential to cause insertional mutations is considered a significant drawback. Therefore, researchers have turned to non-viral vectors for their better biosafety profile. Non-viral vectors offer significant advantages over viral vectors due to their low pathogenicity, low immunotoxicity, low cost, and ease of production. However, their lower gene transfer efficiency and transient transgene expression remain disadvantages (33). The plasma membrane is an amphipathic structure that defines the boundaries of the cell and protects intracellular organelles from dispersion. This structure contains mechanisms that regulate controlled material exchange between the cell and its microenvironment. In contrast, DNA is a sizable, hydrophilic, anionic polymer that is vulnerable to nuclease-mediated degradation in biological matrices. Without the use of specialized methods, DNA molecules cannot cross the physical barrier of the plasma membrane and enter the cell. Theoretically, three major barriers must be overcome for successful DNA transfer into a host cell: first, delivery of DNA to the plasma membrane of target cells; second, transfer of DNA across the plasma membrane into the cytoplasm; and finally, transport of DNA from the nuclear membrane into the nucleus to enable gene expression. Therefore, it is crucial for an ideal vector to meet these requirements to achieve successful transfection (56,57). Several extracellular and intracellular barriers limit the efficient transfer of genes using non-viral methods (58).
FUNDAMENTALS AND METHODS OF GENE TRANSFER 31 One of the most restrictive steps for successful gene transfer is the passage of naked DNA across the cell membrane. A repulsive force is generated as a result of the negative charges present on both DNA and the cell membrane. Therefore, the entry of naked DNA into the cytoplasm can be facilitated either by physical transfection methods (e.g., creating transient pores in the cell membrane) or by chemical transfection methods (e.g., enhancing cellular uptake capacity) (58). 5.1.PhysicalTransfectionMethods The fundamental principle of physical transfection methods is the direct delivery of the transgene into the cytoplasm or nucleus by some type of physical force, without requiring interaction with the plasma membrane. This bypasses the endosomal pathway and thereby limits the amount of damage to the transgene (50). Methods include direct microinjection, particle bombardment, and electroporation (3,5,50). 5.2.Microinjection The microinjection method is a widely used transfection technique that allows the direct injection of a transgene into the cytoplasm or nucleus of a single living cell. This method employs a glass microcapillary pipette (micropipette), a micromanipulator to precisely control the pipette’s movement, and a microinjector to facilitate the flow of the solution. The delivery of genetic material from the micropipette tip relies on hydrostatic pressure, and the entire procedure is performed under controlled conditions using a microscope (3). The microinjection technique, initially reported by Marshall Barber in 1911, only gained widespread application in the 1970s. Later, Gordon et al. produced transgenic mice by microinjecting plasmid DNA into the pronuclei of fertilized mouse oocytes (59). Today, this approach is commonly used to generate transgenic animals such as mice (60), miniature pigs (61) and cattle (62). The most significant advantage of this technique is that it allows the direct delivery of the transgene into the nucleus. Direct nuclear injection protects the transgene from cytoplasmic nuclease activity, which can significantly damage or reduce the amount of transgene and thus affect transgene expression efficiency (63). Additionally, microinjection is a simple, effective, reproducible, and non-toxic method for gene transfer. The main disadvantage of the microinjection technique is that, unlike methods that can deliver a transgene to many cells simultaneously, it only allows injection into a single cell at a time. Microinjection, consequently, is time-consuming and exhibits low efficiency in
32 INNOVATIVE APPROACHES IN MEDICINE AND HEALTH SCIENCES II terms of throughput (64). Furthermore, the procedure requires highly precise micromanipulation equipment. Other important drawbacks include the high cost of the equipment, long duration of the procedure, and relatively low levels and persistence of transgene expression (58,65). 5.3.ParticleBombardment Particle bombardment, also referred to as the gene gun method or microbullet gene transfer, is employed to introduce transgenes or proteins into target cells or tissues. This gene transfer technique was first described by Sanford and colleagues in 1987 and was initially applied successfully to plant tissues (66). It was later adapted to mammalian cells, where successful outcomes were also achieved (4). In this method, gene transfer is accomplished by bombarding target cells with DNA-coated gold particles using pressurized inert gas such as helium or a high-voltage electrical discharge. In short, the principle relies on delivering heavy metal particles coated with the gene of interest into target cells at a specific velocity (67). Typically, heavy metals such as gold, tungsten, or silver are used as carrier particles (68). This technique can target a large number of cells simultaneously and is not limited to nuclear transfer. It can also be applied to other organelles, such as mitochondria (69). The main advantages of this method are the ability to transfer the desired transgene into multiple cells at the same time, and the fact that the transgene crosses the cell membrane directly, avoiding interactions with various receptors and molecules on the cell surface (70). However, the major disadvantage of this technology is that the metal particles, when delivered at a specific velocity, can physically damage target cells or tissues. Therefore, optimization is necessary to minimize tissue or cell damage caused by the impact force of the metal particles (71). 5.4.Electroporation Electroporation is a gene transfer method based on the principle of applying short pulses of high-voltage electric fields to cells or tissues, thereby creating temporary nanometer-sized pores in the cell membrane through which DNA can pass (72). The first evidence that gene transfer could be achieved by electroporation was obtained by Neumann and colleagues using mammalian cells (73). Applying an electric field greater than the membrane capacitance
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45 CHAPTER IV IMMUNOLOGICAL MECHANISMS AND INNATE IMMUNE CELL–TARGETED THERAPIES IN ACETAMINOPHENINDUCED HEPATOTOXICITY Zeynep KARAKOY1 & Zekai HALICI2 & Elif ÇADIRCI3 1Erzincan Binali Yıldırım University, Department of Pharmacology, E-mail: [email protected], ORCID: 0000-0002-4281-0103 2(Prof. Dr.), Atatürk University, Department of Pharmacology, E-mail: [email protected] ORCID: 0000-0001-6854-6059 3(Prof. Dr.), Atatürk University, Department of Pharmacology, E-mail: ecadir[email protected] ORCID: 0000-0003-0836-7205 1. Introduction The liver, recognized as a central organ in the immune system, is notable not only for its metabolic and detoxification functions but also for its active role in regulating immune responses. Liver tissue harbors a wide variety of immune cell populations, and these cells are responsible for maintaining the balance between immune tolerance and inflammatory response in an environment constantly exposed to antigens and microorganisms from the circulation (1). Intrahepatic lymphocytes, in particular, constitute approximately 25% of the total non-parenchymal cell population and represent one of the fundamental components of the hepatic immune system. These cells include natural killer (NK) cells, natural killer T (NKT) cells, T lymphocytes, and B cells. Intrahepatic
46 INNOVATIVE APPROACHES IN MEDICINE AND HEALTH SCIENCES II lymphocytes are involved in the first line of defense and play a key regulatory role in sustaining immunological stability. Therefore, the liver is defined not only as a metabolic organ but also as a dynamic immunological microenvironment at the center of immune surveillance and inflammatory regulation. (2). Beyond conventional lymphocytes, the liver harbors distinct lymphoid cell subsets that utilize alternative mechanisms for antigen recognition. Among these are NKT cells, which recognize glycolipid antigens through invariant receptors, and γδ T cells, which express a γδ T cell receptor (TCR) instead of the conventional αβ TCR found in typical T lymphocytes. Collectively, NKT and γδ T cells account for around 65% of all lymphocytes present in the healthy liver (3). Beyond lymphocytes, the liver encompasses several distinct populations of innate immune cells. Among these, KCs, which constitute approximately 20% of the total non-parenchymal cells, and NK cells, representing about 8% of the non-parenchymal cell population, play prominent roles. These innate immune cell subsets are crucial for maintaining hepatic immune tolerance and orchestrating rapid defense responses against infections (4, 5). In addition to its resident immune cell populations, the liver’s distinctive anatomical position and its highly organized vascular architecture—including the dual blood supply from the hepatic artery and portal vein—(6) enable the efficient recruitment and trafficking of circulating leukocytes such as neutrophils, monocytes, and lymphocytes during episodes of tissue injury or inflammation. This unique vascular connectivity allows immune cells to be rapidly delivered to the hepatic parenchyma and sinusoidal spaces, where they participate in pathogen clearance, cytokine release, and the orchestration of both innate and adaptive immune responses essential for maintaining hepatic homeostasis and initiating tissue repair (3). APAP overdose, hepatocytes initiate an inflammatory response through the secretion of damage-associated molecular patterns as a result of injury caused by reactive metabolites (7). This condition triggers the recognition and activation of innate immune cells in the liver, including macrophages, neutrophils, NK cells, and NKT cells. Whether NK and NKT cells play a direct role in the pathogenesis of acetaminophen-induced liver injury (AILI) remains an open question. Nonetheless, the ability of NK and NKT cells to be activated by a wide range of stimuli—such as lipid antigens, bacterial and viral pathogens, and nicotinamide adenine dinucleotide (NAD‐)—leads to considerable variability in their functional states (8) their variable activation states may represent an important risk factor in the development of AILI or other forms of drug-induced liver injury (3).
IMMUNOLOGICAL MECHANISMS AND INNATE IMMUNE CELL–TARGETED . . . 47 Taken together, these processes indicate that the liver is not merely a metabolic organ but also a dynamic immunological microenvironment that plays a central regulatory role in maintaining immunological balance and in the mechanisms underlying drug-induced liver injury. The effect of innate immune response in APAP-induced liver injury is shown in Figure 1. Figure 1: Innate Immune Mechanisms in AILI 2. Potential Mechanisms of APAP Toxicity APAP is a commonly used analgesic (pain reliever) and antipyretic (fever reducer) agent and is generally considered safe when administered at therapeutic doses. However, if the drug is taken in doses above those recommended for accidental or suicidal purposes, very serious toxic effects may occur. This toxicity is largely concentrated in the liver and is defined as hepatotoxicity. In cases of overdose, the liver’s detoxification capacity is insufficient, and the accumulation of toxic metabolites (particularly N-acetylp-benzoquinone imine; NAPQI) causes oxidative stress in hepatocytes and subsequent cell death. Studies have shown that deaths in these cases mostly occur as a result of acute liver failure characterized by hepatic central lobular necrosis. This condition can be accompanied by serious clinical complications such as irreversible impairment of hepatic functions, coagulation disorders, encephalopathy, and systemic multiple organ failure. Therefore, APAP-related liver damage is considered an important public health issue from both clinical and toxicological perspectives. APAP functions as a pro-toxin, exerting its
48 INNOVATIVE APPROACHES IN MEDICINE AND HEALTH SCIENCES II hepatotoxic effects through the generation of the highly reactive metabolite NAPQI. This metabolite is predominantly produced via the biotransformation of APAP by cytochrome P450 (CYP) enzymes, particularly CYP2E1 and CYP3A4 isoforms. (9). At therapeutic doses, NAPQI is detoxified via conjugation with the major reactive metabolite of APAP, glutathione (GSH), but when a toxic dose of APAP is ingested, the glucuronidation and sulphation pathways are overloaded and liver GSH is depleted. Accumulation of NAPQI then covalently binds to cysteine groups in cellular proteins to form APAP-protein adducts, leading to oxidative stress reactions, mitochondrial dysfunction, and DNA damage (10). During normal therapeutic use of APAP, the decrease in intracellular GSH levels is balanced by endogenous antioxidant regeneration mechanisms. However, in cases of overdose, excessive accumulation of NAPQI depletes hepatic GSH stores, resulting in severe oxidative stress and tissue damage in various organs, primarily the liver, kidneys, and central nervous system (11). 2. Innate Immune Responses in APAP Toxicity 2.1.KupfferCells(KCs) KCs, the specialized macrophages residing in the liver, are key contributors to immune-mediated hepatic injury (12). KCs perform multiple essential functions, such as phagocytosis, endocytosis, immune regulation, and the production and release of various biologically active mediators (13). In general, KCs are divided into two polarization states: M1 and M2. M1 KCs release pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α, which determine the inflammatory signalling pathway (12). The role of KCs in AILI is thought to be mediated by the production of cytokines and reactive oxygen and nitrogen species (10). Recent evidence indicates that KCs exhibit a biphasic role in liver injury, both aggravating liver injury and facilitating liver regeneration (3, 14). The progression of AILI is influenced by the balance between M1 and M2 macrophages (15). M2 macrophages play a crucial role in regulating liver repair and resolving inflammation through the production of anti-inflammatory cytokines such as IL-4, IL-10, and IL-13. In addition, they facilitate tissue regeneration by clearing apoptotic neutrophils and releasing key mediators involved in wound healing (10, 16). There is other evidence that KCs contribute to APAP hepatotoxicity. Inactivation of KCs has been reported to significantly attenuate liver damage and lower transaminase levels in mice exposed to APAP (14, 17).
IMMUNOLOGICAL MECHANISMS AND INNATE IMMUNE CELL–TARGETED . . . 55 11. Josephy PD. The molecular toxicology of acetaminophen. Drug Metab Rev. 2005;37(4):581-94. 12. Liu WH, Zeng XC, Liu YT, Liu JF, Li CP, Chen LL, et al. The Immunological Mechanisms and Immune-Based Biomarkers of Drug-Induced Liver Injury. Front Pharmacol. 2021;12. 13. Kolios G, Valatas V, Kouroumalis E. Role of Kupffer cells in the pathogenesis of liver disease. World J Gastroentero. 2006;12(46):7413-20. 14. Shan Z, Ju C. Hepatic Macrophages in Liver Injury. Front Immunol. 2020;11. 15. Tsuji Y, Kuramochi M, Golbar HM, Izawa T, Kuwamura M, Yamate J. Acetaminophen-Induced Rat Hepatotoxicity Based on M1/M2-Macrophage Polarization, in Possible Relation to Damage-Associated Molecular Patterns and Autophagy. Int J Mol Sci. 2020;21(23). 16. Zeng T, Zhang CL, Xiao M, Yang R, Xie KQ. Critical Roles of Kupffer Cells in the Pathogenesis of Alcoholic Liver Disease: From Basic Science to Clinical Trials. Front Immunol. 2016;7. 17. Michael SL, Pumford NR, Mayeux PR, Niesman MR, Hinson JA. Pretreatment of mice with macrophage inactivators decreases acetaminophen hepatotoxicity and the formation of reactive oxygen and nitrogen species. Hepatology. 1999;30(1):186-95. 18. Laskin DL. Nonparenchymal cells and hepatotoxicity. Semin Liver Dis. 1990;10(4):293-304. 19. Laskin DL, Pilaro AM, Ji S. Potential role of activated macrophages in acetaminophen hepatotoxicity. II. Mechanism of macrophage accumulation and activation. Toxicol Appl Pharmacol. 1986;86(2):216-26. 20. Ju C, Reilly TP, Bourdi M, Radonovich MF, Brady JN, George JW, et al. Protective role of Kupffer cells in acetaminophen-induced hepatic injury in mice. Chem Res Toxicol. 2002;15(12):1504-13. 21. Jaeschke H, Ramachandran A. Mechanisms and pathophysiological significance of sterile inflammation during acetaminophen hepatotoxicity. Food Chem Toxicol. 2020;138. 22. Smith GS, Nadig DE, Kokoska ER, Solomon H, Tiniakos DG, Miller TA. Role of neutrophils in hepatotoxicity induced by oral acetaminophen administration in rats. J Surg Res. 1998;80(2):252-8. 23. Ishida Y, Kondo T, Kimura A, Tsuneyama K, Takayasu T, Mukaida N. Opposite roles of neutrophils and macrophages in the pathogenesis of acetaminophen-induced acute liver injury. Eur J Immunol. 2006;36(4):1028-38.
56 INNOVATIVE APPROACHES IN MEDICINE AND HEALTH SCIENCES II 24. Liu ZX, Han D, Gunawan B, Kaplowitz N. Neutrophil depletion protects against murine acetaminophen hepatotoxicity. Hepatology. 2006;43(6):1220-30. 25. Cover C, Liu J, Farhood A, Malle E, Waalkes MP, Bajt ML, et al. Pathophysiological role of the acute inflammatory response during acetaminophen hepatotoxicity. Toxicol Appl Pharm. 2006;216(1):98-107. 26. Lawson JA, Farhood A, Hopper RD, Bajt ML, Jaeschke H. The hepatic inflammatory response after acetaminophen overdose: Role of neutrophils. Toxicol Sci. 2000;54(2):509-16. 27. Yang T, Wang H, Wang X, Li J, Jiang LF. The Dual Role of Innate Immune Response in Acetaminophen-Induced Liver Injury. Biology-Basel. 2022;11(7). 28. Liu ZX, Govindarajan S, Kaplowitz N. Innate immune system plays a critical role in determining the progression and severity of acetaminophen hepatotoxicity. Gastroenterology. 2004;127(6):1760-74. 29. Masson MJ, Carpenter LD, Graf ML, Pohl LR. Pathogenic role of natural killer T and natural killer cells in acetaminophen-induced liver injury in mice is dependent on the presence of dimethyl sulfoxide. Hepatology. 2008;48(3):889-97. 30. Blazka ME, Elwell MR, Holladay SD, Wilson RE, Luster MI. Histopathology of acetaminophen-induced liver changes: Role of interleukin 1 alpha and tumor necrosis factor alpha. Toxicol Pathol. 1996;24(2):181-9. 31. Blazka ME, Wilmer JL, Holladay SD, Wilson RE, Luster MI. Role of Proinflammatory Cytokines in Acetaminophen Hepatotoxicity. Toxicol Appl Pharm. 1995;133(1):43-52. 32. Ishida Y, Kondo T, Ohshima T, Fujiwara H, Iwakura Y, Mukaida N. A pivotal involvement of IFN-γ in the pathogenesis of acetaminophen-induced acute liver injury. Faseb J. 2002;16(10). 33. Bourdi M, Masubuchi Y, Reilly TP, Amouzadeh HR, Martin JL, George JW, et al. Protection against acetaminophen-induced liver injury and lethality by interleukin 10: Role of inducible nitric oxide synthase. Hepatology. 2002;35(2):289-98. 34. Masubachi Y, Bourdi M, Reilly TP, Graf MLM, George JW, Pohl LR. Role of interleukin-6 in hepatic heat shock protein expression and protection against acetaminophen-induced liver disease. Biochem Bioph Res Co. 2003;304(1):207-12. 35. Reilly TP, Brady JN, Marchick MR, Bourdi M, George JW, Radonovich MF, et al. A protective role for cyclooxygenase-2 in drug-induced liver injury in mice. Chem Res Toxicol. 2001;14(12):1620-8.
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59 CHAPTER V QUANTITATIVE ANALYSIS METHODS IN HEALTHCARE: STEREOLOGY AND CAVALIERI’S PRINCIPLE Muhammet Lütfi SELÇUK1 & Fatma KAYIKÇI HEKİM2 1(Associate Professor), Karamanoğlu Mehmetbey University, Faculty of Health Sciences, Department of Physiotherapy and Rehabilitation, Karaman/ Türkiye E-mail: [email protected] Orcid: 0000-0002-9915-3829 2(Associate Professor), Karamanoğlu Mehmetbey University, Faculty of Health Sciences, Department of Nutrition and Dietetics, Karaman/ Türkiye E-mail: [email protected] Orcid: 0000-0003-0410-5523 1. Introduction Observability, measurability, and reproducibility of data obtained in scientific research are crucial. Therefore, quantitative methods are frequently preferred for the rapid, efficient, and reliable collection of data in healthcare studies. Stereology is a widely accepted method with high scientific value, reliable results, and proven mathematical accuracy. Furthermore, statistical data analysis, using the Cavalieri principle for volume calculations, ensures the reliability and scientific nature of research. 2. Stereology Stereology is the branch of science that uses data obtained from twodimensional cross-sections or projections of three-dimensional objects to make interpretations of their actual three-dimensional properties (1,2). In other words,
60 INNOVATIVE APPROACHES IN MEDICINE AND HEALTH SCIENCES II stereology can also be defined as a method that uses cross-sections of an object to obtain quantitative information about its geometric and statistical structure (3). The term stereology, first used before 1960, derives from the Greek term stereos (three-dimensional object, three-dimensionality). In 1961, biologists, geologists and medical scientists gathered in the Black Forest in Germany to discuss the problems related to the three-dimensional description of matter, and stereology entered the scientific world with the proposal of German Professor Hans Elias (4). Stereology is used in many branches of science, but especially in recent years it has been frequently used in experimental studies in the fields of medicine and biology (5). In recent years, there have been significant changes in scientific approaches in the fields of medicine and biology. Unbiased or objective approaches are more widely accepted than subjective approaches. Quantitative and unbiased data obtained from biological structures using appropriate methods are very important in determining the variation between biological species and in making a definitive diagnosis in the clinic. At the same time, because the data is expressed in numbers that are free from personal bias, comparison is straightforward. Quantitative measurements are easier to store, analyze, highly reliable, and unbiased compared to the subjective approach (6, 7, 8). In stereology, the shape of the structure of interest is not important. Even if the structure of interest lacks regular shapes, if the tissue can be rendered two-dimensional using medical imaging or histological methods, we can easily determine its volume (9, 10). Today, stereology is frequently used in general medicine, astronomy, geology, mathematics, and other engineering sciences. However, it is most useful in fields dealing with biological structures, such as anatomy, histology, physiology, pathology, and botany (11). 2.1.SystematicRandomSampling In scientific studies, the sampling of biological tissue to be studied must be free of systematic bias. In microscopic studies, each part of the sampled object should have an equal impact on the results obtained from the measurement and should have an equal opportunity to be sampled (3). Therefore, it is crucial to adhere to this rule at every stage of the sampling process. Avoiding systematic bias is possible by selecting the correct sampling method and using accurate, calibrated measurement tools (12). Systematic random sampling provides results that are statistically much closer to reality than random sampling (13).
QUANTITATIVE ANALYSIS METHODS IN HEALTHCARE: STEREOLOGY AND . . . 61 The systematic part of the sampling is provided by the predetermined sampling interval in a biological tissue, and the randomness of the sampling is provided by starting from a random point within the first interval. The systematic random sampling method is applied at each stage of stereological research. This includes sampling the tissue to be studied (tissue sampling), during the histological sectioning process (section sampling) (14), and during the examination of these sections under the microscope (area sampling) (15). Statistically, applying this type of sampling to more samples increases the likelihood of obtaining accurate results (11). 2.2.Efficiency One important property of stereology is its efficiency. In stereology, efficiency pertains to obtaining the closest estimation to the true value by utilizing materials, time, and labor at an optimal level while upholding the principle of impartiality (4, 16). Objectivity refers to avoiding a systematic deviation from the true value, while effectiveness means achieving results with the desired accuracy in less time. Mouton (2002) expresses efficiency in terms of time and precision as total observed efficiency GE = precision / time = 1 / CV2 x time (GE = Observed efficiency, CV = Coefficient of Variance, or inter-individual variation) and sampling efficiency OE = precision / time = 1 / CE2 x time (OE = Sampling efficiency, CE = Coefficient of Error or error coefficient). In the light of these explanations, it is seen that the effectiveness is inversely proportional to time and the error coefficient (4). 2.3.Coefficientoferror=CE Determining the error coefficient is a crucial step in stereological research. The quality and precision of numerical measurements obtained from stereological studies, as well as the suitability of the sampling plan, can be assessed by calculating the coefficient of error (CE). In stereological studies, the coefficient of error does not correspond to a real biological value, but it is a value that indicates the quality of the sampling strategy (17). CE is dependent on two factors that can be controlled by the researcher, namely sampling strategy and sample size. These two factors are identified through an initial study (18). In order for the results of stereological studies to be considered reliable, the error coefficient must be 5% or less (13).
62 INNOVATIVE APPROACHES IN MEDICINE AND HEALTH SCIENCES II 2.4.DeterminationofCoefficientofError(CE)inVolumeCalculations In stereological studies, it is recommended that the error coefficient be less than or equal to 5%, particularly for volume measurements. If it is above 5%, the number of sections and the number of points counted must be increased (13, 14, 16, 19). There are multiple methods used to calculate the error coefficient in stereological research. In this section, the error coefficient formula of Gundersen et al (1999) will be discussed. CE = Σvar åP åVar = Noise + VarSRS Noise = 0.0724 x b a x n x P å VarSRS = ( 1 ) n i a = å = (3 x (A-Noise) – 4 x B+C) / 12 VarSRS = ( 1 n i a = å ) = It shows the statistical variability of systematic random sampling across the entire area. In this formula, Noise represents the complexity of the cross-sectional surface areas taken from the tissue of interest, b a represents the edge complexity of the area of interest on the cross-sectional surface. In the formula, b = side length, and a represents the square root of the cross-sectional area. The value 0.0724 in the formula is a constant used to calculate the complexity value, n corresponds to the total number of sections, and ΣP corresponds to the total number of points falling on the sections (14, 16, 20, 21). 2.5.Probes Probes are geometric shapes used to estimate the length, surface area, number and volume of the tissue under study from the appearance of sections taken from the tissue under study. Therefore, it is very important to choose the right probe-parameter combination in studies (4, 12, 22). In stereology, point probes, line probes, surface probes and dissector probes are generally used. The point probe is zero-dimensional and is used in volume calculations, the line probe is one-dimensional and is used in area calculations, the surface probe is
QUANTITATIVE ANALYSIS METHODS IN HEALTHCARE: STEREOLOGY AND . . . 63 two-dimensional and is used in length calculations, and the dissector probe is three-dimensional and is used in calculating the total number of any particle type in biological structures. Since stereological methods are used to evaluate the three-dimensional properties of biological structures, the probe and parameter sum should be three (22). 3. Cavalieri’s Principle There are various methods for calculating the volume of irregularly shaped objects. The most well-known of these is Archimedes’ principle. In this method, the object under investigation is placed in a graduated cylinder filled with water, and the amount of water displaced or lifted by the object is equal to its volume. In this way, the volume of an isolated object can be easily and directly measured (23, 24). Measurements of the volume of small biological structures that cannot be isolated from their surroundings using the Archimedes principle may not yield accurate results due to the capillary effect (25). Recently, the Cavalieri method has been favored for volume calculations in biological structures (10, 26). The Cavalieri method was developed by the 17th-century Italian mathematician and astronaut Bonaventura Cavalieri. Cavalieri demonstrated that the volume of an object can be calculated by cutting the object in parallel at equal intervals and calculating the surface area of each section and then multiplying it by the section thickness (13). The Cavalieri method is widely used today for volume calculations, particularly in the analysis of both macroscopic and microscopic section images of biological tissues. It is also applied to section images obtained through imaging techniques such as CT and MRI (1, 27, 28). In the Cavalieri method, firstly the number of points obtained from each section images is divided by the number of sections and the average crosssectional areas of the structures of interest in the sections are determined. The volumes of the structures of interest in the sections are calculated separately using the formula V= (a/p) x P å x t (2). In this formula, V= the volume of the structure of interest in the sample, a/p = the area of the one point on the grid, ∑P = the total number of points falling on the structure of interest, t = the average section thickness (12, 13, 24, 27, 29). The volume of the entire structure is calculated with the formula VTop=V1+V2……Vn (30). 4. Conclusion Stereology is a valuable quantitative method that yields numerical data for structures that are asymmetrical and cannot be isolated from their surroundings.
64 INNOVATIVE APPROACHES IN MEDICINE AND HEALTH SCIENCES II Its key advantages include the ability to obtain accurate and unbiased data, deliver rapid results, reduce workload, and incur no additional costs. Using stereological methods, quantitative measurements such as volume, volume ratios, and surface area can be calculated with minimal deviation from their true values. In recent years, stereology has become increasingly popular in healthcare research. References 1. Koçyiğit A, Demircioğlu İ, Güzel BC. Stereological and computed tomography calculation of intracranial volume in Hamdani sheep. Anat Histol Embryol. 2024; 53(2): e13021. 2. Mayhew T, Gundersen H. ‘If you assume, you can make an ass out of u and me’: a decade of the disector for stereological counting of particles in 3D space. J Anat. 1996; 188: 1-15. 3. Cruz-Orive LM. Systematic sampling in stereology. Bull Int Stat Inst, 1993; 55: 451-468. 4. Mouton PR. Principles and practices of unbiased stereology, John Hopkins University Press; 2002. 5. Akalan MA, Demirkan AÇ. Stereoloji ve veteriner hekimlikte kullanım alanları. YYU Veteriner Fakültesi Dergisi, 2013; 24(2): 95-100. 6. Black KJ. On the efficiency of stereologic volumetry as commonly implemented for three-dimensional digital images. Psychiatry Research: Neuroimaging, 1999; 90(1): 55-64. 7. Glaser JR, Glaser EM. Stereology, morphometry, and mapping: the whole is greater than the sum of its parts. J Chem Neuroanat. 2000; 20(1): 115-26. 8. James NT. Morphometry and stereology in biology and medicine using confocal microscopy, The institution of Electrical Engineers; 2004. 9. Keleş Aİ. Sağlık alanında kullanılan kantitatif yöntem, stereoloji. Dicle Med J.2019; 46(3): 615-621. 10. Selçuk ML, Tıpırdamaz S. A morphological and stereological study on brain, cerebral hemispheres and cerebellum of New Zealand rabbits. Anat Histol Embryol. 2020; 49(1): 90-96. 11. Russ J, Dehoff R. Practical stereology, New York: Plenum Pub Corp; 2000. 12. Howard V, Reed M. Unbiased stereology: three-dimensional measurement in microscopy. Oxford, Garland Science/Bios scientific Publishers; 2005.
NATURAL PRODUCTS USED IN THE TREATMENT OF PSORIASIS 71 and Diospyros spp. Betulinic acid a naturally occurring pentacyclic triterpenoid, has generated interest in healthcare professionals due to its diverse biological properties, including antiviral, antioxidant, and anticancer, anti-inflammatory. Considering that psoriasis’s pathogenesis, heavily relies on inflammation, betulinic acid’s anti-inflammatory qualities might be particularly relevant to this chronically inflammatory skin disorder (25). By controlling inflammatory pathways and cytokine production, betulinic acid may help reduce psoriasis symptoms such as erythema, scaling, and plaque formation. Through the inhibition of pro-inflammatory mediators and signaling pathways, betulinic acid demonstrates anti-inflammatory effects (26). 2.6.Beta-Sitosterol Portia tree (Thespesia populnea L.), It is a member of the Malvaceae family. It contains various sterols, such as beta-sitosterol. These sterols are known to have immunomodulatory and anti-inflammatory effects, which may help to reduce inflammation and prevent infection in psoriatic wounds (27,28). Beta-sitosterol has demonstrated anti-inflammatory properties by inhibiting the production of proinflammatory cytokines such as IL-6, IL-1 beta (IL-β), and TNF-α. In psoriasis, inflammation is a key driver of disease pathology, leading to the characteristic skin lesions and other symptoms. By dampening the inflammatory response, beta-sitosterol may help reduce the severity of psoriasis symptoms (29,30). 2.7.Charantin Charantin is a chemical compound found in bitter melon (Momordica charantia L.). It is a member of the Cucurbitaceae family, and is one of the bioactive constituents responsible for its medicinal properties (31). The pharmacological profile of M. charantia demonstrates its potential as an immunomodulator, antipsoriatic, cardioprotective, analgesic, anti-inflammatory, antioxidant, anthelmintic, and antimalarial agent, hypotensive, antibacterial, antiviral, anticancer, antifertility, antiulcer, and antimalarial agent. In addition, several studies have indicated that it may be useful in treating many conditions, including gout, eczema, jaundice, kidney stones, pneumonia, rheumatism, scabies, psoriasis, and constipation (32). Prolonged inflammation is essential to the pathophysiology of psoriasis because it promotes the development of psoriatic plaques and increases symptoms. Charantin can reduce skin inflammation and minimize the activity of pro-inflammatory mediators such as enzymes and cytokines or the severity of psoriasis lesions by inhibiting the synthesis (33,34).
72 INNOVATIVE APPROACHES IN MEDICINE AND HEALTH SCIENCES II 2.8.Curcumin Curcumin is a bioactive compound that can be found in the rhizomes of the plant (Curcuma longa L.), belongs to the family Zingiberaceae (35). Commonly known as turmeric, a plant known for its wide spectrum of pharmacological action and medicinal advantages (36). The pharmacological properties of curcumin, including its antioxidant, anti-inflammatory, and anti-vascular remodeling effects, have been extensively studied. Based on available data, curcumin has the potential to be employed as a therapy for various human aliments. A strong foundation for assessing the bioavailability and safety of curcumin in clinical trials has been established by preclinical research. As an adjuvant therapy for psoriasis, curcumin has been shown in numerous clinical trials to be both safe and efficacious. Its low toxicity and poor absorption may be beneficial to psoriasis patients (37,38). 2.9.EllagicAcid Blackberries (Rubus fruticosus L.), belongs to the family Rosaceae. Polyphenolic substance called ellagic acid can be found in a variety of fruits and nuts (39). Its potential therapeutic effects in treating a variety of illnesses, including psoriasis, have been researched. A lot of pharmaceutical products have included it as an active component because of its analgesic, anti-inflammatory, anti-pain, and wound healing qualities. It has been shown to regulate numerous signaling molecules and pathways connected to a range of biological activities, including autophagy, metabolism, apoptosis, cell cycle, regulation, and cell survival and proliferation (40). By suppressing pro-inflammatory cytokines and enzymes, ellagic acid demonstrates anti-inflammatory effects. Inflammation is a key factor in the pathophysiology of psoriasis. By inhibiting the synthesis of inflammatory mediators such as IL-6, IL-1β, and TNF-α ellagic acid may help reduce inflammation (41). 2.10.GlycyrrhetinicAcid Glycyrrhetinic acid (GA), one of the main ingredients of liquorice (Glycyrrhiza glabra L.), belongs to the family Fabaceae (42). It shows to have several pharmacological activities, including, anti-inflammatory activities, anticancer, and antioxidative. Glycyrrhetinic acid prevents immune cells like macrophages and dendritic cells from producing and releasing pro-inflammatory cytokines like TNF-α, IL-1, and IL-6 (43,44).
NATURAL PRODUCTS USED IN THE TREATMENT OF PSORIASIS 73 2.11.Guggulsterone Guggulsterone, a bioactive compound, is extracted from the resin of Guggul (Commiphora wightii L.), belongs to the family Burseraceae (45). It is commonly referred to as the guggul tree. Its potential therapeutic benefits in treating various conditions, such as psoriasis, have been investigated (46). Guggulsterone’s exact mode of action in treating psoriasis is unknown, although several studies have suggested that it possesses anti-inflammatory, antiproliferative, and immunomodulatory properties, which could account for some of the drug’s therapeutic efficacy. Guggulsterone demonstrated that inhibits the synthesis of pro-inflammatory cytokines in a variety of cell types, including keratinocytes and immune cells (47). 2.12.Hesperidin Hesperidin, a bioflavonoid is primarily found in the peel and membranes of citrus fruits, particularly in oranges (Citrus sinensis L.), lemons, grapefruits, and tangerines (48). It belongs to the family Rutaceae. In addition to its wellknown benefits for heart health, and anti-inflammatory activities, recent studies have highlighted hesperidin’s benefits for cutaneous functions like wound healing, UV protection, anti-inflammatory, anti-skin cancer, and antimicrobial. Hesperidin has been found to have anti-inflammatory qualities in numerous studies. IL-1β, TNF-α and other pro-inflammatory cytokines that are connected to the pathophysiology of psoriasis can all be suppressed by it (49,50). 2.13.Kaempferol Kaempferol is a flavonoid present in jasmine flower paste (Jasminum officinale L.), belongs to the family Oleaceae (51). It is also present in various plants, including fruits, medicinal herbs, and vegetables. Its potential therapeutic effects, including antioxidant, anti-inflammatory properties. It has been demonstrated that kaempferol prevents immune cells and keratinocytes from producing and releasing pro-inflammatory cytokines that are involved in the pathophysiology of psoriasis, such as IL-6, IL-1β, and TNF-α (52,53). 2.14.Linalool Linalool is a naturally occurring terpene alcohol found in many essential oils, such as lavender (Lavandula angustifolia L.), belongs to the family Lamiaceae (54). Because of its anti-inflammatory and wound-healing properties, lavender
74 INNOVATIVE APPROACHES IN MEDICINE AND HEALTH SCIENCES II oil an aromatic/essential oil produced from L angustifolia, has long been used as an aromatherapy massage oil (55). It also helps relieve other skin problems like psoriasis, dermatitis, and eczema. Psoriasis is largely influenced by inflammation in its early stages and advancement. Through its inhibition of inflammatory mediators including prostaglandins, leukotrienes, and cytokines like IL-6, TNFα, and others, linalool has demonstrated anti-inflammatory capabilities (56). 2.15.Liquiritin Liquiritin a flavonoid component included in liquorice (G. glabra L.), belongs to the family Fabaceae (57). Root extract, has been investigated for its possible therapeutic applications in treating a range of skin disorders, including psoriasis. The pharmacological properties of Liquiritin, including its antioxidant, anti-inflammatory, antidiabetic, anti-cancer, antiallergy, anti-ulcer, and antiviral properties. The anti-inflammatory characteristics of Liquiritin have been demonstrated through its inhibition of the production of inflammatory cytokines, including IL-6, IL-1β, and TNF-α (58,59). 2.16.Luteolin Luteolin derived from the medicinal plants (Andrographis paniculata L.), belongs to the family Acanthaceae, (Costus speciosus L.), belongs to the family Costaceae, (Justicia adhatoda L.), belongs to the family Acanthaceae, (Plectranthus amboinicus L.), belongs to the family Lamiaceae, and (Tinospora cordifolia L.), belongs to the family Menispermaceae (60). These compounds are utilized in standard medicinal methods. There has been speculation that luteolin, a naturally occurring anti-inflammatory flavonoid, may have therapeutic benefits for psoriasis due to its well-established inhibitory effect on keratinocyte growth. Luteolin is a flavonoid that can be found in herbs, flowers, vegetables, and spices (61). By partially absorbing UVA and UVB light, it serves a significant function in protecting plants from UV radiation. Thus, by serving as a first line of defence, luteolin can help lessen harmful photobiological effects in the skin. Additionally, luteolin has been shown to have anti-inflammatory and antioxidative properties on fibroblasts, keratinocytes, and a variety of immune cells, including neutrophils, mast cells, dendritic cells, and T cells. Proinflammatory mediators such as IL-1β, IL-6, IL-8, IL-17, IL-22, TNF-α, and COX-2 can be suppressed by luteolin, which can also control other signaling pathways such as the TLR signaling pathways, JAK-STAT, and NF-κB. Luteolin has been proposed as a possible chemical target for future treatment studies of inflammatory skin conditions such as psoriasis (62).
NATURAL PRODUCTS USED IN THE TREATMENT OF PSORIASIS 75 2.17.Matricin Chamomile plants (Matricaria chamomilla L.), belongs to the family Asteraceae, is related to most plants that contain matricin (63). These extracts can be derived from the flowers, leaves, and stems of the plant and used topically as lotions, ointments, or creams to affected parts of the skin. Psoriasis sufferers may benefit from these herbs’ traditional medical uses because of their antioxidant, anti-inflammatory, immunomodulatory, and woundhealing properties. Studies have shown that chamomile extracts inhibit proinflammatory mediators such as IL-1, IL-6, and TNF-α, which are significant in the inflammation related to psoriasis (64,65). 2.18.Nicotine Tobacco (Nicotiana tabacum L.), belongs to the family Solanaceae, in tobacco alkaloid that moderates the addictive properties of tobacco products is nicotine (66). More than 7000 different chemicals are found in tobacco, and smoking is the leading preventable cause of death globally. It is known to increase the risk of developing several diseases in people, such as lung and cardiovascular conditions, as well as various malignancies. Psoriasis is one of the inflammatory immune-related diseases that has been linked to smoking. Psoriasis onset is influenced by smoking (67).Nicotine, which is mostly present in tobacco products, has been linked to immune response and inflammatory modulation, two important processes in the onset and aggravation of psoriasis. By regulating the release of cytokines and chemokines involved in the inflammatory response, nicotine has been demonstrated to have antiinflammatory effects. It inhibits pro-inflammatory cytokines including IL-6, TNF-α, and IL-1β via acting on nicotinic acetylcholine receptors (nAChRs) on immune cells like macrophages and T cells (68). 2.19.Quercetin Quercetin is a common dietary flavonoid present in a wide variety of natural plants. Its many biological properties, including cardio-protective, antioxidant, liver-protective, anti-inflammatory, and anti-cancer activities, vasodilatory, have been shown by numerous studies (69). Quercetin strong anti-inflammatory properties are evidenced by its inhibition of pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-6. These cytokines are crucial to understanding the pathophysiology of psoriasis because they promote keratinocyte proliferation and inflammation (70,71).
76 INNOVATIVE APPROACHES IN MEDICINE AND HEALTH SCIENCES II 2.20.RosmarinicAcid Rosmarinic acid is a phenolic acid that is found in many herbs, including oregano (Origanum vulgare L.), Oregano oil is derived from the leaves and flowers of the oregano plant, which belongs to the mint family (Lamiaceae) (72). Common skin pathology psoriasis is characterized by persistent inflammation and deregulation of epidermal keratinocyte function, indicating that molecules with anti-inflammatory potential may be useful in managing the condition. One naturally occurring bioactive compound with anti-inflammatory properties is rosmarinic acid. Natural polyphenolic chemical rosmarinic acid is present in many plants, such as some mints, rosemary, and lemon balm. Its possible medicinal benefits, and anti-inflammatory, antioxidant, and immunomodulatory characteristics, have drawn interest (73). Rosmarinic acid content as a therapeutic agent for inflammation linked to psoriasis in human keratinocytes. When Interferon-gamma (IFN-γ), IL-17, and IL-22 were combined, regulatory genes from the NF-κB and Janus kinase / signal transducer and activator of transcription (JAK/STAT) signaling pathways were activated (74). 2.21.Solanine Solanine is a glycoalkaloid found in a wide variety of plants, including black nightshade (Solanum nigrum L.), belongs to the family Solanaceae (75). It is family members like potatoes, tomatoes, and eggplants. It has historically been used to treat a variety of cancers as well as dermatitis, eczema, acute nephritis, urethritis, leucorrhoea, sore throat, psoriasis, toothache, carbuncles, and furuncles. It is unclear if solanine truly treats psoriasis, despite research on solanine’s immunomodulatory and anti-inflammatory effects. Studies have demonstrated that solanine reduces inflammation by blocking inflammatory mediators. Furthermore, through affecting T-cell activity and cytokine production, solanine has been demonstrated to modify immune responses (76,77). 2.22.Silymarin Silymarin is a flavonoid obtained from plants that is taken from the fruits and seeds of the Asteraceae family plant known as milk thistle (Silybum marianum L.) (78). Silymarin comes in topical and oral forms, and it has been used to treat a variety of dermatological disorders, including wound healing, cosmeceuticals, melanoma and nonmelanoma skin malignancies, melasma, rosacea, psoriasis, atopic dermatitis, acne, and wound healing (79). Through the
NATURAL PRODUCTS USED IN THE TREATMENT OF PSORIASIS 77 inhibition of several inflammatory mediators and pathways linked to psoriasis, silymarin has anti-inflammatory effects. It can inhibit the synthesis of proinflammatory cytokines that are important in the pathophysiology of psoriasis, TNF-α, IL-1β and IL-6) (80). 2.23.Thymol Thymol (Thymus vulgaris L.), belongs to the family Lamiaceae. The antifungal activity of essential oil is the main source of thymol, a naturally occurring substance that may have therapeutic benefits for psoriasis (81). Utilizing steam distillation or alternative extraction techniques. It is a potential substance in the treatment of inflammation and wound healing because of its antibacterial, antioxidant, and antiseptic effects. Thymol has anti-inflammatory qualities that may help reduce the symptoms of psoriasis (82,83). 2.24.Wrightial Wrightial is the principal active ingredient extracted from (Wrightia tinctoria L.), belonging to the family Apocynaceae. W. tinctoria, also referred to as Dyer’s Oleander or Sweet Indrajao, has long been utilized in Ayurvedic medicine to treat a variety of skin issues (84). Including gynaecological issues, wounds, leukaemia, dandruff, diarrhea, psoriasis, eczema, and scabies, have long been treated with this plant. Multiple pharmacological actions were demonstrated by W tinctoria, including anti-microbial, anti-helminthic, antioxidant, anti-cancer, anti-psoriatic, anti-inflammatory, anti-diabetic, diuretic, hepatoprotective, and anti-ulcer properties (85). Studies on phytochemicals have identified triterpenoids, alkaloids, steroids, lipids, and carbohydrates. These medications may lessen inflammation and improve psoriatic symptoms by inhibiting cytokines and enzymes that cause inflammation (86). 2.25.Zeaxanthin Capsicum (Capsicum annuum L.), belongs to the family Solanaceae. In this rich in carotenoids, including Zeaxanthin. Zeaxanthin is an organic pigment that is produced by plants, algae, and some microbes. It is a member of the carotenoid family (87). It is especially prevalent in a variety of vegetables, fruits, and other foods made from plants. The main benefits of zeaxanthin are that it has anti-inflammatory and antioxidant effects. Zeaxanthin has antiinflammatory effects that might help those with psoriasis. Zeaxanthin could
78 INNOVATIVE APPROACHES IN MEDICINE AND HEALTH SCIENCES II reduce the intensity of psoriatic inflammation and improve related symptoms by regulating the inflammatory response (88,89). 3. Conclusion Despite advances in treatment options for psoriasis, many patients seek alternative or complementary therapies due to the limitations and side effects of conventional treatments. Plants-derive phytoconstituents have attracted attention for their potential in the treatment of psoriasis due to their diverse pharmacological properties and relatively fewer side effects. This systematic review aimed to critically evaluate the existing literature on phytoconstituents for the treatment of psoriasis. The reviewed studies demonstrated a broad spectrum of phytoconstituents with promising antipsoriatic effects. Phytoconstituents such as azadirachtin, boswellic acid, curcumin, ellagic acid, hesperidin, kaempferol, luteolin, quercetin, and thymol. These phytoconstituents exerted their effects through several mechanisms, including anti-inflammatory, immunomodulatory, antioxidant, and antiproliferative activities, which are key pathways involved in the pathogenesis of psoriasis. Biological therapies (IL-17 inhibitors, IL-23 inhibitors, and TNF-α inhibitors), these drugs target cytokines involved in the immunological response of psoriasis, reducing inflammation, and stopping aberrant proliferation of psoriasis. In conclusion phytoconstituents represent a promising avenue for the treatment of psoriasis, offering potential benefits in terms of efficacy, safety, and tolerability. Considering the identified limitations and challenges, future research should focus on these gaps to realize the full therapeutic potential of phytoconstituents in the treatment of psoriasis. Collaboration between researchers, clinicians and industry stakeholders is essential to advance our understanding and application of phytoconstituentbased therapies in clinical practice, thereby improving outcomes and quality of life for affected patients. References 1. Krueger JG. The immunologic basis for the treatment of psoriasis with new biologic agents. J Am Acad Dermatol. 2002;46(1). 2. Wang Y, Li J, Guo C, Yang G, Lin H, Zhang Y. Bibliometric analysis and description of research trends in the treatment of psoriasis with biologic agents in the past two decades (2004–2023). J Dermatolog Treat. 2024;35(1):2346282. 3. Griffiths CE, Barker JN. Pathogenesis, and clinical features of psoriasis. Lancet. 2007;21(9583):263.
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87 CHAPTER VII THE ANTI-AGEING ROLE OF BIOACTIVE COMPOUNDS DERIVED FROM SEAWEED Mehmet BERKÖZ (Assoc. Prof. Dr.), Van Yuzuncu Yil University, Faculty of Pharmacy, Department of Biochemistry, Van, TÜRKİYE, E-mail: [email protected] ORCID: 0000-0003-4219-8054 1. Introduction Cellular senescence is a permanent state of cell cycle arrest that occurs when cells are subjected to various forms of stress. This process is characterized by the cessation of cellular proliferation, changes in cell morphology, alterations in gene expression, and the secretion of proinflammatory cytokines, growth factors, and proteases, collectively known as the senescence-associated secretory phenotype (SASP). Senescence serves as a critical mechanism to prevent the propagation of damaged cells, thereby acting as a tumor-suppressive measure. However, the accumulation of senescent cells over time contributes to tissue dysfunction and the progression of age-related diseases (1). Aging is a complex, multifactorial process driven by various interconnected biological mechanisms and pathways. Key pathways involved in aging regulation include sirtuins, AMP-activated protein kinase (AMPK), insulin-like growth factor (IGF) pathway, autophagy, and nuclear factor erythroid 2-related factor 2 (Nrf2). Sirtuins are a family of NAD+ dependent deacetylases that regulate cellular processes such as metabolism, stress response, and aging, promoting longevity by enhancing DNA repair, reducing oxidative stress, and modulating mitochondrial function (2). AMPK is an energy-sensing enzyme that maintains cellular energy homeostasis; its activation promotes autophagy, enhances mitochondrial biogenesis, and inhibits mTOR (mechanistic target of rapamycin) signaling, thereby extending lifespan. The IGF pathway, involved in growth and
88 INNOVATIVE APPROACHES IN MEDICINE AND HEALTH SCIENCES II development, influences aging by modulating cellular growth, metabolism, and survival, with reduced IGF signaling being associated with increased lifespan and delayed aging (3). Autophagy, a cellular degradation process, removes damaged organelles and proteins, maintaining cellular homeostasis and function, with enhanced autophagy linked to increased longevity and resistance to agerelated diseases. Nrf2 is a transcription factor that regulates the expression of antioxidant proteins; its activation enhances cellular resistance to oxidative stress, mitigating aging and promoting longevity (4). Aging significantly impacts health by increasing susceptibility to a variety of chronic diseases. The decline in cellular and tissue function associated with aging is a major risk factor for the development of cancer, cardiovascular disease, diabetes, and neurodegenerative diseases (5). The accumulation of genetic mutations and a decline in immune surveillance contribute to increased cancer incidence with age. Age-related changes in the cardiovascular system, such as arterial stiffening and endothelial dysfunction, lead to a higher risk of hypertension, atherosclerosis, and heart failure. Impaired glucose metabolism and insulin resistance are more prevalent in older adults, increasing the risk of type 2 diabetes (3). Additionally, aging is a major risk factor for neurodegenerative disorders such as Alzheimer’s and Parkinson’s disease, which are characterized by the accumulation of damaged proteins and neuronal loss (6). Understanding the cellular and molecular mechanisms underlying aging and cellular senescence is crucial for developing therapeutic strategies to combat agerelated diseases and promote healthy aging. This review will explore the potential of seaweed-derived bioactive compounds in modulating these aging-related pathways and their implications for extending lifespan and improving health span. 2. Seaweed Derived Bioactive Compounds with Anti-Aging Effects Seaweed, also known as marine macroalgae, comprises a diverse group of photosynthetic organisms found in marine environments worldwide. Classified into three main types based on pigmentation brown algae (Phaeophyceae), red algae (Rhodophyta), and green algae (Chlorophyta). Brown algae like Laminaria (kelp), Macrocystis (giant kelp), and Sargassum are predominantly found in colder, temperate waters and often form extensive underwater forests (7). They are typically found in intertidal and subtidal zones, attached to rocky substrates or floating freely in the water column. Brown algae are significant for coastal ecosystems as they provide habitat and food for various marine organisms (8). They are also commercially valuable for their bioactive compounds and
THE ANTI-AGEING ROLE OF BIOACTIVE COMPOUNDS DERIVED FROM SEAWEED 89 industrial uses. Red algae like Porphyra (nori), Gracilaria, Gelidium are characterized by their red to purplish coloration due to the presence of pigments like phycoerythrin and phycocyanin, which mask the green chlorophyl ll. They are typically found in deeper waters but can also thrive in intertidal zones. They are commonly found in tropical and subtropical regions, attached to rocks or other substrates. Green algae share characteristics with land plants, including chlorophylls a and b, which give them a green coloration. They can range in size from microscopic unicellular forms to large multicellular species (9). 2.1.Fucoidan Seaweed is renowned for its rich array of bioactive compounds that contribute to its nutritional, medicinal, and industrial value. Among these compounds, fucoidan stands out prominently. Primarily sourced from brown algae Phaeophyceae such as Fucus vesiculosus, Undaria pinnatifida (wakame), and various Sargassum species, fucoidan is a sulfated polysaccharide known for its diverse biological activities (10). It exhibits potent antioxidant properties, scavenging free radicals and reducing oxidative stress. Moreover, fucoidan demonstrates anti-inflammatory effects by modulating inflammatory pathways and enhancing immune responses. These characteristics make fucoidan a promising candidate for various therapeutic applications, including cancer treatment, cardiovascular health promotion, and immune system support. In the cosmetic industry, fucoidan is utilized for its moisturizing and anti-aging properties, contributing to skincare products aimed at improving skin elasticity and reducing wrinkles (11-13). 2.2.Phlorotannins Another significant group of bioactive compounds found in seaweed is phlorotannins, exclusive to brown algae. Synthesized through the polymerization of phloroglucinol units, phlorotannins are potent antioxidants that surpass terrestrial polyphenols like catechins and flavonoids in radicalscavenging capacity (14,15). They are known for their anti-inflammatory properties, inhibiting inflammatory enzymes and reducing cytokine production. Additionally, phlorotannins exhibit antimicrobial activity against bacteria and fungi, suggesting potential applications in food preservation and pharmaceuticals. Their promising role in cancer prevention and treatment is under exploration, highlighting their potential as natural compounds for therapeutic interventions (16).
90 INNOVATIVE APPROACHES IN MEDICINE AND HEALTH SCIENCES II 2.3.Carotenoids Carotenoids, found in various seaweed types including brown algae (fucoxanthin), red algae (astaxanthin), and green algae (beta-carotene), contribute significantly to seaweed’s bioactive profile. These pigments provide antioxidant protection against oxidative stress, enhancing cellular defense mechanisms and supporting overall health (17). Fucoxanthin and astaxanthin offer photoprotective properties, shielding skin cells from UV-induced damage and contributing to skincare formulations aimed at sun protection and antiaging effects. Carotenoids also exhibit anti-inflammatory properties, potentially benefiting inflammatory skin conditions and promoting immune function. Studies on fucoxanthin further explore its role in promoting fat metabolism and aiding in weight management, highlighting its potential as a functional ingredient in dietary supplements and nutritional products (18). 2.4.Polyphenols Polyphenols, ubiquitous in all types of seaweed, encompass a diverse group of compounds with significant biological activities. These include antioxidant properties, which protect cells from oxidative damage and contribute to overall health maintenance. Seaweed polyphenols also possess anti-inflammatory effects, mitigating inflammation and supporting cardiovascular health. Their antimicrobial properties offer potential applications in food preservation and hygiene products (19,20). Polyphenols found in seaweed are increasingly studied for their therapeutic potential in chronic diseases such as cancer and metabolic disorders. In the food industry, these compounds serve as natural preservatives and enhancers of nutritional value, while in cosmetics, they contribute to skincare products aimed at anti-aging and skin protection (21). 2.5.Polysaccharides Polysaccharides, such as agar, carrageenan, ulvan, and alginate, represent another vital group of bioactive compounds abundant in seaweed. These complex carbohydrates contribute to seaweed’s gelling, thickening, and stabilizing properties, widely utilized in food, pharmaceuticals, and biotechnology (19). Beyond their structural roles, seaweed polysaccharides exhibit diverse biological activities including antioxidant, immunomodulatory, and antiviral effects. They are integral to wound healing processes and are explored for their potential in drug delivery systems and tissue engineering. Polysaccharides like agar and carrageenan are essential in food products for their gelling properties,
THE ANTI-AGEING ROLE OF BIOACTIVE COMPOUNDS DERIVED FROM SEAWEED 91 while ulvan and alginate find applications in biomedical research and industrial processes due to their biocompatibility and sustainability (22). 3. Mechanisms of Cellular Senescence and Aging Cellular senescence is a state of irreversible growth arrest characterized by altered cellular morphology and function, playing a pivotal role in aging and agerelated diseases. Understanding the mechanisms underlying cellular senescence is crucial for elucidating the biological processes driving aging phenotypes and identifying potential therapeutic interventions (23). 3.1.TelomereShortening Telomeres, protective DNA-protein complexes at chromosome ends, play a critical role in maintaining genomic stability. During cellular replication, telomeres progressively shorten due to the end replication problem and insufficient telomerase activity. Shortened telomeres trigger a DNA damage response, leading to cellular senescence or apoptosis. Studies have demonstrated that telomere dysfunction contributes significantly to replicative senescence and age-related pathologies (24,25). 3.2.OxidativeStress Reactive oxygen species (ROS), byproducts of cellular metabolism, induce oxidative damage to cellular components, including proteins, lipids, and DNA. Chronic oxidative stress overcome cellular antioxidant defenses, leading to persistent DNA damage and activation of senescence-associated pathways such as p53 and p16INK4a. Oxidative stress-mediated senescence is implicated in aging processes and age-related diseases, highlighting the role of redox homeostasis in cellular aging (26,27). 3.3.DNADamage Accumulation of DNA damage, both from endogenous sources (replication errors, ROS) and exogenous insults (UV radiation, environmental toxins), triggers a DNA damage response (26,27). This response activates signaling pathways involving ATM, ATR, and p53, promoting cellular senescence or apoptosis to prevent the propagation of damaged DNA. Persistent DNA lesions and genomic instability in senescent cells contribute to aging phenotypes and age-related diseases (26,28,29).
92 INNOVATIVE APPROACHES IN MEDICINE AND HEALTH SCIENCES II 3.4.EpigeneticAlterations Epigenetic modifications, including DNA methylation, histone modifications, and chromatin remodeling regulate gene expression patterns and cellular identity.30 During aging, alterations in epigenetic marks disrupt normal gene regulation, contributing to cellular senescence and functional decline. Studies have implicated epigenetic dysregulation in the maintenance of senescent cell states and tissue aging, underscoring the role of chromatin remodeling complexes and histone modifying enzymes in aging processes (31,32). 3.5.InflammationandtheSenescence-AssociatedSecretoryPhenotype Senescent cells exhibit a senescence-associated secretory phenotype (SASP), characterized by the secretion of pro-inflammatory cytokines, chemokines, growth factors, and matrix metalloproteinases (2). SASP components create a microenvironment of chronic low-grade inflammation, known as inflammaging, which contributes to tissue dysfunction and age-related pathologies such as cancer, cardiovascular diseases, and neurodegeneration. Understanding the regulation of SASP and its impact on neighboring cells is crucial for targeting senescent cell clearance and inflammation in aging interventions (2,33). 4. Effects of Seaweed Derives on Lifespan Extension and Inhibition of Cellular Senescence In animal models such as yeasts and flies, the lifetime extension effects of marine macroalgal chemicals have been reported. The carotenoid fucoxanthin, which is a significant photosynthetic pigment derived from brown algae, has been shown to extend the longevity of Caenorhabditis elegans and Drosophila melanogaster. A sulfated polysaccharide called porphyran is derived from the red algae Porphyra haitanensis and there have been reports of the lifespan extension effect of both natural and degraded porphyran in Drosophila (34). Porphyrans have also been observed to boost the vitality of middle-aged flies in addition to their lifespan. These results imply that porphyran, at least in Drosophila, can lengthen life and improve health. In a similar way, polysaccharides derived from Saccharina japonica, a brown alga, have the ability to considerably increase the mean lifespan of Drosophila in both males and females. However, among females, the rise was more significant (34). Conversely, an algal substance called fucoidan protects cells against stress-induced senescence as well as replicative senescence. According to some
THE ANTI-AGEING ROLE OF BIOACTIVE COMPOUNDS DERIVED FROM SEAWEED 93 studies, fucoidan alleviated the senescence-induced senescence-associated betagalactosidase (SA-β-Gal) activity in long-term cultivated endothelium colonyforming cells. Senescent cell identification is most commonly achieved with the use of the SA-β-Gal activity biomarker (35). Additionally, fucoidan promotes the expression of the anti-senescence protein regucalcin while decreasing the expression of the pro-senescence protein p21. According to a different study, fucoidan therapy also lessens the SA-β-Gal activity in mesenchymal stem cells that is brought on by p-cresol, a significant uremic toxin. Furthermore, in stress-induced senescent fibroblasts, some chemicals derived from algae, such porphyran, demonstrated the anti-SA-β-Gal action. Furthermore, a class of red photosynthetic pigments found in red algae is called phycoerythrin.36 The red alga Pyropia yezoensis synthesized phycoerythrin-derived peptide reduced the activity of SA-β-Gal in aged primary hippocampal neuron cells and lessened age-dependent neurite degeneration (36). The anti-senescence capacity and lifespan extension effect of seaweed derivatives in lower organisms encourage studies on the anti-aging effect of algal compounds in higher animals. Although the effects of algal derivatives on major pathways that regulate lifespan have been reported, to the best of our knowledge, the lifespan extension effect of seaweed-derived bioactive compounds has not been evaluated using mammalian models (35). This chapter investigates the anti-aging and regulatory effects of bioactive compounds detected in seaweeds using evidence from in vitro and in vivo studies. This chapter aims to explore the possible anti-aging advantages of these drugs and evaluate their application in future research. This study analyzes the regulatory roles of the anti-aging pathway using in vitro and in vivo studies on bioactive compounds derived from seaweeds. The goal is to understand the potential anti-aging impact of these compounds and explore their feasibility for future research. 5. Regulation of Anti-Aging Pathways by Seaweed Derived Bioactive Compounds The SIRT (sirtuins), AMPK (5′ AMP-activated protein kinase), autophagy, and IGF (Insulin-like growth factor) signaling pathways have been identified as the main anti-aging-associated pathways. Other recognized regulators of aging include nuclear factor erythroid 2-related factor 2 (NRF2) and the antioxidant protein expression regulator. These routes are not, however, independent of one another. They typically collaborate in an integrative way, controlling the beginning or course of aging through mutual interactions (36,37).
94 INNOVATIVE APPROACHES IN MEDICINE AND HEALTH SCIENCES II 5.1.AMPKandSIRTPathways A class of protein deacetylases dependent on nicotinamide adenine dinucleotide (NAD+) is known as sirtuins (SIRT1–SIRT7). While SIRT2 is exclusively found in the cytoplasm, SIRT1 travels back and forth between the cytoplasm and the nucleus. While SIRT6 and SIRT7 are found in the nucleus and nucleolus, respectively, SIRT3, SIRT4, and SIRT5 are proteins found in the mitochondria (38). Numerous transcription factors, histones, signaling molecules, and other enzymes are among their downstream deacetylation targets. The most closely related mammalian homologue to yeast SIR2, the most extensively researched sirtuin, is SIRT1 (37). In human fibroblasts, endogenous SIRT1 protein expression decreases with replicative aging. SIRT1 expression declined with ageing in the epidermis and kidneys of mice as well. Its additional genetic copies can replicate the effects of calorie restriction and have been demonstrated to increase the longevity of a variety of creatures, such as mice, flies, and yeasts (38). Additionally, in yeasts and mice, SIRT1 small molecule activators like resveratrol and SRT1720 also demonstrate the ability to extend lifespans. SIRT1 substrates associated with cellular senescence include, but are not limited to, the autophagy negative regulator mammalian target of rapamycin (mTOR), the transcription factor forkhead box protein O (FOXO), which regulates cell development, and the tumor repressor p53. In addition to SIRT1, other sirtuins affect longevity and age-related illnesses. While resveratrol itself is not found in seaweeds, several seaweed-derived polyphenols and bioactive compounds, such as phlorotannins and fucoxanthin, have been shown to exhibit SIRT1-modulatory activity and similar anti-senescence effects. These compounds may mimic resveratrol’s mechanism of action, positioning seaweeds as promising natural sources for anti-aging and cancer-preventive agents (35). Mice with overexpressed SIRT6 have a much longer lifetime than wild-type mice. However, this lifespan extension effect was only seen in male mice (35). In addition to SIRT, calorie restriction can activate AMPK, which has been shown to be crucial for the lifespan extension effect. Both SIRT1 and AMPK are affected by cellular energy status. Increased NAD+/NADH ratio activates SIRT1, whereas stressors that enhance the AMP/ATP ratio activate AMPK. SIRT1 partially inhibits senescence by activating AMPK via hepatic kinase B1. Simultaneously, AMPK promotes SIRT1 action by boosting cellular NAD+ levels (38). When AMPK is phosphorylated, its activity is at its highest. As a serine/ threonine protein kinase, AMPK inhibits the synthesis of proteins and fatty acids while promoting the oxidation of fatty acids and glycolysis to produce ATP. It
THE ANTI-AGEING ROLE OF BIOACTIVE COMPOUNDS DERIVED FROM SEAWEED 95 does this by phosphorylating specific metabolic enzymes and controlling the expression of relevant genes. Through an integrated signaling network, AMPK controls the aging process. It can improve cells tolerance to stress by activating signaling pathways like p53, Nrf2, and FOXO (38). In the adipose tissue of older mice, the expression levels of both total AMPK and phospho-AMPK were lower than those of their younger counterparts. There is growing evidence that AMPK signaling becomes less sensitive as people age (39). Numerous investigations have documented the lifespan extension function of AMPK. While lowered AMPK expression by RNA interference shortened the lifetime of Drosophila, transgenic expression of AMPK in adult muscle or fat bodies increased lifespan. Furthermore, mice with metformin, an AMPKactivating chemical, have longer lifespans and better health. More notably, male mice have shown similar health benefits when supplementing begins in middle life (40). Adipose tissue ages more quickly in obese people. Genetically obese mice’s adipose tissue displayed signs of early aging, including elevated p53 expression, proinflammatory cytokine secretion, and SA-β-Gal activity. In addition to persons with normal body weight, overweight subjects can also benefit from the anti-aging effects of the SIRT1 and AMPK pathways (39,40). In mice fed a high-fat diet, resveratrol administration increases the activity of both SIRT1 and AMPK, hence increasing the mice’s survival. Overweight male individuals have also been shown to activate AMPK and SIRT1 in response to calorie restriction (41). 5.2.Seaweed-BasedSIRTandAMPKActivators The anti-aging properties of sirtuin-activating chemicals have been investigated. Resveratrol, a naturally occurring phenol found in berries and grapes, is the most well-known sirtuin-activating substance. Under a variety of circumstances, certain bioactive substances found in seaweeds have demonstrated the capacity to activate sirtuin (42). The edible marine brown algae Ecklonia cava is found along the Korean and Japanese coasts. Its polyphenol extract has been shown to activate AMPK and SIRT1 in mice that have been made obese by high fat. The polyphenol extract supplementation dramatically raised the amount of hepatic phosphorylated AMPK and the expression of its downstream genes, while also mitigating the decline in hepatic SIRT1 protein level brought on by a high-fat diet (43). According to a recent study, the methanol extract of Eucheuma cava, abundant
96 INNOVATIVE APPROACHES IN MEDICINE AND HEALTH SCIENCES II in polyphenols, such as eckol, dieckol, and phloroglucinol derivatives, has shown significant biological activity. These phlorotannins are known for their potent antioxidant and anti-senescence properties, and have been reported to modulate cellular signaling pathways including SIRT1 activation, suppression of mTOR, and regulation of FOXO transcription factors, enhances AMPK phosphorylation in C2C12 mouse myoblasts after only one hour of treatment. Mice with streptozotocin-induced type I diabetes showed improved fasting glucose levels and recovered plasma insulin concentrations when they fed with methanol extract of E. cava. In mice fed a high-fat diet, it was discovered that an ethanol extract containing meroterpenoid-rich fraction from Sargassum serratifolium, another brown alga, raised the amount of phospho-AMPK in the liver. This fraction reduced obesity and non-alcoholic fatty liver disease brought on by a high-fat diet because it contains high quantities of sargahydroquinoic acid, sargachromenol, and sargaquinoic acid (44). Additionally, conjunctival epithelial cells that have been treated with the brown alga A. nodosum extract have enhanced SIRT1 activity. Similarly, some red algae have demonstrated the ability to activate AMPK and/or SIRT1. For example, in differentiated 3T3-L1 mouse adipocytes, lipid formation was reduced by a phenol-rich extract of the red algae Gracilaria verrucosa. Additionally, during 3T3-L1 development, G. verrucosa extract enhanced glucose absorption and more significantly AMPK phosphorylation. Certain bioactive compounds from different algae such as phlorotannins (eg, dieckol), fucoxanthin, and algal sterols have also been found to demonstrate AMPKand/or SIRT-activation capabilities, in addition to the ethanol and methanol extracts of algae, which contain a variety of diverse chemicals (35). Phloroglucinol and dieckol, two substances that were isolated from E. cava, have demonstrated a notable capacity to increase AMPK. Cellular phospho-AMPK levels rose when immortalized human hepatocytes HepG2 were treated with phloroglucinol, a significant phenolic component in E. cava. Phloroglucinol given orally also markedly enhanced glucose tolerance in male mice given a high-fat diet (35,45). The brown algae Ecklonia stolonifera and E. cava contain the phlorotannin dieckol. Dieckol treatment of 3T3-L1 adipocytes during differentiation demonstrated the potential to activate AMPK, which resulted in the suppression of adipogenesis. Dieckol has also been shown to have an AMPK-activating action in db/db mice with type II diabetes. After 14 days of intraperitoneal dosing, the muscle tissues of the dieckol-administered group had a higher degree of AMPK phosphorylation than those of the saline-
THE ANTI-AGEING ROLE OF BIOACTIVE COMPOUNDS DERIVED FROM SEAWEED 103 on increased levels of FOXO3 and daf16 by seaweed derivatives have been conducted under stressful settings, including heat stress, pathogen infection, and oxidative stress, much as those on AMPK and/or SIRT activation. More research is necessary to determine whether these substances can block insulin/IGF-1 and activate FOXOs as people age (35). 8. Seaweed Bioactive Compounds Activate the NRF2 Pathway 8.1.SignalingPathwayofNRF2 In addition to the previously discussed important pathways, antiinflammatory and antioxidant properties are also thought to help to prevent aging. Low-grade chronic inflammation and excessive oxidative stress may be factors in the beginning of aging. One of the main signs of aging is increased oxidative stress, which is linked to a number of age-related diseases. While the amount of different antioxidant enzymes falls with age, the generation of oxidants rises (38). The overabundance of oxidative products damages cells. A transcription factor called NRF2 promotes cellular defenses against oxidative stress through its signaling pathway. The NRF2 repressor Kelch-like ECH-associated protein 1 (Keap1) is particular. NRF2 stays in the cytoplasm by interacting with Keap1. Keap1 conjugation also facilitates NRF2 degradation by functioning as an NRF2-specific E3 ligase adaptor protein (58). Nrf2 separates from Keap1 and moves into the nucleus in a free and stable state when oxidative stress or other triggers are present. Nrf2 functions as a transcription factor inside the nucleus. The antioxidant response element (ARE), a DNA promoter found in the genes of many antioxidant proteins and detoxifying enzymes, is activated by NRF2 by dimerization with a small Maf protein. In addition to detoxifying enzymes like hemoxygenase1 (HO-1), glutathione S-transferase (GST), and NADPH: quinone oxidoreductase (NQO1), the Nrf2-ARE pathway can trigger antioxidant enzymes like superoxide dismutase (SOD) and catalase (CAT) (38,58). Calorie restriction requires NRF2. NRF2 signaling guards against oxidative stress’s after effects, including as aging and illnesses linked to aging. When exposed to acute stress, aging flies gradually lose their capacity to activate NRF2 targets. As people age, their ability to respond to stimuli like exercise by activating their NRF2-ARE downstream genes is compromised. Age-related functional loss can be countered by maintaining NRF2 signaling competence (38,58). It has been demonstrated that Keap1 loss-of-function mutations increase Drosophila lifespan, confirming NRF2 significance in regulating longevity. A
104 INNOVATIVE APPROACHES IN MEDICINE AND HEALTH SCIENCES II comparison of mouse lifespans revealed a favorable correlation between lifespan and constitutive NRF2-signaling activity (ARE-binding activity). Reactivation of NRF2 can repair cellular HGPS abnormalities, while impaired activity of the NRF2 antioxidant pathway is a driving mechanism of Hutchinson-Gilford progeria syndrome (HGPS), a rare and always fatal premature aging disorder. Additionally, NRF2 exhibits tissue-specific anti-aging properties. For instance, compared to wild-type mice, the outer retina of Nrf2-deficient mice was more susceptible to age-related macular degeneration (38,58). 8.2.Seaweed-BasedNRF2Activators It has been observed that certain chemicals extracted from seaweeds displayed an NRF2 activation effect. IMR-32 neuroblastma and LNCaP prostate cancer cells’ NRF2-ARE pathway was strongly activated by extracts and a number of fractions made from cultivated green algae. The chosen fractions caused transcription of NQO-1, an NRF2 target gene, and nuclear translocation of NRF2. Furthermore, RAW 264.7 macrophages expressed the NRF2 protein in response to an ethanol extract of the marine brown algae S. serratifolium. In contrast to the ethanol extract’s dose-dependent suppression of the Keap1 protein level, a downstream target, HO-1, boosted its protein expression. Furthermore, in the presence and absence of lipopolysaccharide, a phlorotannin-rich extract of another brown alga, E. cava, also stimulated the production of HO-1 and NRF2 in macrophages. Lipopolysaccharides encourage oxidative stress by activating macrophages’ membrane-bound NADPH oxidase (59,60). It has been demonstrated that a variety of algal substances, including polysaccharides, unsaturated fatty acids, sargaquinoic acid, and carotenoids, have the ability to activate NRF2. Isolated unsaturated fatty acid (C18:1(n-11)) from the green alga Ulva lactuca, which in human neuroblastoma IMR-32 cells activated cytoprotective genes controlled by the NRF2/ARE pathway, such as NQO1 and HO1. The ARE-activation activity of Ulva extract enriched with C18:1(n–11) was further confirmed by evaluation of several mouse organs, including the brain, heart, lung, liver, and stomach (35). By encouraging NRF2-dependent cytoprotection, polysaccharides from the brown algae Sargassum fusiforme enhanced antioxidant defense and reduced stress insult in aging mice. As male mice aged, their livers’ levels of the Nrf-2 protein decreased in both the cytoplasm and the nucleus. Two months after middle-aged mice (9 months old) were given polysaccharide, the mice’s livers showed enhanced nuclear accumulation of NRF2 and total protein expression. At the same time, the level of the NQO1 protein rose as well (35).
THE ANTI-AGEING ROLE OF BIOACTIVE COMPOUNDS DERIVED FROM SEAWEED 105 By activating the Nrf-2 signaling pathway, indole-6-carboxaldehyde (I6CA), which was extracted from Sargassum thunbergii, reversed oxidatively induced cell cycle arrest in Chinese Hamster lung fibroblasts. When cells were treated with both H2O2 and I6CA, the phosphorylation of Nrf-2 and the protein expressions of HO-1 and Nrf-2 were much higher than when cells were treated with H2O2 alone. Mycosporine-like amino acids (MAAs) are water-soluble metabolites that absorb ultraviolet light and are generated by seaweed. The NRF2-ARE pathway may be activated by two MAAs, shinorine and porphyra-334, which are competitive inhibitors of Keap1-NRF2 binding. Furthermore, when LPS was stimulated in RAW 264.7 cells, sargaquinoic acid, which was extracted from the brown algae Myagropsis myagroides, had antiinflammatory properties. Treatment with sargaquinoic acid raised the levels of the nucleus NRF2 and total HO-1 protein in response to LPS (61). Using the NRF2/ARE pathway, dieckol, which was isolated from Ecklonia stolonifera, activated NRF2 and raised the expression of NRF2 target proteins in HepG2 cells, such as HO-1, NQO-1, and GST. It was also discovered that eckol, another phlorotannin isolated from E. stolonifera, stimulated the nuclear translocation of NRF2 and caused HepG2 cells to produce HO-1. The brown algae Dictyopteris undulata contains zonarol, a para-hydroquinone-type proelectrophilic chemical activated the NRF2/ARE pathway and induced NRF2 target genes like HO-1 and NQO1 in HT22 cells, which are neurons in the hippocampus (35,61). Furthermore, in reaction to glutamate, which causes cell death, zonarol improved the survival of HT22 cells. The activated NRF2/ARE pathway is at least partially responsible for zonarol’s neuroprotective effects. In mouse hepatocytes BNL CL.2, fucoxanthin also enhanced the transcription of HO-1 and NQO1 and the accumulation of nuclear NRF2. The NRF2 activation effects of several classes of seaweed derivatives provide compelling evidence for the antioxidant properties of algal substances. Along with other agingregulating mechanisms, such an NRF2-stimulating action may also have antiaging effects (35,61). 9. Conclusions and Future Prospects The necessity to develop safe, dependable natural products with healthpromoting qualities has increased due to the growing demand for nutraceuticals. Despite the fact that many macroalgae species have been used for millennia as food and natural remedies, there is no many research examining their potential antiaging benefits. The possible effects of seaweed extracts or chemicals on pathways linked to aging are compiled in this review. At the cellular and tissue levels,
106 INNOVATIVE APPROACHES IN MEDICINE AND HEALTH SCIENCES II the regulation of these pathways points to a potential function for macroalgal bioactive chemicals in anti-aging, occasionally, they also show neuroprotective, anti-obesity, and anti-diabetic actions. Senescence has been linked to a number of illnesses, including type II diabetes, obesity, and cognitive decline. The potential for using seaweed-derived bioactive chemicals in anti-aging applications is further supported by their ability to protect against these disorders. To learn more about the roles of these substances, research on the lifespan and health span extension effects of seaweed derivatives on elderly animals is required. In the meanwhile, it is important to investigate how sex affects the anti-aging properties of these compounds generated from seaweed. Several substances, including fucoxanthin and meroterpenoid, have demonstrated the capacity to control several anti-aging pathways. More research is needed on these substances to determine whether they have anti-aging properties, whether they have the ability to protect against age-related illnesses like cardiovascular and neurological disorders, and prolong life expectancy, particularly in higher animal models. References 1. Li Z, Zhang Z, Ren Y, et al. Aging and age-related diseases: from mechanisms to therapeutic strategies. Biogerontology. 2021;22(2):165-187. 2. Correia-Melo C, Hewitt G, Passos JF. Telomeres, oxidative stress and inflammatory factors: partners in cellular senescence? Longev Healthspan. 2014;3(1):1-9. 3. Wang K, Liu H, Hu Q, et al. Epigenetic regulation of aging: implications for interventions of aging and diseases. Signal Transduct Target Ther. 2022;7(1):374. 4. Prasanna PG, Citrin DE, Hildesheim J, et al. Therapy-Induced senescence: opportunities to improve anticancer therapy. J Natl Cancer Inst. 2021;113(10):1285-1298. 5. Almeida AD, Ribeiro TP, Medeiros ID. Aging: Molecular Pathways and Implications on the Cardiovascular System. Oxid Med Cell Longev. 2017;2017:1-19. 6. Bailly C. The traditional Chinese medicine WuJiaPi (acanthopanacis cortex) and its main anti-inflammatory terpenoids. Longhua Chinese Med. 2021;4:14-14. 7. Kumarasinghe HS, Gunathilaka TL, Jayasooriya RGPT, Samarakoon KW. Impact of algal research and its potential for industrial applications: a review. Sri Lankan J Biol. 2023;8(2):4-19.
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111 CHAPTER VIII CONTEMPORARY APPROACHES TO ADRENAL DISEASE AND SURGERY Timuçin SİPAL1 & Çağlar SARIOĞLU2 1(Ass. Prof. Dr. and Lecturer), Kirikkale University Faculty of Medicine, Department of Urology Kirikkale / Turkiye E-mail: [email protected] Orcid No: 0000-0003-39992-2013 2(Urologist MD.), University of Health Sciences Bilkent Training and Education City Hospital, Department of Urology Ankara/ Turkiye E-mail: [email protected] Orcid No: 0000-0001-5136-9690 1. Introduction Adrenal disorders present a broad spectrum of clinical challenges, ranging from incidentally discovered, nonfunctioning nodules to hormonally active tumors and aggressive malignancies. Advances in imaging, biochemical testing, and surgical techniques have greatly improved our ability to diagnose and manage these conditions. Surgical treatment remains the cornerstone of management for most functional tumors and for lesions with features suspicious for malignancy. The evolution from traditional open operations to laparoscopic and, more recently, robotic approaches has transformed adrenal surgery into a safe and effective procedure with reduced morbidity and faster recovery. Nevertheless, open surgery continues to play a critical role in the treatment of large, invasive, or malignant tumors. This chapter provides an overview of current strategies in the management of adrenal disease, with a focus on surgical approaches, perioperative considerations, and evolving techniques that continue to shape practice.
112 INNOVATIVE APPROACHES IN MEDICINE AND HEALTH SCIENCES II 1.1.AdrenalAnatomy The adrenal gland has dual embryologic origins: cortex from mesoderm (urogenital ridge) and medulla from neural crest cells; their convergence explains the organ’s mixed endocrine functions (1). The cortex forms ~85% of the gland and comprises three concentric zones—zona glomerulosa (outer), zona fasciculata (middle), and zona reticularis (inner)—surrounding the medulla (2). Paired glands sit superior to the kidneys: the right is triangular and predominantly suprarenal; the left is crescentic and more prerenal. Typical dimensions are ~5 cm (height) × 2–3 cm (width) × 1 cm (AP), with a weight of ~2–6 g each. They are yellowgrey, firm, encapsulated by thick connective tissue, surrounded by perirenal fat, and bounded by Gerota’s fascia (except in the plane with the kidney). Movement with respiration reflects diaphragmatic attachments. Ectopic cortical rests (ovary, spermatic cord, testis) and extraadrenal medullary tissue (neck, bladder, paraaortic path) may occur (3,4). Right adrenal gland has a medial/anterior relation with the IVC (sometimes completely anterior); superolateral contact with the liver’s bare area; inferolateral peritoneal/liver/hepatic flexure; inferior surface against the right renal upper pole; anterior to IVC lie foramen of Winslow, secondpart duodenum, and pancreatic head. Left gland has anterior relations to the omental bursa (superior), splenic vessels, pancreatic body/tail, medial spleen edge (inferior); more anteriorly, stomach and transverse mesocolon; lateral contact with medial upper pole of left kidney; close to renal vessels (inferior) and aorta/celiac trunk (medial). Both glands have relations posteriorly to diaphragm and crura; medially inferior phrenic vessels and celiac ganglia. 1.2.ArterialSupply. Adrenal glands are highly vascular organs (~10 mL/min blood flow). Classically, three sets of arteries supply each gland—superior (from inferior phrenic), middle (from aorta), and inferior (from renal)—but origins vary (e.g., superior from aorta/celiac; middle from inferior phrenic/renal/SMA/celiac; inferior from aorta/gonadal/inferior phrenic). Additional supply may arise from gonadal/subcostal arteries. Intraglandular, capsular arterioles, cortical sinusoids (cortex→ medulla), and medullary arterioles feed sinusoids, conferring dual medullary inflow. Figure 1 1.3.VenousDrainage. Typically, a single central vein exits each gland, with asymmetric drainage: the left adrenal vein (longer) joins the inferior phrenic vein then the left renal
CONTEMPORARY APPROACHES TO ADRENAL DISEASE AND SURGERY 119 Molecular studies have shown that up to 40% of cases carry germline mutations in susceptibility genes, making these tumors among the most heritable of all neoplasms (21) . Mutations can be grouped into two main biological clusters: · Cluster 1 (pseudohypoxia pathway): Genes such as VHL, SDHx, EPAS1, and FH activate hypoxia-inducible factor signalling, increasing angiogenesis and tumorigenesis. · Cluster 2 (kinase signalling pathway): Mutations in RET, NF1, TMEM127, and MAX drive abnormal MAPK and PI3K-AKT pathways, promoting proliferation and survival (2) (2). This classification not only clarifies tumor biology but also informs prognosis, as certain mutations correlate with aggressive behaviour or higher metastatic risk. 4.2.ClinicalFeaturesandBiochemicalDiagnosis The hallmark of PCC/PGL is catecholamine excess. Classic symptoms include episodic headache, palpitations, and diaphoresis; however, many patients present with resistant hypertension or incidentally discovered adrenal masses (2) (3) . Normotensive cases occur in up to 15%, emphasising the importance of biochemical testing rather than relying solely on symptoms. The recommended biochemical approach is measurement of plasma free metanephrines or urinary fractionated metanephrines, which reflect continuous catecholamine metabolism and have the highest sensitivity (23 ,24) . False positives may occur with stress, certain medications, or improper sampling; thus, confirmatory testing and repeat sampling under controlled conditions are often needed. Elevated chromogranin A can support diagnosis but is nonspecific. 4.3.ImagingandLocalization Once biochemical evidence is established; imaging is performed for localization. CT and MRI are standard first-line modalities, with MRI favored in younger patients and for head and neck PGLs due to superior soft-tissue contrast (24) . Functional imaging is crucial in metastatic, multifocal, or hereditary disease. 123 I-MIBG scintigraphy remains widely used, particularly for adrenergic tumors, while PET tracers such as 1 8 F-FDOPA, 1 8F-FDG, and 6 8 Ga-DOTATATE provide higher sensitivity in certain genetic subgroups (25) . Selecting the optimal modality is increasingly genotype-driven: for example, 18 F-FDG PET is particularly effective in SDHB-mutated tumors. Figure 1 and Figure 2.
120 INNOVATIVE APPROACHES IN MEDICINE AND HEALTH SCIENCES II 4.4.Treatment Surgical resection is the definitive treatment for localized disease. Preoperative management requires meticulous alpha-adrenergic blockade to prevent intraoperative catecholamine crises, with beta-blockade added only after adequate alpha control Table 1 (26) . Laparoscopic adrenalectomy is the preferred approach for PCCs <6 cm without local invasion, while open resection is indicated for larger or invasive tumors. For PGLs, resection strategy depends on tumor location and surgical accessibility. For unresectable, metastatic, or recurrent disease, several modalities are available. High-specific-activity 131 I-MIBG therapy can achieve disease control in MIBG-avid tumors, while peptide receptor radionuclide therapy (PRRT, e.g., 177 Lu-DOTATATE) offers benefit in somatostatin receptor-positive disease (25,26 ). Systemic therapies such as tyrosine kinase inhibitors (e.g., sunitinib, cabozantinib) or chemotherapy (CVD regimen: cyclophosphamide, vincristine, dacarbazine) are reserved for progressive cases (27) . 4.5.Follow-UpandPrognosis Despite optimal treatment, recurrence occurs in up to 16% of patients, often many years later (27) . Lifelong follow-up with periodic biochemical testing and imaging is therefore essential, particularly in patients with high-risk genotypes such as SDHB. Malignancy is defined not histologically but by the presence of local invasion or distant metastasis. Prognosis varies by genotype, tumor size, location, and completeness of resection. Early recognition through genetic testing and family screening plays a crucial role in improving long-term outcomes (21 ,22) .
CONTEMPORARY APPROACHES TO ADRENAL DISEASE AND SURGERY 121 Table 4.1. Preoperative Medication for Pheochromocytoma and Paraganglioma Drug Class Examples Purpose / Notes α-blockers (firstline) Phenoxybenzamine (non-selective, longacting) Doxazosin, Prazosin, Terazosin (selective, shorter-acting) Start 7–14 days pre-op. Control hypertension and prevent intraoperative crisis. Phenoxybenzamine: more effective but higher risk of post-op hypotension; selective blockers: fewer side effects, easier titration. β-blockers (only after α-blockade) Propranolol, Atenolol, Metoprolol Used if tachycardia or arrhythmia persists after adequate α-blockade. Never start before α-blockade (risk of unopposed vasoconstriction & hypertensive crisis). Calcium channel blockers Amlodipine, Nifedipine, Nicardipine Alternative or adjunct for blood pressure control, especially if α-blockers not tolerated. Metyrosine — Catecholamine synthesis inhibitor. Reserved for large tumors, severe hypertension, or refractory cases. Often combined with α-blockers. Volume expansion High-salt diet + fluids Begin 2–3 days pre-op. Restores intravascular volume depleted by chronic catecholamine excess and α-blockade; reduces post-op hypotension risk.
122 INNOVATIVE APPROACHES IN MEDICINE AND HEALTH SCIENCES II synthesis inhibitor. Reserved for large tumors, severe hypertension, or refractory cases. Often combined with αblockers. Volume expansion High-salt diet + fluids Begin 2–3 days pre-op. Restores intravascular volume depleted by chronic catecholamine excess and α-blockade; reduces post-op hypotension risk. Figure 1. Arrow indicates 5.2 cm in size adrenal mass in noncontrast CT scan. Figure 2. 18F-FDG PET CT confirms the metastatic adrenal mass from right lung. 5. Adrenal Metabolic Workup synthesis inhibitor. Reserved for large tumors, severe hypertension, or refractory cases. Often combined with αblockers. Volume expansion High-salt diet + fluids Begin 2–3 days pre-op. Restores intravascular volume depleted by chronic catecholamine excess and α-blockade; reduces post-op hypotension risk. Figure 1. Arrow indicates 5.2 cm in size adrenal mass in noncontrast CT scan. Figure 2. 18F-FDG PET CT confirms the metastatic adrenal mass from right lung. 5. Adrenal Metabolic Workup Figure 1. Arrow indicates 5.2 cm in size adrenal mass in non-contrast CT scan. Figure 2. 18F-FDG PET CT confirms the metastatic adrenal mass from right lung. 5. Adrenal Metabolic Workup 5.1.Introduction Adrenal incidentalomas are increasingly common, detected in about 7% of imaging studies and more frequently with advancing age (17) . Once an adrenal mass is identified, clinicians must determine whether it is malignant, hormonally active, and whether intervention is needed. Biochemical evaluation is advised for all nodules ≥1 cm, since nearly half of adenomas demonstrate some degree of hormone autonomy (28) 5.2.PhysiologicBasis The adrenal cortex produces aldosterone, cortisol, and androgens, while the medulla generates catecholamines. Functional testing is designed to identify excess secretion from each pathway. 5.2.1.FunctionalTumors 5.2.1.1.Pheochromocytoma These tumors account for about 4–5% of adrenal nodules in population studies (29) . Patients may present with headache, palpitations, and sweating,
CONTEMPORARY APPROACHES TO ADRENAL DISEASE AND SURGERY 123 although many are asymptomatic. Plasma metanephrines are the most sensitive test, with 24-hour urine assays as confirmation. Posture is critical; supine sampling reduces false positives (2) (3) . Screening before intervention is essential, as in one series of oncology patients undergoing adrenalectomy, 24% of resected lesions proved to be pheochromocytomas (30) . The clonidine suppression test, first described in 1981, remains useful when results are inconclusive(31) . 5.2.1.2.Cortisol-ProducingLesions Mild autonomous cortisol secretion (MACS) is present in 20–50% of adenomas (32) . Even subtle hypercortisolism carries cardiovascular and metabolic risks. The most reliable tool is the 1-mg overnight dexamethasone suppression test, with a morning cortisol ≤1.8 µg/dL regarded as normal (20) , Table 2. Low DHEA-S or suppressed ACTH can support diagnosis, and in bilateral disease, adrenal venous sampling may assist localisation. 5.2.1.3.PrimaryHyperaldosteronism Primary aldosteronism is estimated to affect 5–10% of patients with hypertension(33) . Screening uses the aldosterone-to-renin ratio, ideally measured under controlled conditions. In those with spontaneous hypokalaemia, suppressed renin, and aldosterone >20 ng/dL, confirmatory testing can be omitted (8) . For others, confirmatory strategies include salt loading, saline infusion, fludrocortisone suppression, or captopril challenge. In a Mayo Clinic series of 194 patients, reliance on CT alone would have led to inappropriate management in nearly 25%, underscoring the value of adrenal venous sampling (34) . 5.2.1.4.AdrenocorticalCarcinoma(ACC) ACC is rare, representing about 0.3% of incidentalomas (1) (7) . These tumors often secrete multiple hormones and present as large, suspicious lesions. Urine steroid metabolomics have shown promise for distinguishing ACC, with one prospective study reporting a positive predictive value of 76% and negative predictive value of 99.7%(17) . Biopsy should be avoided, as it risks tumour spread and worsened prognosis. 5.2.2.NonfunctionalLesions About half of adrenal incidentalomas are nonfunctional (28) . Adenomas may require surveillance as some later develop cortisol autonomy. Myelolipomas larger than 6 cm may cause compression, and 14% in this size range have been
124 INNOVATIVE APPROACHES IN MEDICINE AND HEALTH SCIENCES II reported to become hemorrhagic (35) . Ganglioneuromas account for roughly 2% of adrenalectomies in surgical series (36) . Metastases make up about 8% of adrenal nodules, especially in patients with known cancer (37) . 5.3.AdrenalInsufficiency Bilateral adrenal infiltration by metastases or other processes may cause adrenal insufficiency, reported in up to 8% of cases (38) . Symptoms are nonspecific but early recognition is vital, as adrenal crisis can be life-threatening. Morning cortisol and ACTH levels are first-line, with cosyntropin stimulation used when results are uncertain. 5.4.ClinicalCarePoints 1. All nodules ≥1 cm warrant hormonal evaluation. 2. Pheochromocytoma should be excluded before biopsy or surgery. 3. ARR remains the cornerstone test for primary aldosteronism; AVS guides surgical management. 4. Urine steroid profiling is valuable in suspected ACC. 5. Biopsy should generally be avoided unless it will directly alter management.
CONTEMPORARY APPROACHES TO ADRENAL DISEASE AND SURGERY 125 Table 5.1. Adrenal Metabolic Workup Condition Single Best Test Additional / Confirmatory Tests Special Considerations Pheochromocytoma Plasma metanephrines 24-h urine metanephrines or catecholamines; clonidine suppression if equivocal False positives with medications (TCAs, levodopa, MAOIs); seated vs. supine sampling important Cortisol autonomy (Adrenal Cushing/ MACS) 1-mg overnight dexamethasone suppression test ACTH, DHEA-S, 24-h urine cortisol if needed Altered dexamethasone metabolism, oral estrogen, pseudoCushing states may confound results Primary Hyperaldosteronism Morning plasma renin activity + aldosterone (ARR) 24-h urine aldosterone after salt load; saline/ fludrocortisone suppression; captopril test Medication interference (ACEi, ARB, diuretics, MR antagonists); AVS for subtyping Adrenocortical Carcinoma Multihormonal elevations + suspicious imaging 24-h urine steroid profiling, DHEA-S, sex steroids Avoid biopsy unless diagnosis will change management; resection preferred 6. Summary of Adrenal Mass Diagnosis Adrenal masses are increasingly encountered in urologic practice due to the widespread use of abdominal imaging. The majority are incidentally detected (“incidentalomas”), with prevalence reported at around 4% in adults and up to 10% in the elderly (Mantero 2000). Every lesion larger than 1 cm requires careful evaluation to determine two critical questions: is the mass malignant, and is it hormonally active (39) 6.1.BackgroundandClinicalAssessment Most adrenal masses are benign adenomas, making up roughly 70–75% of incidentalomas. Other benign lesions include myelolipomas (~8%),
126 INNOVATIVE APPROACHES IN MEDICINE AND HEALTH SCIENCES II ganglioneuromas (~4%), and cysts (~5%) (4) (0) . However, more than 10% of adrenal lesions demonstrate hormonal activity, and approximately 20% of incidentalomas ultimately undergo resection(41) . A structured history and examination can identify features suggestive of cortisol, aldosterone, or catecholamine excess. For instance, resistant hypertension may indicate Conn’s syndrome, paroxysmal palpitations point to pheochromocytoma, and virilization or gynecomastia suggests sex-steroid excess. 6.2.RadiologicEvaluation 6.2.1.First-LineImaging Noncontrast CT is the cornerstone for adrenal mass assessment. Adenomas, rich in intracellular lipid, typically measure <10 Hounsfield Units (HU), which carries near 100% specificity for benignity (42) . Lesions >10 HU require further characterization. 6.2.2.SizeCriteria Tumor size is strongly correlated with malignancy risk: <4 cm carries ~0% risk, 4–6 cm about 2.4%, and >6 cm nearly 20% risk of carcinoma(43) . 6.2.3.AdditionalImagingFeatures · Macroscopic fat → diagnostic of myelolipoma. · Rim enhancement, heterogeneity, necrosis, invasion → concerning for malignancy. · Calcifications → usually benign but may be present in carcinoma. 6.2.2.AlternativeImaging · CT Washout Studies: Adenomas show rapid contrast washout; >60% absolute washout is strongly suggestive of benignity. Figure 2 · Chemical-Shift MRI: Identifies intracellular fat; an alternative in young, pregnant, or renally impaired patients. · Growth Kinetics: Lesions growing >5 mm/year or >20% in diameter warrant resection. · PET (18F-FDG): Useful for staging adrenocortical carcinoma (ACC) or differentiating adenoma from metastasis in cancer patients.
CONTEMPORARY APPROACHES TO ADRENAL DISEASE AND SURGERY 127 6.3.HormonalEvaluation All lesions >1 cm merit biochemical assessment. Around 10–15% of adrenal masses are functional (41) . Key tests include: · 1-mg overnight dexamethasone suppression test (DST): Cortisol ≤1.8 µg/dL rules out hypercortisolism. · Aldosterone-to-Renin Ratio (ARR): Best screening for Conn’s syndrome. ARR >20 with aldosterone >15 ng/dL is strongly suggestive (8) . · Plasma metanephrines or urinary fractionated metanephrines: Standard tests for pheochromocytoma (23) . · Sex steroids (DHEA-S, testosterone, estradiol, 17-OH progesterone): Measured only if ACC is suspected. Figure 1 Table 6. 1. Hormonal Workup Condition First-line Test Key Thresholds Notes Cortisol autonomy 1-mg DST ≤1.8 µg/dL = ruled out; >5 µg/dL = diagnostic Consider drug effects and false positives Primary aldosteronism Morning ARR ARR >20; aldosterone >15 ng/dL Hold MRAs, correct potassium Pheochromocytoma Plasma or urine metanephrines >3× ULN or both metabolites elevated Supine testing preferred Suspected ACC Sex steroids Elevated DHEA-S, estradiol, testosterone Marker for follow-up 6.4.Biopsy Adrenal biopsy plays a limited role. It cannot differentiate adenoma from carcinoma nor assess hormone activity. Its only role is in suspected metastasis from extra-adrenal cancer, provided pheochromocytoma has been excluded. Even then, 4–8% of biopsies are nondiagnostic, and complications occur in up to 13% of cases (17) . Management · Benign nonfunctional adenomas <4 cm → no intervention. · Functional or suspicious lesions → surgical resection.
128 INNOVATIVE APPROACHES IN MEDICINE AND HEALTH SCIENCES II · ACC → aggressive en-bloc resection, open surgery for tumors >5 cm, possible lymph node dissection. · Large benign lesions (e.g., myelolipoma >7–10 cm, cyst >5 cm) → surgery if symptomatic or uncertain. 6.5.Follow-Up Guidelines vary, but common principles include: · Re-image indeterminate benign masses at 3–12 months. · Monitor growth kinetics (>5 mm/year threshold). · After resection of pheochromocytoma: imaging at 12 weeks, then every 6–12 months for 5 years, annually thereafter. · ACC: surveillance every 3–12 months for 5 years. Special Populations · Pregnancy: Prefer MRI or low-dose CT; urinary free cortisol is recommended for Cushing’s evaluation, with trimester-specific cut-offs. · Bilateral Masses: Usually hyperplasia or benign adenomas, but metastasis and lymphoma are possible. Workup should include 17-OH progesterone to exclude congenital adrenal hyperplasia. · Young Patients: Masses are rare but more likely malignant; expedite workup. · Elderly Patients: Most lesions are benign; weigh surgery against frailty and comorbidities. · Extra-Adrenal Malignancy: In known cancer patients, up to 75% of adrenal lesions are metastases (44) . PET or biopsy may assist if imaging is equivocal. 6.6.SummaryandKeyPoints Evaluation of adrenal masses requires systematic assessment of malignancy risk and hormonal activity. Noncontrast CT is the first imaging tool, with size and HU attenuation providing strong predictors. Functional evaluation must include cortisol, aldosterone, and catecholamine screening, with selective sex steroid testing. Most lesions are benign and nonfunctional, but timely detection of functional adenomas, pheochromocytomas, and carcinomas is critical to reduce morbidity and mortality. Management should be multidisciplinary, balancing surgical intervention with conservative follow-up based on risk stratification.
CONTEMPORARY APPROACHES TO ADRENAL DISEASE AND SURGERY 135 Robotic adrenalectomy provides equivalent outcomes, with possible advantages in larger or complex tumors. The choice of approach should be individualized based on tumor characteristics, surgeon experience, and institutional expertise. Figure 8.1. Laparoscopic Right Adrenelectomy. Blue star: Adrenal mass, Red star: Liver, Yellow star: Hepatorenal Ligament Figure 8.2: Adrenal adenoma has been revealed after peritoneal dissection. Figure 8. 3. The Right surrenal vein has been exposed. Blue star: The Right adrenal vein, green Star: The Vena cava Figure 8.1. Laparoscopic Right Adrenelectomy. Blue star: Adrenal mass, Red star: Liver, Yellow star: Hepatorenal Ligament Figure 8.1. Laparoscopic Right Adrenelectomy. Blue star: Adrenal mass, Red star: Liver, Yellow star: Hepatorenal Ligament Figure 8.2: Adrenal adenoma has been revealed after peritoneal dissection. Figure 8. 3. The Right surrenal vein has been exposed. Blue star: The Right adrenal vein, green Star: The Vena cava Figure 8.2: Adrenal adenoma has been revealed after peritoneal dissection. Figure 8.1. Laparoscopic Right Adrenelectomy. Blue star: Adrenal mass, Red star: Liver, Yellow star: Hepatorenal Ligament Figure 8.2: Adrenal adenoma has been revealed after peritoneal dissection. Figure 8. 3. The Right surrenal vein has been exposed. Blue star: The Right adrenal vein, green Star: The Vena cava Figure 8. 3. The Right surrenal vein has been exposed. Blue star: The Right adrenal vein, green Star: The Vena cava
136 INNOVATIVE APPROACHES IN MEDICINE AND HEALTH SCIENCES II Figure 8. 4. Adrenalectomy has been completed. Blue arrow: The adrenalectomy piece, yellow star: The Right Kidney, green star: The Adrenal bed, blue star: The Liver, red star: The diaphragm. 9. Conclusion Adrenal disease presents with a wide clinical spectrum, from incidentally discovered, nonfunctional nodules to hormonally active lesions and aggressive malignancies. Accurate evaluation requires a careful balance of imaging, biochemical assessment, and clinical judgment. While minimally invasive approaches have become the standard for most benign and functional adrenal tumors, open surgery continues to play a vital role in the management of large, invasive, or malignant lesions. Advances in laparoscopy, retroperitoneoscopic access, and robotic platforms have expanded surgical options, offering reduced morbidity without compromising safety in properly selected patients. Optimal outcomes are achieved through multidisciplinary care, meticulous perioperative preparation, and treatment in experienced centers. Acknowledgements All images and figures included in this chapter were obtained from the archives of the Department of Urology Library Kirikkale University Faculty of Medicine. Figure 8. 4. Adrenalectomy has been completed. Blue arrow: The adrenalectomy piece, yellow star: The Right Kidney, green star: The Adrenal bed, blue star: The Liver, red star: The diaphragm. 9. Conclusion Adrenal disease presents with a wide clinical spectrum, from incidentally discovered, nonfunctional nodules to hormonally active lesions and aggressive malignancies. Accurate evaluation requires a careful balance of imaging, biochemical assessment, and clinical judgment. While minimally invasive approaches have become the standard for most benign and functional adrenal tumors, open surgery continues to play a vital role in the management of large, invasive, or malignant lesions. Advances in laparoscopy, retroperitoneoscopic access, and robotic platforms have expanded surgical options, offering reduced morbidity without compromising safety in properly selected patients. Optimal outcomes are achieved through multidisciplinary care, meticulous perioperative preparation, and treatment in experienced centers. Acknowledgements All images and figures included in this chapter were obtained from the archives of the Department of Urology Library Kirikkale University Faculty of Medicine. References 1. Miller WL, White PC. History of Adrenal Research: From Ancient Anatomy to Contemporary Molecular Biology. Endocr Rev. 2023 Jan 12;44(1):70–116.
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141 CHAPTER IX SPLENIC ARTERY ANEURYSM Mehmet Burak ÇİLDAй & Tuğba ÖZTÜRK² ¹(Prof Dr.), Aydın Adnan Menderes University E-mail: [email protected] ORCID : 0000-0003-2371-3540 ² (Dr.), Aydın Adnan Menderes University E-mail: [email protected] ORCID : 0009-0001-7714-3582 1. Introduction An aneurysm is defined as a focal arterial dilatation measuring between 1.5 and 2 times the normal vessel diameter. This pathology can manifest as a true aneurysm, which involves all three layers of the arterial wall, or as a pseudoaneurysm, which does not involve all wall layers. In most aneurysms, degenerative changes in the medial layer are observed. These changes are characterized by the loss or fragmentation of elastic fibers and a reduction in the number of smooth muscle cells. Etiological factors include atherosclerosis, fibromuscular dysplasia, connective tissue disorders, portal hypertension, and pregnancy. Pseudoaneurysms, on the other hand, may develop due to secondary causes such as trauma, inflammation, infection, or vasculitis. Visceral artery aneurysms (VAAs) are a rare condition, with an incidence ranging from 0.01% to 0.2% in routine autopsies. However, these aneurysms are clinically significant and possess life-threatening potential (1,2). Approximately 22% of reported VAA cases present with rupture, a condition associated with a mortality rate of 8.5% (3). Splenic artery aneurysms (SAAs) are considered the most prevalent type of VAAs, accounting for up to 60% of these lesions. SAAs are frequently discovered incidentally, with a reported incidence of 0.78% in arteriograms and a prevalence ranging from 0.1% to 10.4% in autopsy studies (4). The splenic artery, the largest and widest branch of the celiac trunk, typically has a diameter
142 INNOVATIVE APPROACHES IN MEDICINE AND HEALTH SCIENCES II of 6 to 10 mm and an average length of 13 cm. Its primary anatomical function is to provide blood supply to the spleen. The main branches originating from the splenic artery include the pancreatic, posterior gastric, short gastric, and left gastroepiploic arteries (5). Pregnancy, portal hypertension, medial fibrodysplasia, arteritis, collagen vascular diseases, alpha-1 antitrypsin deficiency, and pancreatitis have been reported as risk factors for the development of aneurysms. Unlike other visceral aneurysms, atherosclerosis has not been implicated in the development of SAAs, and in cases where atherosclerosis is present, it has been reported to occur subsequent to aneurysm formation (6). SAAs are most often asymptomatic and are usually detected incidentally during radiological imaging. Rarely, unruptured aneurysms may present with epigastric pain or left upper quadrant pain radiating to the left shoulder (7). In uncommon cases, they may manifest with signs of extrahepatic portal hypertension. In certain cases, splenic infarction may ocur due to embolization of thrombus with in the aneurysm. Erosion of the aneurysm into the stomach may result in upper gastrointestinal bleeding (8). Spontaneous rupture of the aneurysm presents with acute abdominal symptoms due to intraperitoneal hemorrhage and hypovolemic shock. 2. Diagnosis Conventional angiography is considered the gold standard in the diagnosis of SAAs; however, its major drawback is the requirement for arterial catheterization. Although ultrasonography can be used for diagnosis, it is a challenging modality due to its operator dependency and patientrelated limitations. Computed tomographic angiography (CTA) and magnetic resonance angiography (MRA) provide high-resolution images of the artery, allowing the detection of aneurysms, stenosis, or occlusion. In MRA examinations, the imaging time is relatively long, requiring the patient to remain stil for an extended period. This modality is also highly sensitive to motion artifacts. With the use of modern multidetector computed tomography, CTA imaging has shown improved performance, reducing examination time and slice thickness while allowing for an increased volume of coverage (figure 1). The shorter examination time reduces motion artifacts, thereby improving image quality. A shorter imaging time also enables the evaluation of parenchymal organs in different phases of contrast enhancement, facilitating lesion characterization. Moreover, the reduction in scanning time has decreased the amount of contrast material required for the examination. By
SPLENIC ARTERY ANEURYSM 143 reducing slice thickness, isotropic imaging has be come possible, providing comparable spatial resolution in all planes (9,10). The mortality rate associated with SAA rupture is reported to be approximately 25% (11). Rupture occurs more frequently during pregnancy and is linked to high rates of both maternal and fetal mortality (12,13). Additionally, rupture has been observed to ocur more often in pseudoaneurysms (14). Current vascular surgery clinical practice guidelines (15) recommend treatment for nonruptured splenic artery pseudoaneurysms of any size, and for non-ruptured true splenic artery aneurysms of any size in women of childbearing age. Treatment is also advised for non-ruptured true splenic artery aneurysms that are 3 cm or larger, particularly those that show interval growth or are associated with symptoms in patients with acceptable surgical risk, due to the potential for rupture. Conversely, observation is suggested for small (<3 cm), stable, and asymptomatic true splenic artery aneurysms, as well as in patients with significant medical comorbidities or limited life expectancy. 3. Treatment Treatment can be performed either through open surgery or by endovascular approaches. The splenic artery has a well-developed collateral circulation. Therefore, proximal and distal ligation of the aneurysmal segment represents a feasible surgical option. Endovascular treatment may also be achieved using a stent graft or coil embolization. The choice of treatment should be individualized based on the underlying disease and anatomical considerations. The conventional surgical management of SAAs varies depending on the location of the lesion. For aneurysms located in the proximal or mid-portion of the artery, treatment involves aneurysmectomy combined with proximal and distal ligation. Revascularization is typically not necessary because the distal splenic artery is also perfused by the short gastric arteries. For more distal lesions near the splenic hilum or for mycotic aneurysms, the traditional approach has been excision, either with or without splenectomy. Splenectomy becomes mandatory if the aneurysm involves the intrasplenic branches. Furthermore, in some cases of distally located aneurysms, distal pancreatectomy may also be required (16). In recent years, laparoscopic treatment options have also gained importance. Particularly for retro-pancreatic aneurysms, laparoscopic surgery can be combined with coil embolization (17,18). Endovascular treatment options involve coil embolization of both the proximal and distal segments of the aneurysm. However, for specific types, such
144 INNOVATIVE APPROACHES IN MEDICINE AND HEALTH SCIENCES II as saccular aneurysms, it is also possible to directly embolize the aneurysm sac using coils or cyanoacrylate-based adhesives (19) (figure 2). In addition, stent grafting is utilized for SAAs located in the proximal and middle segments, particularly in the proximal portion. In true SAAs with a wide neck or those involving major vessels at the origin of the splenic artery, bare stent-assisted coil embolization has also been recommended. For distal splenic artery and intrasplenic aneurysms, coil embolization alone is typically performed (20). However, splenic infarction or pancreatitis may ocur following distal splenic artery embolization Ruptured SAAs constitute a true surgical emergency. The splenic artery should be ligated both proximally and distally, and splenectomy is generally performed without vascular reconstruction (15). In emergency surgical cases requiring splenic artery ligation and splenectomy, vaccination is recommended either prior to the procedure or at least 14 days before hand in planned procedures where the splenic artery cannot be preserved, due to the risk of post-operative sepsis (15). 4. Conclusion Splenic artery aneurysms are the most common type of visceral artery aneurysms. They are often asymptomatic. Altough conventional angiography is the gold standard for diagnosis, diagnosis is usually made incidentally using multidetector computed tomography devices. Due to the high mortality rate following SAA rupture, caution is necessary. Rupture is particularly dangerous in pregnant patients. Treatment can be performed either through open surgery or by endovascular approaches. Improved endovascular methods can be preferred as a treatment option, in selected patient group,due to their minimally invasive method feature. References: 1. Røkke, O., Søndenaa, K., Amundsen, S., Bjerke-Larssen, T., &Jensen, D. The Diagnosis and Management of Splanchnic Artery Aneurysms. Scandinavian Journal of Gastroenterology, 1996 31(8), 737–743. 2. Nosher JL, Chung J, Brevetti LS, et al. Visceral and Renal Artery Aneurysms: A Pictorial Essay on Endovascular Therapy. RadioGraphics 2006; 26:1687–1704 3. Stanley JC, Thompson NW, Fry WJ. Splanchnic artery aneurysms. ArchSurg. 1970 Dec;101(6):689-97.