Kiwicha (Amaranthus caudatus L.) protein hydrolysates reduce intestinal inflammation by modulating the NLRP3 inflammasome pathway

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This is an Accepted Manuscript of an article published in FOOD & FUNCTION on 2022, available at: https://doi.org/10.1039/d2fo02177c” 1 Kiwicha (Amaranthus Caudatus L.) protein hydrolysates reduce intestinal 1 inflammation by modulating NLRP3 inflammasome pathway 2 Alicia Martinez-Lopez1, Fernando Rivero-Pino2, Alvaro Villanueva3, Rocio Toscano1, 3 Elena Grao-Cruces1, Elvira Marquez-Paradas1, Maria E. Martin4, Sergio Montserrat-de 4 la Paz*,1, Maria C. Millan-Linares1 5 1 Department of Medical Biochemistry, Molecular Biology, and Immunology, School of 6 Medicine, University of Seville, Av. Sanchez Pizjuan s/n, 41009 Seville, Spain. 7 2 European Food Safety Authority (EFSA), Via Carlo Magno, 1A, 43126 Parma, Italy. 8 3 Department of Food & Health, Instituto of Fat (IG-CSIC), Ctra. Utrera Km 1, 41013 Seville, 9 Spain. 10 4 Department of Cell Biology, Faculty of Biology, University of Seville, Av. Reina Mercedes 11 s/n, 41012 Seville, Spain. 12 *Corresponding Author: 13 Prof. Sergio Montserrat-de la Paz 14 Department of Medical Biochemistry, Molecular Biology, and Immunology, School of 15 Medicine, Universidad de Sevilla, Av. Sanchez Pizjuan s/n, 41009 Seville, Spain. 16 Tel: +34 955 421 051 17 E-mail: [email protected] 18 Short title: Kiwicha protein hydrolysates prevent intestinal inflammation 19 20 21 2 ABSTACT 1 2 The increase of world population, along with the new policies going towards a more 3 sustainable world has led to the need of searching for new food sources, which are 4 environmentally friendly and implying healthy and nutritious diets. This study explores the 5 biological activity of two Kiwicha (Amaranthus Caudatus L.) protein hydrolysates obtained 6 with Bioprotease LA-660 regarding the anti-inflammatory response at intestinal level, 7 employing the CACO-2 cell line. The results obtained showed that in vitro administration of 8 these hydrolysates decreases the expression of proinflammatory cytokines, increases the 9 expression of anti-inflammatory cytokines, and decreases the gene expression of major 10 components of the inflammasome in the intestinal CACO-2 cell model. To the author’s 11 knowledge, this is the first study evaluating the antiinflammatory activity of Kiwicha 12 hydrolysates at intestinal level, employing the CACO-2 cell model and its ultrastructural 13 characterization by scanning electron microscopy. We conclude that the Amaranthus 14 caudatus hydrolysates are a valuable source of active peptides able to take part as functional 15 ingredients into food and nutraceutical preparations. 16 17 Keywords: bioactive peptides, ultrastructure, inflammasome, immunonutrition, protein 18 hydrolysates. 19 20 21 22 23 24 25 26 27 28 29 30 31 3 Introduction 1 Kiwicha (Amaranthus caudatus L.) is a pseudocereal crop which provides macro and 2 micronutrients of quality, including a relevant amount of proteins (13-19%), mainly albumins 3 and globulins, with a complete set of amino acids.1,2 The amino acid content of this protein 4 source encompasses isoleucine, lysin, tryptophan, and threonine as major compounds, as well 5 as sulfur-containing amino acids.3 Consequently, the protein quality of kiwicha has been 6 proved to be higher than other cereals, based on the digestibility and availability of the 7 proteins4 making this protein an adequate source as recommended by FAO/WHO.2 8 In addition, A. caudatus has been investigated as a source of bioactive peptides, potentially 9 exerting anti-diabetic, anti-hyperlipidemic, and antihypercholesteraemic effects and 10 antioxidant and antimicrobial activities.1,5,6 The bioactivity of these peptides is due to their 11 similarity to the structure of human regulatory molecules, making them suitable to interact 12 with enzymes and receptors. These peptides can be obtained by enzymatic hydrolysis, as the 13 most commonly employed technique due to the advantages compared to chemical hydrolysis 14 or fermentation. The main advantage of bioactive peptides is their potential use as functional 15 ingredients to prevent or pre-treat diseases without presenting the side effects that 16 medications can exert,7 and it implies consequently, adding value to the product in terms of 17 potential health attributes. To exert biological activities after oral administration, active 18 peptides must first be resistant to degradation by digestive enzymes and, once absorbed, must 19 be resistant to degradation by plasma peptidases until they reach their target tissues. 20 Inflammatory bowel disease (IBD), which includes Crohn's disease and ulcerative colitis, is a 21 chronic relapsing inflammation of the gastrointestinal tract. Little is known about the 22 pathogenesis, but IBD is characterized by loss of epithelial barrier integrity due to excessive 23 inflammatory responses. The intestinal epithelium is a critical barrier that controls the 24 transportation of water and electrolytes and inhibits the passage of pathogens. This 25 epithelium is a cell monolayer regulating permeability, which is increased during active 26 phases of IBD. Currently, anti-inflammatory drugs, antibodies against TNFα, steroids, or 27 immunomodulators that inhibit the proliferation of cells of the immune system are used as 28 treatment for these diseases. However, these drugs only relieve symptoms, but also exert 29 serious side effects, and in the most severe cases, surgery is still required. Thus, there is no 30 treatment to reduce inflammation and restore intestinal barrier function.8 In this regard, 31 inflammasomes are intracellular proteins in charge of initiating the cellular inflammatory 32 4 signalling pathway. Specifically, the NLRP3 inflammasome is one of the main protein 1 complexes involved in the development of inflammatory events.9 2 The relationship between health and nutrition highlights the possibility of using functional 3 ingredients as a pre-treatment for this disease. In fact, nutritional supplements, including 4 bioactive peptides, have been extensively studied in cell culture and animal models, and some 5 of them have shown promising results in clinical studies.10 For instance, low molecular 6 weight peptides have proved to exert radical scavenging activity, while higher size peptides 7 can exert cytotoxic effects against colon cancer cells.6 Currently, there is little scientific 8 evidence of the use of plant sources as a precursor of peptides with an antiinflammatory 9 effect at the intestinal level,11–13 and in general, there is still little evidence on human 10 studies.12 Consequently, further research employing alternative sources scarcely studied is 11 relevant in terms of discovering new approaches that could be employed in the near future to 12 overcome the upcoming limitation of food supply and to tackle diverse diseases from a 13 prevention strategy. 14 The aim of this work was to evaluate the potential anti-inflammatory activity exerted by two 15 kiwicha protein hydrolysates at intestinal level, employing the CACO-2 cell model, and thus, 16 to provide evidence on the potential of bioactive peptides from this source, to be employed as 17 functional ingredient to prevent intestinal inflammation. 18 19 Materials and Methods 20 Chemicals and Samples 21 Amaranthus caudatus seed flour was provided by STURLA, S.R.L. (Buenos Aires, 22 Argentina). Bioprotease LA-660 was purchased from BIOCON (Barcelona, Spain). All the 23 chemicals (reagents and solvents) were of analytical grade and provided by Sigma Chemical 24 Co., Bachem AG (Bubendorf, CH, EU), and Gibco (Waltham, MA, USA). 25 Preparation of Protein Isolate and protein hydrolysates 26 Kiwicha protein isolates (KPI) from the raw material were obtained following the method 27 described by Lqari et al.14 The flour was solubilised in Na2SO3 (0.25% w/v) for 1 h at pH 11. 28 Then, the extract was centrifuged at 9500 g for 15 min. The supernatant was recovered, and 29 the pellet was subjected to the extraction procedure again. The supernatants obtained in both 30 centrifugations were adjusted to the isoelectric point (pI) of kiwicha proteins (pH 4 during 30 31 5 min).15,16 The precipitate was washed with distilled H2O, adjusted to pH 4 and centrifuged in 1 order to remove other non-protein compounds and residual salts. At the end, the proteins 2 from the precipitate were lyophilized and stored at room temperature. 3 The isolate was subjected to hydrolysis under continuous stirring, using a LAMBDA 4 MINIFOR fermenter-bioreactor (Zurich, Switzerland), at controlled conditions of pH and 5 temperature. To this end, the isolate was diluted in distilled water (10% w/v) at 50 ºC 6 adjusted to pH 8. The protease employed for the hydrolysis was Bioprotease LA-660, added 7 at a ratio enzyme/substrate = 0.3 AU/g protein. Based on the previous results from our 8 research group, considering the degree of hydrolysis, its time, and the antioxidant activity 9 from a battery of several hydrolysates,16 the hydrolysis time was fixed to 15 and 30 min. In 10 both cases, the enzymes were deactivated by heating at 85 °C for 20 min in a heating plate. 11 The product obtained was centrifuged during 15 min at 9500 g and the supernatant was 12 collected for the following experimental procedures, named as Kiwicha protein 13 hydrolysates15 (KPH15) and KPH30, where the number indicates the hydrolysis time in 14 minutes. 15 Compositional analysis of kiwicha protein products 16 The concentration of protein was determined as % nitrogen content × 6.25 by elemental 17 microanalysis using a LECO TRUSPEC MICRO (Leco Corporation, St. Joseph, MI, USA). 18 The gravimetric method was used to determine the total dietary fiber. According to the 19 ignition method (550 °C during 36 h) ash content was determined. Measure of polyphenols 20 and soluble sugars was done using standard curves of chlorogenic acid for the first and 21 glucose for the second. UHPLC (Ac quity Arc, Waters, USA) was used for quantification of 22 amino acid. Besides, tryptophan content was assessed according to described in Montserrat- 23 de la Paz et al.16 Hydrolysis degree (HD) was calculated by a method which determine free 24 amino acid groups, the TNBS method. 25 Ultrastructural characterization by scanning electron microscopy analysis 26 Ultrastructural characterization of the lyophilized kiwicha deffated flour and protein 27 hydrolysates obtained by Bioprotease LA-660 were performed using Scanning Electron 28 Microscopy (SEM). A thin layer of each sample was mounted using sticky conductive carbon 29 tape on an aluminium stub, removing any unattached particle. The specimen holder was then 30 sputter-coated under vacuum with approximately 20 nm gold-palladium using a Leica EM 31 ACE600 instrument (Leica Microsystems, Wetzlar, Germany). Images were obtained with a 32 6 Zeiss Crossbeam 550 scanning electron microscope (Zeiss, Madrid, Spain) at 2.00 kV 1 accelerating voltage and different magnifications. 2 Cell culture 3 CACO-2 cells, as an in vitro intestinal absorption model, were obtained from the Cell 4 Biology Unit of the Fat Institute (Seville, Spain). Cells were maintained in high glucose 5 Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% of heat-inactivated 6 foetal bovine serum (FBS) and 1% penicillin/streptomycin. They were incubated in a 5% CO2 7 atmosphere at 37 °C. Experiments were performed in twelve-well plates, seeding CACO-2 8 cells at a density of 5 x 105 cells per well. Different conditions were used: untreated cells 9 (negative control), cells exposed to LPS at 1 μg/mL (positive control) and cells exposed to 10 LPS at 1 μg/mL together with the different treatments (KPH15 and KPH30) at 50 and 100 11 µg/mL. For this, LPS (E. coli 055:B5, Sigma-Aldrich) was first added for 1 hour and then the 12 kiwicha hydrolysates were added. After incubation for additional 24 hours, cells were 13 harvested to perform a set of determinations. 14 Cell viability 15 The cells were incubated, in this case, with different concentrations of kiwicha hydrolysates 16 in 96-well plates (1 x 105 cells per well) for 24 hours. The concentrations used were the 17 following: 10, 25, 50, 100 and 200 μg/mL. Next, 3-(4,5-dimethylthiazol-2-yl)-2,5- 18 diphenyltetrazol bromide (MTT, Sigma-Aldrich) solution was added to the wells and 19 incubated for 3-5 hours until a purple precipitate was visible. This assay is based on the 20 metabolic reduction of MTT, carried out by the mitochondrial enzyme succinate 21 dehydrogenase, in a purple-coloured compound (formazan), allowing the mitochondrial 22 functionality of the treated cells to be determined. The number of living cells is proportional 23 to the amount of formazan produced. MTT-formazan crystals were solubilized with dimethyl 24 Sulfoxide (DMSO, Sigma-Aldrich) and measured with a Thermo Scientific Fluoroskan 25 Ascent® microplate reader at 570 nm corrected to 650 nm. Cell viability was expressed as 26 the percentage of absorbance compared to that obtained in the control, that is, in the untreated 27 cells. 28 RNA extraction and RT-qPCR 29 RNA was extracted from CACO-2 cells using TRIsure reagent (Bioline), according to the 30 manufacturer's instructions. The quantity and purity of the RNA was evaluated by calculating 31 the ratio of absorbances between the wavelengths 230, 260 and 280 nm, through the ratio 32 7 (A260/A280) and (A260/A230) using a NanoDrop ND-1000 spectrophotometer (Thermo 1 Scientific). RNA (1 μg) was reverse transcribed using the iScript kit (Bio-Rad, Madrid, 2 Spain) following the manufacturer's instructions. An amount of 10 ng of the resulting cDNA 3 was used as a template for real-time PCR amplifications. The mRNA levels for specific genes 4 were determined in a CFX96 system (Bio-Rad). For each PCR reaction, cDNA template was 5 added to Brilliant SYBR green QPCR Supermix (Bio-Rad) containing the primer pairs for 6 either gene or for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as housekeeping 7 genes (Supplementary Table 1). All amplification reactions were performed in triplicate and 8 average threshold cycle (Ct) numbers of the triplicates were used to calculate the relative 9 mRNA expression of candidate genes. The magnitude of change of mRNA expression for 10 candidate genes was calculated by using the standard 2-(ΔΔCt) method. All data were 11 normalized to endogenous reference (GAPDH) gene content and expressed as percentage of 12 controls. 13 Extracellular quantification of cytokines 14 The levels of TNFα, IL-1β, IL-6, and IL-10 in the culture supernatants were determined by 15 enzyme-linked immunoassay (ELISA), following the manufacturer's instructions (Diaclone, 16 Besançon, France). The concentrations of these cytokines were expressed in pg/mL, 17 calculated from the calibration curves of the serial dilution of human and mouse recombinant 18 standards in each assay. 19 Statistical Analysis 20 All values are presented as the means ± standard deviations (SD). Data were evaluated using 21 Graph Pad Prism version 9.1.2 (San Diego, CA, USA). The statistical significance of any 22 difference in parameters between the groups was assessed using two-way analysis of variance 23 (ANOVA), followed by Tukey’s multiple-comparison test. P values less than 0,05 were 24 considered statistically significant. 25 26 Results and Discussion 27 Kiwicha protein products contain amino acid requirements in adults established by 28 FAO/WHO/UNU 29 Chemical composition of kiwicha protein products was showed in Table 1. The KPI was 30 extracted from KDF in 30.87% yield by basic extraction and then by acidic precipitation. The 31 8 resulting KPI showed 84.52% protein content. These results are similar to those obtained in 1 other pseudocereals of emerging crops as wheat and higher than others as quinoa. The amino 2 acid content in KDF, KPI, and KPHs is reported in Table 2, being the highest concentrations 3 those related to glutamic acid (Glu), aspartic acid (Asp) and arginine (Arg). These profiles 4 have similarities with the amino acid profiles reported in the literature. The importance of 5 these amino acids lies in that Asp is involved in the correct blood circulation, detoxification 6 of toxins in liver and kidneys, and different steps of cellular metabolism and Glu is involved 7 in the neurotransmission, energy transport, and nutrient absorption.16 8 Enzymatic hydrolysis remodel ultrastructure of kiwicha protein products 9 The surface morphology of three kiwicha protein products (KDF, KPH15, and KPH30) 10 observed by SEM is shown in Figure 1. From SEM images, it has observed that the protein 11 presented in KDF has degraded into small fragments after enzyme hydrolysis, increasing by 12 reaction time, which led to reduction in particle size of the KPH compared to KDF under 13 the same SEMparameters (Mag = 90x and AV = 2.0 kV / Mag = 345x and AV = 2.0 kV). 14 These findings are closely associated to Islam et al.17 who mentioned that protein has 15 degraded into small fragments and particle size reduction after treatment by enzyme and 16 reported that reduced the particle size may attributed to the high solubility of protein 17 hydrolysates. 18 Kiwicha protein products do not affect to viability of CACO-2 cells 19 As a first approach, to evaluate the cytotoxic effects of kiwicha products on the viability of 20 CACO-2 cells, they were treated with the kiwicha protein isolate, and the hydrolysates 21 (KPH15 and KPH30), up to 200 μg/mL for 24 h. The cell viability evaluated by the MTT 22 method was not affected by any of the selected concentrations of kiwicha products (Data not 23 shown). These results are in line with the absence of effects usually proved from protein 24 hydrolysates of diverse nature and treated with different proteases.18,19 25 Kiwicha protein hydrolysates down-regulate proinflammatory cytokine gene expression 26 in CACO-2 cells 27 In order to evaluate the anti-inflammatory properties of the HPKs at the intestinal level, the 28 gene expression of the cytokines TNFα, IL-1β, IL-6, and IL-10 was first measured by RT- 29 qPCR in CACO-2 cells, using LPS as a proinflammatory stimulus, and as treatment, the two 30 selected hydrolysates (KPH15 and KPH30) at 50 and 100 μg/mL for 24h. Regarding the 31 expression of TNFα, as can be seen in Figure 2A, the LPS showed a tendency to increase the 32 15 References 1 1 F. Janssen, A. Pauly, I. Rombouts, K. J. A. Jansens, L. J. Deleu and J. A. 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Surface characteristics of (A,D) kiwicha deffated flour (KFD) and kiwicha protein 2 hydrolysates after hydrolysis with Bioprotease LA-660 during (B,E)15 min (KPH15) and 3 (C,F) 30 min (KPH30) by Scanning Electron Microscopy (SEM) at magnifications (Mag.). 4 Upper SEM images (A, B, and C) were taken at Mag = 90x and AV = 2.0 kV. Lower SEM 5 images were taken at Mag = 345x and AV = 2.0 kV. 6 Figure 2. Gene expression of TNFα (A), IL-1β (B), IL-6 (C), and IL-10 (D) in CACO-2 cells 7 after 24 h incubation with LPS (1 µg/mL) in presence or absence of KPH15 and KPH30 at 50 8 and 100 µg/mL. Values are relative to untreated CACO-2 cells (control). Values are 9 presented as means ± SD (n = 3) and those marked with * are significantly different (* p < 10 0.05; ** p < 0.01; *** p < 0.001). 11 Figure 3. Protein release (pg/mL) of TNFα (A), IL-1β (B), IL-6 (C), and IL-10 (D) in 12 CACO-2 cells after 24 h incubation with LPS (1 µg/mL) in presence or absence of KPH15 13 and KPH30 at 50 and 100 µg/mL. Untreated CACO-2 cells as named as control. Values are 14 presented as means ± SD (n = 3) and those marked with * are significantly different (* p < 15 0.05; ** p < 0.01; *** p < 0.001). 16 Figure 4. Gene expression of NLRP3 (A), ASC (B), CASP1 (C), and IL-18 (D) in CACO-2 17 cells after 24 h incubation with LPS (1 µg/mL) in presence or absence of KPH15 and KPH30 18 at 50 and 100 µg/mL. Values are relative to untreated CACO-2 cells (control). Values are 19 presented as means ± SD (n = 3) and those marked with * are significantly different (* p < 20 0.05; ** p < 0.01; *** p < 0.001). 21 22 23 24 25 26 27 28 29 19 Table 1. Chemical composition of kiwicha protein products (KDF, kiwicha deffated flour; 1 KPI, kiwicha protein isolate; KPH, kiwicha protein hydrolysates). 2 Fat (seed flour, %) 5.730.12 KDF KPI KPH15 KPH30 Proteins 15.88±0.13 84.52±0.55 80.30±0.36 76.89±0.01 Moisture 7.01±0.17 0.96±0.10 1.29±0.09 4.54±0.04 Ash 2.18±0.09 1.75±0.05 8.40±0.16 9.13±0.59 Fiber 10.63±0.16 7.86±0.08 8.31±0.31 8.09±0.24 Sugar 1.73±0.22 0.78±0.12 0.46±0.02 0.30±0.04 Polyphenols 0.01±0.00 0.03±0.00 0.04±0.00 0.04±0.00 Others a 62.56 4.1 1.2 1.01 a Others: 100- (proteins + moisture + ash + fiber + sugar + polyphenols) 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Table 2. Amino acid composition of kiwicha protein products (KDF, kiwicha deffated flour; 1 KPI, kiwicha protein isolate; KPH, kiwicha protein hydrolysates) and adult requirements. 2 AA (mg/g prot) KDF KPI KPH15 KPH30 FAO a,b Indispensable amino acids His 24.300.0 27.600.0 31.200.1 31.100.0 15 Ile 30.80±0.3 43.80±0.0 44.90±0.2 44.90±0.2 30 Leu 61.20±0.0 72.00±0.1 74.00±0.1 74.30±0.1 59 Lys 62.30±0.1 53.10±0.1 55.00±0.1 55.00±0.0 45 Met+Cys 32.50±0.1 34.60±0.1 34.5±0.1 37.10±0.1 22 Met 13.1±0.1 20.90±0.2 20.0±0.2 22.7±0.2 Cys 19.40±0.1 13.70±0.0 14.50±0.1 14.40±0.1 Phe+Tyr 77.90±0.1 92.60±0.1 93.70±0.1 95.50±0.0 38 Thr 39.8±0.1 41.0±0.0 43.0±0.0 43.0±0.0 23 Trp 9.2±0.0 11.5±0.0 15.2±0.0 12.6±0.1 6 Val 35.3±0.3 46.10±0.1 48.1±0.2 47.30±0.2 39 Total 405.80 456.90 474.10 477.90 277 Dispensable amino acids Asp+Asn 94.3±0.4 92.6±0.2 97.1±0.1 96.8±0.0 - Glu+Gln 183.0±0.6 155.1±0.1 162.4±0.2 160.8±0.0 - Ser 79.1±0.4 55.4±0.1 58.5±0.1 58.4±0.1 - Gly 83.1±0.4 49.3±0.1 52.5±0.1 51.4±0.0 - Arg 87.3±0.2 89.4±0.1 93.8±0.1 93.7±0.1 - Ala 41.4±0.2 41.1±0.0 43.3±0.1 43.0±0.0 - Pro 58.5±0.7 94.9±0.6 52.7±0.5 54.8±0.6 - Tyr 34.9±0.1 40.8±0.2 40.4±0.2 42.3±0.0 - a FAO/WHO/UNU. Scoring pattern mg/g protein requirements in adults. 3 b FAO and FINUT, 2017. Dietary protein quality evaluation in human nutrition. FAO Food 4 and Nutrition Paper NO. 92. 5 6 7 8 21 Figure 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 22 Figure 2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 23 Figure 3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 24 Figure 4 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20