Evaluation of anti-inflammatory and atheroprotective properties of wheat gluten protein hydrolysates in primary human monocytes

foods Article Evaluation of Anti-Inflammatory and Atheroprotective Properties of Wheat Gluten Protein Hydrolysates in Primary Human Monocytes Sergio Montserrat-de la Paz 1, Noelia M. Rodriguez-Martin 2, Alvaro Villanueva 2, Justo Pedroche 2, Ivan Cruz-Chamorro 1,3 , Francisco Millan 2and Maria C. Millan-Linares 2,4,* 1Department of Medical Biochemistry, Molecular Biology and Immunology, School of Medicine, Universidad de Sevilla, Av. Dr. Fedriani 3, 41071 Seville, Spain; [email protected] (S.M.-d.l.P.); [email protected] (I.C.-C.) 2Department of Food & Health, Instituto de la Grasa, CSIC. Ctra. de Utrera Km. 1, 41013 Seville, Spain; [email protected] (N.M.R.-M.); [email protected] (A.V.); [email protected] (J.P.); [email protected] (F.W.) 3Instituto de Biomedicina de Sevilla, IBiS (Universidad de Sevilla, HUVR, Junta de Andalucía, CSIC), Av. Manuel Siurot s/n, 41013 Seville, Spain 4Cell Biology Unit, Instituto de la Grasa, CSIC. Ctra. de Utrera Km. 1, 41013 Seville, Spain *Correspondence: [email protected]; Tel.: +34-954-61-15-50 Received: 21 May 2020; Accepted: 29 June 2020; Published: 30 June 2020   Abstract: Bioactive protein hydrolysates have been identified in several sources as possible agents in the prevention and treatment of many diseases. A wheat gluten (WG) concentrate was hydrolyzed by Alcalase under specific conditions. The resulting hydrolysates were evaluated by in vitro cell-free experiments leading to the identification of one bioactive WG protein hydrolysate (WGPH), which was used at 50 and 100 µ g/mL on primary human monocytes. Reactive oxygen species (ROS) and nitrite levels and RT-qPCR and ELISA techniques were used to analyze the functional activity of WGPH. Our results showed that WGPH hydrolyzed in 45 min (WGPH45A) down-regulated gene expression of Interleukin (IL)-1 β ,IL-6,IL-17, and Interferon gamma (IFN γ ) and reduced cytokine release in lipopolysaccharide (LPS)-stimulated monocytes. In addition, WGPH45A down-regulated gene-related to atherosclerotic onset. Our results suggest that WGPH45A has a potent anti-inflammatory and atheroprotective properties, reducing the expression of gene-related inflammation and atherosclerosis that could be instrumental in maintaining cardiovascular homeostasis. Keywords: wheat gluten; protein hydrolysates; atherosclerosis; monocytes; celiac disease 1. Introduction Proteins are essential nutrients for the smooth operation organisms. The protein requirement in the diet is needed for several cell functions such as in the building and repairing of tissues, or such as for signaling molecules among others. Plant proteins are an interesting source to find new lead compounds of good-quality that are cheaper, and with a lesser environmental impact than animal proteins [ 1 ]. The substitution of animal proteins with plant proteins in the food industry or in our own diet is another important issue for health [ 2 ]. Within plant proteins, the study of their enzymatic hydrolysates is of special interest as, they have many bioactive functions, some of which have been shown to exhibit anti-inflammatory, antioxidant, or other bioactive activities [3–6]. Wheat is consumed worldwide and is a cultivated cereal and it is the basic food in the human diet through bread consumption. Wheat gluten (WG) is an economically important co-product in the recovery of wheat starch in wet milling of wheat flour [ 7 ]. Currently, wheat protein is used in animal Foods 2020,9, 854; doi:10.3390/foods9070854 www.mdpi.com/journal/foods Foods 2020,9, 854 2 of 16 and human nutrition. Approximately 80% of the production of wheat is for human consumption [ 8 ]. The main food application for gluten is bread, but it is also used to enrich other foods such as breakfast cereals, pasta, flour-based foods, and vegetable-based meat alternatives that are on the rise [ 8 ]. Gluten is a complex mixture of hundreds of related but distinct proteins, mainly gliadin and glutenin, which are highly resistant to hydrolysis and are mediated by proteases of the human gastrointestinal tract. Being able to generate oligopeptides, they remain in the small intestine for longer periods of time and can produce inflammation and consequently celiac disease and allergy in genetically predisposed people [ 9 , 10 ]. Nowadays, the increasing incidence of celiac disease has shifted the focus on to the development of WG-derivative healthy food ingredients [ 11 ]. WG enzymatic hydrolysis, which is essentially appropriate for expanding applications in food products, could produce functional WG protein hydrolysates (WGPHs). WG is composed of a high amount of glutamine and nonpolar amino acids (proline and glycine), which render the protein insoluble in aqueous media [ 12 ]. The low water solubility of gluten at a neutral pH limits its application as a functional component in the food industry, even though its cost is low compared to that of other protein isolates, such as those from peas, soy, or animal sources [ 13 ]. Several chemical and enzymatic modification strategies for improving the water solubility of gluten have been explored. Enzymatic hydrolysis tweaks the molecular mass and the openness of hydrophobic regions to the surrounding aqueous phase. Atherosclerosis is a common multifactorial disease with an important nutritional role. It is responsible for a high fraction of cardiovascular diseases [ 14 ]. A feature of atherosclerosis is chronic inflammation and increased activation of monocytes in the systemic blood circulation. Monocytes are cells with a myeloid origin, which are actively engaged in systemic and chronic inflammation and have distinct contributions to the formation, progression, and destabilization of the atherosclerotic plaque [ 15 ]. The metabolism of monocytes can be reprogrammed in the atherogenic microenvironments, which consist of hypoxia, molecular damage, and modified lipoproteins [ 16 ]. It is widely known that monocytes have an important role in the early stages of atherosclerosis. Monocytes are recruited to the endothelial layer; monocytes squeeze into the wall and are differentiated into macrophages mediated by adhesion molecules and cytokines [ 17 ]. Human monocytes are classified as different phenotypes depending on the surface membrane antigen expression, which is especially important for the disease progression. The M1 phenotype highly expresses the CD14 antigen in the cell membrane and relates to the pro-inflammatory stage with inflammatory marker production. Alternatively, the M2 phenotype highly expresses the CD16 antigen in its membrane and relates to anti-inflammatory marker production and inflammation resolution. This plastic character may define the monocyte cells phenotype, which is predicted on the chemical environment. For instance, the atherogenic microenvironment highly expresses the M1 phenotype, which is related to unstable plaque formation, while an anti-atherogenic microenvironment expresses the M2 phenotype and is related to stable plaques (17). An endogenous material addition such as lipopolysaccharide (LPS) thrives in a negative environment of monocyte and triggers the activation of the M1 phenotype. Despite this, the negative environment can be reversed with treatment of bioactive natural food chemicals such as small peptides, which triggers the activation of the M2 phenotype. Activated monocytes express high levels of chemokine receptors (CCRs) infiltrating into the sub-endothelial space upon binding to the adhesion molecules on the endothelium surface [ 18 ]. In addition, activated monocytes also produce high amounts of reactive oxygen species (ROS) and pro-inflammatory cytokines, including tumor necrosis factor (TNF)- α , interleukin (IL)-1 β , and IL-6 in response to LPS in vitro [ 19 ]. They also have a greater capacity for trans-endothelial migration, the ingestion of oxidized low-density lipoprotein (ox-LDL) via CD36 within the intima of the atherosclerotic lesion and become “foam cells” [ 20 ]. Taken together, the purpose of this work was to study the potential anti-inflammatory and atheroprotective activity of new WGPHs in primary human monocytes. Foods 2020,9, 854 3 of 16 2. Materials and Methodology 2.1. Hydrolysis of Wheat Gluten Protein Concentrate Hydrolysis of WG protein concentrate (WGPC, chemical, and amino acidic characterizations are shown in Tables S1 and S2, respectively) was carried out in a bioreactor in uninterrupted stirring at a restricted pH and temperature. WGPC was suspended in distilled water (10% w/v) and pH and temperature were adjusted to 8 and 50 ◦ C, respectively. Alcalase 2.4 L (2.4 AU/g was kindly donated by Novozymes, Bagsvaerd, Denmark) was added at enzyme/substrate =0.3 AU/g protein. Samples were taken at 0, 15, 30, 45, and 60 min. After that, the resulting hydrolysates were boiled at 85 ◦ C for 15 min in order to stop enzymatic reaction. The supernatants obtained after centrifuging at 19107.7 g for 15 min constituted the WGPHs. The resulting hydrolysates were composed of a mixture of amino acids and small peptides. The WGPHs were designated with the suffix: 0A, 15A, 30A, 45A, and 60A, which indicates in min the hydrolysis time. 2.2. Hydrolysis Degree Determination The hydrolysis degree (HD), defined as the percentage of peptide bonds cleaved, was evaluated by the TNBS (Sigma-Aldrich, St. Louis, MO, USA) method according to Adler-Nissen (1979) [ 21 ]. The amino group total number was examined in a sample that had been 100% hydrolyzed at 110 ◦ C for 24 h in 6 N HCl. 2.3. Inhibitory Activity of Angiotensin-Converting Enzymes Angiotensin-converting enzyme (ACE) activity was evaluated in harmony with the method described by Sentandreu and Toldr á [ 22 ]. Briefly, 40 µ L of WGPHs (at 1 µ g/ µ L) were added to 40 µ L of ACE (at 0.04 U/mL) in a 96-well multiplate. The addition of 160 µ L of Abz-Gly-p-nitro-Phe-Pro-OH (Bachem AG, Bubendorf, Switzerland) started the reaction at 0.45 mM in 150 mM Tris-base buffer (pH 8.3) with 1.125 M NaCl. After 30 min of incubation at 37 ◦ C, fluorescence was assessed using 340 and 535 nm as excitation and emission wavelengths, respectively. 2.4. Inhibitory Activity of Thrombin Thrombin activity was evaluated according to Ialenti et al. [ 23 ] Briefly, 50 µ L of WGPHs at a concentration of 20 µ g protein/ µ L, was previously incubated at room temperature for 1 h with 5 µ L of enzymes (at 0.25 U/ µ L). Then the mixture was resuspended in 1.45 mL of 50 mM Tris and 150 mM NaCl (pH 8.3). The addition of 0.5 mL of 0.3 mM Chromozym started the reaction. The increase of absorbance at 405 nm was obtained after 5 min. A mixture composed by 30 µL of AT III (4.04 µg/mL) and 15 µL of heparin (3.74 g/mL) was used as a positive control. 2.5. WGPH45A Characterization and Amino Acid Profile In the selected hydrolyzed WGPH45A, after 45 min of hydrolysis with Alcalase of WGPI, the protein concentrations were assessed by elemental microanalysis as % nitrogen content × 6.25 using a Leco CHNS932 analyzer (St. Joseph, MI, USA). Total dietary fiber was obtained by the gravimetric method [ 24 ]. Ash content was measured according to the direct ignition method (550 ◦ C for 36 h) and oil content was measured using the AOAC method 945.16 [ 25 ]. Polyphenols were determined using standard curves of chlorogenic acid, while soluble sugars were obtained using standard curves of glucose [ 26 , 27 ]. Amino acid quantification was determined according to the Alainz et al. method by High Performance Liquid Chromatography (HPLC) [ 28 ]. To finish, tryptophan quantification was measured according to the Yust et al. method [29]. Foods 2020,9, 854 4 of 16 2.6. Analysis of Molecular Profile by Fast Protein Liquid Chromatography (FPLC) FPLC Akta purifier 10 (GE Healthcare Bio-sciences AB, Uppsala, Switzerland) was used in order to determined the molecular profile of each sample using the Superose 12 HR 10/300 GL colum, which is a prepacked column for high-performance size exclusion chromatography. Hence, the fractionation range broad for molecules with molecular weights between 1 and 300 kDa, was used. The column was previusly calibrated with different well-known and defined proteins. From the logarithms of the molecular weights of these proteins and their elution volumes, the calibration line was made. The elution was carried out using a flow of 1 mL/min with 50 mL of sodium phosphate buffer (0.05 M), sodium chloride (0.5 M), and sodium azide (0.02% w/v) at pH 7.5. The injected volume of each sample was 500 µ L and its concentrations were of 1 mg/mL of protein. The elution of the proteins was reported at 280 nm of absorbance. 2.7. Isolation of Primary Human Monocytes This study was managed according to the Good Clinical Practice Guidelines and in line with the principles delimited in the Helsinki Declaration of the World Medical Association. Blood was obtained from healthy donors in Centro Regional de Transfusion Sanguinea de Sevilla-Huelva y Banco de Tejidos. Firstly, the peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation over a Ficoll-Histopaque (Sigma-Aldrich, Madrid, Spain) gradient from buffy coat. CD14 microbeads and LS columns on a midiMACS system (Miltenyi Biotec, Madrid, Spain) were used in order to isolate monocytes from PBMCs according to the specified protocol of the manufacturer. The purity for CD14 monocyte isolations was routinely >95% by flow cytometry (FACScanto II flow cytometer and FACSDiva software, versi ó n 5.0.1., Becton Dickinson, Erembodegem, Belgium) [ 30 ]. After that, monocytes were cultivated in a supplemented (L-glutamine, 1% penicilin/streptomicin, 10% foetal bovine serum) Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma-Aldrich, St. Louis, MO, USA). Finally, the in vitro treatments were performed over purified monocytes in a density of 5 × 10 5 in a 12-well plate. Monocytes were previously stimulated with or without 0.1 µ L/mL of LPS, and were exposed to WGPH45A for 24 h at 50 and 100 µg/mL. 2.8. Cell Viability Assay (MTT) Monocytes were seeded in 96-well plates at a density of 1 × 10 4 cells/well and incubated with WGPH45A at different concentrations up to 200 µ g/mL for 24 h. Next, the MTT solution (Sigma-Aldrich, St. Louis, MO, USA) was added to each well until a purple precipitate was visible. MTT-formazan crystals were finally solubilized with DMSO (Sigma-Aldrich, St. Louis, MO, USA). The outcomes were obtained in a microplate reader at 570 nm corrected to 650 nm [ 31 ]. Cell survival was calculated as the percentage of absorbance contrasted with that obtained in control, non-treated cells. 2.9. Reactive Oxygen Species (ROS) Generation The intracellular ROS was obtained using the CellROX Green Reagent (ThermoFisher Scientific, Madrid, Spain). After in vitro stimulation with LPS at 100 ng/mL, primary human monocytes were exposed to 50 µ g/mL and 100 µ g/mL of the WGPH45A for 24 h and then with CellROX Green Reagent (5 µ M) for 30 min. Cells were washed with PBS and fixed with 3.7% formaldehyde, and the fluorescence signal was analysed in a Fluoroskan Microplate Fluorometer (ThermoFisher Scientific) equipped with a 485/555 excitation/emission filter set. The auto-fluorescence of cells was measured under the same conditions but without adding CellROX Green Reagent. Data shown refers to the % of intracellular ROS production and to the comparison with a positive control (100% ROS production) after cell treatment in the presence of LPS. Foods 2020,9, 854 5 of 16 2.10. Nitic Oxide (NO) Generation Primary human monocytes (10 5 cells/well in 24 well plates), after being stimulated with LPS at 100 ng/mL, were incubated for 24 h in the existence of 50 µ g/mL and 100 µ g/mL of WGPH45A. The Griess reagent (Sigma-Aldrich) was used to calculate the production of nitrite, considered an NO generation indicator. We had previously transferred 100 µ L of the culture supernatant to a 96 well plate, and a volume of 100 µ L of Griess reagent was added. A BioTek plate reader measured absorbance at 540 nm wavelength, using a sodium nitrite standard curve to estimate concentration. 2.11. RNA Isolation and qRT-PCR Analysis RNA was totally extracted by using Trisure Reagent (Bioline GmbH, Luckenwakde, Germany), following the manufacturer’s instructions. The measure of A 260 /A 280 ratio in a NanoDrop ND-1000 Spectrophotometer (Thermo Scientific, Madrid, Spain) was used to determinate the RNA grade. Then, the reverse transcription (iScript, Bio-Rad, Madrid, Spain) was performed to obtain cDNA from RNA (1 µ g). The resulting cDNA (10 ng) was used as a template for real-time PCR amplifications. The mRNA levels for specific genes were determined in a CFX96 system (Bio-Rad). For each PCR reaction, a cDNA template was added to Brilliant SYBR green QPCR Supermix (Bio-Rad, Hercules, CA, USA) containing the primer pairs for either genes or for housekeeping genes such as glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Table S3). All amplification reactions were performed at least thrice and average threshold cycle (Ct) numbers of these triplicates were used to obtain the relative mRNA expression of candidate genes. The magnitude of change of mRNA expression for candidate genes was calculated by using the mathematical method of 2 −(∆∆Ct) . Briefly, all candidate genes were normalized to housekeeping genes (GAPDH) and expresed as the control’s percentage. 2.12. Cytokine Quantification The levels of IL-1 β , IL-6, IL-17, and IFN γ in culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA), following the protocol of the manufacturer (Thermo Fisher Scientific, Waltham, MA, USA). The cytokine concentrations were expressed in pg per mL for IL-1 β , IL-6, and IL-17 and in ng per mL for IFN γ , as calculated from the calibration curves from serial dilution of human recombinant standards in each assay. 2.13. Statistical Analysis All analysis was performed in triplicate. The outcomes obtained are mathematically represented as arithmetic means ± standard deviations (SD) and assessed with Graph Pad Prism Version 5.01 software (San Diego, CA, USA). The one-way analysis of variance (ANOVA) was used to evaluated the statistical significance of any difference in each parameter among the groups, also the Tukey multiple comparisons test was used as a post hoc test. Pvalues considered statistically significant were less than 0.05. 3. Results and Discussion The hydrolysis length may be assessed by the HD, which also can indicate longer or shorter lengths of the peptide chain. Previous studies showed <30% values of HD in others protein hydrolysates by the use of a single enzyme [ 7 ]. The HD outcomes of WGPC treated with Alcalase after 0, 15, 30, 45, and 60 min is shown in Figure 1. Analysis of results showed that Alcalase performance increased up to 28% the HD in 15 min (WGHP15A), rising up to 36% after 60 min (WGHP60A) of hydrolysis. A high reaction rate has also been showed for the hydrolysis of other vegetable proteins using Alcalase [ 32 , 33 ]. Foods 2020,9, 854 6 of 16 Foods 2020, 9, x FOR PEER REVIEW 6 of 16 Figure 1. Time-course of Hydrolysis Degree (HD) of WGPC (WGPH0A) during hydrolysis with enzyme Alcalase at 15 (WGPH15A), 30 (WGPH30A), 45 (WGPH45A), and 60 (WGPH60A) min. All values are expressed as the percentage of peptide bonds cleaved, and are mean ± standard deviation of triplicates. The resulting hydrolysates were evaluated by in vitro cell-free experiments leading to the identification of one bioactive WGPH. ACE activity, which plays an important role in regulating blood pressure in the renin-angiotensin system, atherosclerosis, and inflammation [34], was determined in the presence of WGPHs. The ACE-inhibitory activity of WGPHs are reported in Figure 2A. Whereas WGPC (WGPH0A) did not show ACE inhibition, all WGPHs reduced ACE activity by more than 60%. These results are better than those obtained by Motoi and Kodama [35] who hydrolyzed WG with six different proteases, not including Alcalase, and found ACE inhibition rates between 17% and 52%. In fact, Alcalase has been previously reported to be the most effective protease to yield ACE-inhibitory peptides [36,37]. In addition, thrombin, which exerts the formation of blood clots, pro-inflammatory effects by stimulating the production of pro-inflammatory cytokines, and the onset of atherosclerosis [38] was determined in order to evaluate the thrombin-inhibitory activity of WGPHs (Figure 2B). WGPC (WGPH0A) inhibited 18.5% of thrombin activity. This result was only improved with Alcalase hydrolysis after 45 min (WGPH45A), which showed 25% thrombininhibitory activity. Although this value is not very high, similar results with Alcalase were reported in lupine protein hydrolysates [33], and in blue mussel proteins [39]. Taking together HD, ACE, and thrombin-inhibitory activity, we selected WGPH45A to analyze chemical and amino acidic composition and to evaluate biological activity in primary human monocytes. Figure 2. Inhibition of the angiotensin-converting enzyme (ACE) (A) and thrombin (B) activity by WGPHs performed using Alcalase in cell-free in vitro experiments. Data are showed as means ± SD (n = 3) and those marked with different letters are significantly different (p < 0.05). Figure 1. Time-course of Hydrolysis Degree (HD) of WGPC (WGPH0A) during hydrolysis with enzyme Alcalase at 15 (WGPH15A), 30 (WGPH30A), 45 (WGPH45A), and 60 (WGPH60A) min. All values are expressed as the percentage of peptide bonds cleaved, and are mean ± standard deviation of triplicates. The resulting hydrolysates were evaluated by in vitro cell-free experiments leading to the identification of one bioactive WGPH. ACE activity, which plays an important role in regulating blood pressure in the renin-angiotensin system, atherosclerosis, and inflammation [ 34 ], was determined in the presence of WGPHs. The ACE-inhibitory activity of WGPHs are reported in Figure 2A. Whereas WGPC (WGPH0A) did not show ACE inhibition, all WGPHs reduced ACE activity by more than 60%. These results are better than those obtained by Motoi and Kodama [ 35 ] who hydrolyzed WG with six different proteases, not including Alcalase, and found ACE inhibition rates between 17% and 52%. In fact, Alcalase has been previously reported to be the most effective protease to yield ACE-inhibitory peptides [ 36 , 37 ]. In addition, thrombin, which exerts the formation of blood clots, pro-inflammatory effects by stimulating the production of pro-inflammatory cytokines, and the onset of atherosclerosis [ 38 ] was determined in order to evaluate the thrombin-inhibitory activity of WGPHs (Figure 2B). WGPC (WGPH0A) inhibited 18.5% of thrombin activity. This result was only improved with Alcalase hydrolysis after 45 min (WGPH45A), which showed 25% thrombin-inhibitory activity. Although this value is not very high, similar results with Alcalase were reported in lupine protein hydrolysates [ 33 ], and in blue mussel proteins [ 39 ]. Taking together HD, ACE, and thrombin-inhibitory activity, we selected WGPH45A to analyze chemical and amino acidic composition and to evaluate biological activity in primary human monocytes. Foods 2020, 9, x FOR PEER REVIEW 6 of 16 Figure 1. Time-course of Hydrolysis Degree (HD) of WGPC (WGPH0A) during hydrolysis with enzyme Alcalase at 15 (WGPH15A), 30 (WGPH30A), 45 (WGPH45A), and 60 (WGPH60A) min. All values are expressed as the percentage of peptide bonds cleaved, and are mean ± standard deviation of triplicates. The resulting hydrolysates were evaluated by in vitro cell-free experiments leading to the identification of one bioactive WGPH. ACE activity, which plays an important role in regulating blood pressure in the renin-angiotensin system, atherosclerosis, and inflammation [34], was determined in the presence of WGPHs. The ACE-inhibitory activity of WGPHs are reported in Figure 2A. Whereas WGPC (WGPH0A) did not show ACE inhibition, all WGPHs reduced ACE activity by more than 60%. These results are better than those obtained by Motoi and Kodama [35] who hydrolyzed WG with six different proteases, not including Alcalase, and found ACE inhibition rates between 17% and 52%. In fact, Alcalase has been previously reported to be the most effective protease to yield ACE-inhibitory peptides [36,37]. In addition, thrombin, which exerts the formation of blood clots, pro-inflammatory effects by stimulating the production of pro-inflammatory cytokines, and the onset of atherosclerosis [38] was determined in order to evaluate the thrombin-inhibitory activity of WGPHs (Figure 2B). WGPC (WGPH0A) inhibited 18.5% of thrombin activity. This result was only improved with Alcalase hydrolysis after 45 min (WGPH45A), which showed 25% thrombininhibitory activity. Although this value is not very high, similar results with Alcalase were reported in lupine protein hydrolysates [33], and in blue mussel proteins [39]. Taking together HD, ACE, and thrombin-inhibitory activity, we selected WGPH45A to analyze chemical and amino acidic composition and to evaluate biological activity in primary human monocytes. Figure 2. Inhibition of the angiotensin-converting enzyme (ACE) (A) and thrombin (B) activity by WGPHs performed using Alcalase in cell-free in vitro experiments. Data are showed as means ± SD (n = 3) and those marked with different letters are significantly different (p < 0.05). Figure 2. Inhibition of the angiotensin-converting enzyme (ACE) ( A ) and thrombin ( B ) activity by WGPHs performed using Alcalase in cell-free in vitro experiments. Data are showed as means ± SD (n=3) and those marked with different letters are significantly different (p<0.05). The chemical composition of WGPH45A is reported in Table 1. The main difference it has with WGPC is the increase in ash content, which is due to the addition of NaOH to maintain a Foods 2020,9, 854 7 of 16 constant pH during the hydrolytic process, a usual result after the hydrolysis of several protein sources [ 40 ]. The amino acid content in WGPH45A is reported in Table 2. WGPH45A showed 38% glutamic acid/glutamine, 7% leucine, and 7% proline. WGPH45A complies with Food and Agriculture Organization/World Health Organization (FAO/WHO) recommendations for branched amino acids as leucine, isoleucine, and valine, and sulfur amino acids such as methionine and cysteine. In addition, histidine content exceeds what is recommended (>1.9). Several studies have shown that a diet rich in histidine can help suppress the pro-inflammatory response. For instance, histidine-rich supplementary meals analyzed in vivo have shown a sepsis status improvement by the inhibition of systemic inflammation [ 41 ]. Besides this, histidine-dipeptides and small peptides rich in histidine may have the ability to reduce oxidative stress in chronic disease [ 42 ]. Additionally, plasma protein that is rich in histidine was negatively associated with oxidative stress and inflammation in a clinical trial with obese women [ 43 ] and was also associated with a general inflammatory status, which relates to the pathophysiological role of this amino acid [44]. Table 1. Chemical composition of WGPH45A. Values, showed as a percentage in dry basis, are mean ±standard deviation of triplicates. (%) WGPH45A Protein 81.36 ±0.44 Ash 6.19 ±0.15 Fibre 1.51 ±0.05 Oil 0.92 ±0.04 Soluble sugars 0.01 ±0.00 Polyphenols 0.06 ±0.01 Other compounds 9.95 ±0.63 Table 2. Amino acid composition of WGPH45A. Values, showed as a percentage of total amino acid content, are mean ±standard deviation of triplicates. Amino Acid WGPH45A FAO/WHO Aspartic acid +asparragine 4.03 ±0.09 Glutamic acid +Glutamine 38.27 ±0.06 Serine 5.73 ±0.03 Histidine 1.98 ±0.01 1.9 Glycine 3.70 ±0.11 Threonine 3.05 ±0.02 3.4 Arginine 3.69 ±0.03 Alanine 3.03 ±0.02 Proline 7.12 ±0.63 Tyrosine 3.49 ±0.14 Valine 4.03 ±0.04 3.5 Methionine 0.94 ±0.14 2.5 a Cysteine 1.60 ±0.24 Isoleucine 3.67 ±0.00 2.8 Tryptophan 0.87 ±0.00 Leucine 7.40 ±0.01 6.6 Phenylalanine 5.20 ±0.02 6.3 b Lysine 2.20 ±0.15 5.8 aMetionine +Cysteine; bPhenilalanine +Tyrosine. As depicted in Figure 3, the analysis of the protein molecular profile by HPLC determined that WGPC is constituted mainly by peptides of three sizes in the range of 1–300 kDa (30.08, 5.36, and 2.19 kDa). The WGPH45A protein molecular profile showed that all peptides are in a value next to 1.90 kDa. Results indicated that enzymatic hydrolysis by Alcalase clearly reduced the high molecular weight of WGPC, and that WGPH45A obtains low molecular weight peptides Foods 2020,9, 854 8 of 16 (<2 kDa). Reportedly, low molecular weight peptides (2–20 amino acids) are more biologically active compared to their parent polypeptide/proteins, which are larger [45]. Foods 2020, 9, x FOR PEER REVIEW 8 of 16 As depicted in Figure 3, the analysis of the protein molecular profile by HPLC determined that WGPC is constituted mainly by peptides of three sizes in the range of 1–300 kDa (30.08, 5.36, and 2.19 kDa). The WGPH45A protein molecular profile showed that all peptides are in a value next to 1.90 kDa. Results indicated that enzymatic hydrolysis by Alcalase clearly reduced the high molecular weight of WGPC, and that WGPH45A obtains low molecular weight peptides (<2 kDa). Reportedly, low molecular weight peptides (2–20 amino acids) are more biologically active compared to their parent polypeptide/proteins, which are larger [45]. Figure 3. Molecular weight profiles by size-exclusion Fast Protein Liquid Chromatography (FPLC) of WGPC and WGPH45. The inflammatory stage has been associated with several chronic diseases related to cardiovascular and atherogenic risk and the activation of circulating blood cells, as primary human monocytes, is an accepted method to validate the in vitro inflammation assays [19]. Oxidative stress and ROS production are directly implicated at the beginning of inflammatory progression. Together, the oxidative stress and specific pro-inflammatory molecule release allows the triggering of events involved in the atherosclerosis origin, such as circulating lipoprotein oxidation from the bloodstream [20]. In previous studies, plant protein hydrolysates have exhibited anti-inflammatory activities in the THP-1 cell line [46] and primary human monocytes [30]. Herein, to our knowledge, we now report for the first time that a new WGPH has the ability to prevent the pro-inflammatory activation of primary human monocytes. Cells were incubated with WGPH45A at concentrations up to 200 μg/mL for 24 h. The cell viability evaluated by the MTT method was not affected by any WGPH45A concentration (data not shown). Quantification of both ROS (Figure 4A) and nitrite (Figure 4B) concentrations in primary human monocytes let us analyze the preventive role of WGPH45A on oxidative conditions. LPS remarkably increased both intracellular ROS and nitrite, compared to nonstimulated cells. The ROS and nitrite production induced in the presence of WGPH45A was significantly lower than LPS control. In addition, LPS down- and up-regulated Heme oxygenase (HO)-1 (Figure 4C) and inducible Nitric oxide synthase (iNOS) (Figure 4D) mRNA expression, respectively. Interestingly, HO-1 levels are lowly expressed in macrophages that are enriched in progressing plaques [47]. Recently, rice bran protein hydrolysates have been shown to restore HO-1 expression [48]. We have no evidence of previous studies regarding the effect of WGPHs on HO-1 and iNOS expression; however, gluten-related disorders are mediated by an up-regulation of iNOS [49]. WGPH45A could be an appropriate food ingredient in celiac patients. Figure 3. Molecular weight profiles by size-exclusion Fast Protein Liquid Chromatography (FPLC) of WGPC and WGPH45. The inflammatory stage has been associated with several chronic diseases related to cardiovascular and atherogenic risk and the activation of circulating blood cells, as primary human monocytes, is an accepted method to validate the in vitro inflammation assays [ 19 ]. Oxidative stress and ROS production are directly implicated at the beginning of inflammatory progression. Together, the oxidative stress and specific pro-inflammatory molecule release allows the triggering of events involved in the atherosclerosis origin, such as circulating lipoprotein oxidation from the bloodstream [ 20 ]. In previous studies, plant protein hydrolysates have exhibited anti-inflammatory activities in the THP-1 cell line [ 46 ] and primary human monocytes [ 30 ]. Herein, to our knowledge, we now report for the first time that a new WGPH has the ability to prevent the pro-inflammatory activation of primary human monocytes. Cells were incubated with WGPH45A at concentrations up to 200 µ g/mL for 24 h. The cell viability evaluated by the MTT method was not affected by any WGPH45A concentration (data not shown). Quantification of both ROS (Figure 4A) and nitrite (Figure 4B) concentrations in primary human monocytes let us analyze the preventive role of WGPH45A on oxidative conditions. LPS remarkably increased both intracellular ROS and nitrite, compared to non-stimulated cells. The ROS and nitrite production induced in the presence of WGPH45A was significantly lower than LPS control. In addition, LPS down- and up-regulated Heme oxygenase (HO)-1 (Figure 4C) and inducible Nitric oxide synthase (iNOS) (Figure 4D) mRNA expression, respectively. Interestingly, HO-1 levels are lowly expressed in macrophages that are enriched in progressing plaques [ 47 ]. Recently, rice bran protein hydrolysates have been shown to restore HO-1 expression [ 48 ]. We have no evidence of previous studies regarding the effect of WGPHs on HO-1 and iNOS expression; however, gluten-related disorders are mediated by an up-regulation of iNOS [ 49 ]. WGPH45A could be an appropriate food ingredient in celiac patients. Foods 2020,9, 854 9 of 16 Foods 2020, 9, x FOR PEER REVIEW 9 of 16 Figure 4. Intracellular ROS (A) and nitrite (B) production, expressed as a percentage of fluorescence/absorbance, and HO-1 (C) and iNOS (D) mRNA levels after 24 h incubation with or without LPS (100 ng/mL) and WGPH45A at 50 and 100 μg/m. Values are presented as means ± SD (n = 3) and those marked with different letters are significantly different (p < 0.05). In gaining a deeper insight into the role of WGHPs on human monocyte inflammation, we observed that LPS induced IL-1β, IL-6, IL-17, and IFNγ gene expression (Figure 5) and release of these pro-inflammatory cytokines (Figure 6) were diminished by WGPH45A in LPS-treated primary human monocytes. The cytokine environment of these monocytes could indicates that M1 phenotypes related to pro-inflammatory progression had been lowered, while M2 phenotype, which is associated with resolution and tissue repairs, had been enhanced. In the literature, it possible to find a large number of examples where plant-derived protein and peptides have been used as antiinflammatory or antioxidant compounds. In 2017, Kan et al. demonstrated that a combination of wheat peptides and fucoidan attenuates oxidation and inflammation of ethanol-induced gastric mucosal damage in rats [50]. Other protein hydrolysates isolated from nutritional seeds have also showed anti-inflammatory properties. For instance, the case of hemp seed (Cannabis sativa L.) protein hydrolysates, which inhibited LPS-induced inflammation in the BV2 cell line [6], such as in the case of amaranth (Amaranth hypochondriacus) hydrolysates, which also inhibited LPS-induced inflammation in human and mouse myeloid cells by preventing activation of NF-κB signaling [51]. Our study can improve the efficacy of existing WGPHs that may delay or prevent immune- and inflammatory-related diseases. Figure 4. Intracellular ROS ( A ) and nitrite ( B ) production, expressed as a percentage of fluorescence/ absorbance, and HO-1 ( C ) and iNOS ( D ) mRNA levels after 24 h incubation with or without LPS (100 ng/mL) and WGPH45A at 50 and 100 µ g/m. Values are presented as means ± SD (n=3) and those marked with different letters are significantly different (p<0.05). In gaining a deeper insight into the role of WGHPs on human monocyte inflammation, we observed that LPS induced IL-1 β ,IL-6,IL-17, and IFN γ gene expression (Figure 5) and release of these pro-inflammatory cytokines (Figure 6) were diminished by WGPH45A in LPS-treated primary human monocytes. The cytokine environment of these monocytes could indicates that M1 phenotypes related to pro-inflammatory progression had been lowered, while M2 phenotype, which is associated with resolution and tissue repairs, had been enhanced. In the literature, it possible to find a large number of examples where plant-derived protein and peptides have been used as anti-inflammatory or antioxidant compounds. In 2017, Kan et al. demonstrated that a combination of wheat peptides and fucoidan attenuates oxidation and inflammation of ethanol-induced gastric mucosal damage in rats [ 50 ]. Other protein hydrolysates isolated from nutritional seeds have also showed anti-inflammatory properties. For instance, the case of hemp seed (Cannabis sativa L.) protein hydrolysates, which inhibited LPS-induced inflammation in the BV2 cell line [ 6 ], such as in the case of amaranth (Amaranth hypochondriacus) hydrolysates, which also inhibited LPS-induced inflammation in human and mouse myeloid cells by preventing activation of NF- κ B signaling [ 51 ]. Our study can improve the efficacy of existing WGPHs that may delay or prevent immune- and inflammatory-related diseases. Finally, the scientific literature has demonstrated that the inflammatory component of atherosclerosis is represented by various cells and mediators in the immune system [ 52 ]. The progression of the plaque formation was thrived by several inflammatory mediators, which change over the cell environment in the release of the above-mentioned cytokines (mainly IL-1 β and IL-6) and induce CCR production that has been directly involved in plaque development. The transfer of monocytes into a plaque requires three sequential steps: the capture, rolling, and transmigration of monocytes. CCL5 have an important role in the capture and rolling steps, which are attached to the luminal surface of the endothelium mediated by adhesion molecules (glycosaminoglycans, P-selectin [ 47 ]). CCR2 are involved in the transmigration activity between vascular endothelial cells and intima layers. Additionally, CCR7 promote migratory activity in LPS stimulated cells [ 53 ]. Apart from CCRs chemokynes, M1 phenotype macrophages have the ability to recognize and process modified LDLs by scavenger receptors such as CD36 which bind to ox-LDL and differentiate to foam cells [ 47 ]. Hence, for the first time, a WGPH has showed atheroprotective properties in primary human monocytes. To observe atheroprotective properties of WGPH45A after LPS-stimulated monocytes, we extracted mRNA and performed RT-qPCR of atherosclerosis-related genes (Figure 7). LPS increased mRNA Foods 2020,9, 854 16 of 16 56. Malaguti, M.; Dinelli, G.; Leoncini, E.; Bregola, V.; Bosi, S.; Cicero, A.F.; Hrelia, S. Bioactive peptides in cereals and legumes: Agronomical, biochemical and clinical aspects. Int. J. Mol. Sci. 2014 ,15, 21120–21135. [CrossRef] 57. Nongonierma, A.B.; FitzGerald, R.J. The scientific evidence for the role of milk protein-derived bioactive peptides in humans: A review. J. Func. Foods. 2015,17, 640–656. [CrossRef] 58. Ejima, A.; Nakamura, M.; Suzuki, Y.A.; Sato, K. Identification of food-derived peptides in human blood after ingestion of corn and wheat gluten hydrolysates. JFB 2018,2, 104–111. [CrossRef] © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).