A lupin protein hydrolysate protects the central nervous system from oxidative stress in WD-fed ApoE

RESEARCH ARTICLE www.mnf-journal.com A lupin protein hydrolysate protects the central nervous system from oxidative stress in WD-fed ApoE−/−mice Guillermo Santos-Sánchez, Eduardo Ponce-España, Ana Isabel Álvarez-López, Justo Pedroche, María del Carmen Millán-Linares, María-Soledad Fernández-Pachón, Patricia Judith Lardone, Ivan Cruz-Chamorro,* and Antonio Carrillo-Vico* Oxidative stress plays a crucial role in neurodegenerative diseases like Parkinson’s and Alzheimer’s. Studies indicate the relationship between oxidative stress and the brain damage caused by a high-fat diet. It is previously found that a lupin protein hydrolysate (LPH) has antioxidant effects on human leukocytes, as well as on the plasma and liver of Western diet (WD)-fed ApoE−/−mice. Additionally, LPH shows anxiolytic effects in these mice. Given the connection between oxidative stress and anxiety, this study aimed to investigate the antioxidant effects of LPH on the brain of WD-fed ApoE−/−mice. LPH (100 mg kg−1) or a vehicle is administered daily for 12 weeks. Peptide analysis of LPH identified 101 amino acid sequences (36.33%) with antioxidant motifs. Treatment with LPH palliated the decrease in total antioxidant activity caused by WD ingestion and regulated the nitric oxide synthesis pathway in the brain of the animals. Furthermore, LPH increased cerebral glutathione levels and the activity of catalase and glutathione reductase antioxidant enzymes and reduced the 8-hydroxy-2’-deoxyguanosine levels, a DNA damage marker. These findings, for the first time, highlight the antioxidant activity of LPH in the brain. This hydrolysate could potentially be used in future nutraceutical therapies for neurodegenerative diseases. 1. Introduction Bioactive peptides are defined as peptide sequences of between 2 and 20 amino acids in length, that are present within the food protein and exert physiological health benefits, beyond its known nutritional value.[1]They are released during enzymatic G. Santos-Sánchez, E. Ponce-España, A. I. Álvarez-López, P. J. Lardone, I. Cruz-Chamorro, A. Carrillo-Vico Instituto de Biomedicina de Sevilla IBiS/Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla Seville 41013, Spain E-mail: [email protected];[email protected] The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/mnfr.202300503 © 2024 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH GmbH. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. DOI: 10.1002/mnfr.202300503 proteolysis and food processing.[2]Nowadays, there are numerous peptides derived from vegetable and animal food products that have demonstrated several health effects (antidiabetic, antihypertensive, cholesterol-lowering, antiinflammatory, etc.).[2,3] Oxidative stress is an imbalance between the production of reactive oxygen species (ROS) and antioxidant defenses. A large body of evidence shows that oxidative stress is responsible for the onset and progression of several diseases,[4]and has been linked to several neurodegenerative conditions such as Alzheimer’s disease, mild cognitive impairment in Parkinson’s disease, amyotrophic lateral sclerosis, and Huntington’s disease.[5]For this reason, the potential beneficial role of antioxidants from a food source in reducing oxidative stress at the systemic level, and especially in the central nervous system, is a field of great interest. In this line, food-derived peptides such as those of walnut have been shown to increase superoxide dismutase (SOD) and catalase (CAT) activities and reduce the malonaldehyde levels (marker of lipid peroxidation) in the brain of mice treated with lipopolysaccharide (LPS).[6]Phytochemicals from medicinal plants have long been known to often offer a safer alternative to synthetic G. Santos-Sánchez, E. Ponce-España, A. I. Álvarez-López, M. del C. Millán-Linares, P. J. Lardone, I. Cruz-Chamorro, A. Carrillo-Vico Departamento de Bioquímica Médica y Biología Molecular e Inmunología Facultad de Medicina Universidad de Sevilla Seville 41009, Spain J. Pedroche, M. del C. Millán-Linares Department of Food & Health Instituto de la grasa CSIC, Ctra, Utrera Km 1, Seville 41013, Spain M.-S. Fernández-Pachón Área de Nutrición y Bromatología Departamento de Biología Molecular e Ingeniería Bioquímica Universidad Pablo de Olavide Ctra. Utrera Km 1, Sevilla 41013, Spain Mol. Nutr. Food Res. 2024,68, 2300503 2300503 (1 of 11) © 2024 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH GmbH www.advancedsciencenews.com www.mnf-journal.com medications. They possess neuroprotective activity targeting various elements in pathogenic pathways due to their antioxidant, anti-inflammatory, and other properties.[7] Lupin is a non-starchy grain legume characterized by a high protein content (31–52%) compared to other legumes such as chickpea (19%) or lentils (26%). Thus, in recent decades lupin seeds have gained attention as an important source of bioactive peptides. In this regard, we have recently demonstrated that a Lupinus angustifolius protein hydrolysate (LPH) improves antioxidant and anti-inflammatory status both in vitro[8]and in vivo,[9] as well as reduces abdominal adiposity and improves metabolicassociated fatty liver disease in mice.[10]Moreover, we have shown that the ingestion of a beverage based on LPH for 28 days improves the antioxidant status and reduces the cardiovascular risk ratio in humans.[11]Furthermore, LPH shows anxiolytic-like effects in Western Diet (WD)-fed ApoE−/−mice.[12]In this line, oxidative stress has been described as a key risk factor for the anxiety process and memory impairment.[13]LPHs biological effects have been recently revised in Cruz-Chamorro et al.[14] Although we have evidence that LPH improves plasmatic and hepatic antioxidant capacity, there is no evidence of the effects of LPH on the oxidative status of the central nervous system (CNS) that could justify the anxiolytic effects of LPH. Therefore, the aim of this multidisciplinary study was to evaluate whether LPH can exert antioxidant effects on the CNS. First, a peptidomic characterisation of the LPH was performed, and the antioxidant motifs, present in the peptide sequences, were identified. Subsequently, in vitro tests were carried out to evaluate the antioxidant capacity of LPH. Finally, the effect of LPH on the CNS oxidative status was evaluated in ApoE−/−mice fed a WD. ApoE is a key protein related to the lipid metabolism whose deficiency has been shown to increase oxidative stress in the brain tissue,[15]and, furthermore, WD consumption increases oxidative stress in this mouse model.[16] 2. Experimental Section 2.1. LPH Preparation and Characterization LPH was prepared at the “Instituto de la Grasa” (CSIC, Seville, Spain) by hydrolysis of a lupin (Lupinus angustifolius) seedderived protein isolate. Hydrolysis was carried out with the foodgrade Alcalase 2.4L (Novozymes, Bagsvaerd, Denmark) enzyme (2.4 AU g−1) for 15 min, pH 8, 50°C. LPH was lyophilized, dissolved in a saline solution, filtered, autoclaved, and stored at −80°C until use, as previously described.[8] The molecular weight profile of LPH was determined by molecular exclusion chromatography, as previously described.[8] Molecular weight estimation was performed by gel filtration chromatography in an Akta purifier system (GE Healthcare) equipped with a Superose 12 HR 10/30 column with an optimal separation range of 300–1000 Da. The elution was developed in 50 mM sodium phosphate 0.5 M NaCl, 0.02% (w/v) sodium azide buffer, and monitored at 280 nm. The analytical flow was 1.0 mL min−1, and the protein concentrations of the samples were 1 mg protein mL−1, with an injection volume of 0.5 mL in two elution column volume (50 mL). From the total sequences identified in LPH, sequences associated with the L. angustifolius species were selected by using the L angustifolius database (31386 sequences) from UnitProt. Identification of raw data spectra was performed using Proteome Discoverer v1.3 (Thermo) with the Mascot search engine v2.3.02. Furthermore, the BIOPEP-UWM database[17]was used to identify LPH peptide sequences with previously demonstrated bioactive motifs.[18]The hydrophobicity of these sequences was calculated using ToxinPred software.[19] Protein concentrations were determined by elemental microanalysis as a percentage of nitrogen content ×6.25 using a Leco CHNS-932 analyzer (St. Joseph, MI, USA). The ash content was determined using the direct ignition method (550°C for 25 h). Total dietary fiber was determined according to Lee.[20]The oil content was measured using the AOAC method 945.16.[21]Soluble sugars and polyphenols were measured using standard glucose curves and chlorogenic acid, respectively.[22,23] 2.2. In vivo Experimental Approach Twelve ApoE−/−mice (4-week-old male) were housed in the animal facility of the “Instituto de Biomedicina de Sevilla” (IBiS) under standard conditions (12/12 light/dark cycles, temperature 22 ±2°C, and humidity <55%) with free access to water and diet. Of these, four mice were fed a standard diet (SD) and eight mice with WD (Test Diet 58v8, 45% energy from fat), provided by the Special Diet Production Section of the University of Granada (Granada, Spain). The composition of diets is reported in Table S1 (Supporting Information). After 2 weeks (6-week-old mice) the WD-fed mice were divided into two groups: 1) animals intragastrically treated with LPH (100 mg kg−1)(WD+LPH group, n=4) and 2) mice intragastrically treated with saline (WD group, n=4). SD-fed mice were also intragastrically treated with saline (SD group, n=4). Treatment for each group was 12 weeks (up to mice were 18-weeks-old) and 5 days per week. Individual body weight was recorded weekly. At the end of the experiment, 12 h fasted mice were sacrificed, each brain was collected, and the hypothalamus was separated, which was rapidly frozen, and stored at −80°C until use. The whole frozen brain (without hypothalamus) was homogenized in 4 mL of PBS containing a protease inhibitor cocktail (Sigma Aldrich, Darmstadt, Germany) using the Tissue Ruptor II (Qiagen, Hilden, Germany). The samples were then centrifuged at 12 000 ×gfor 15 min at 4°C and the supernatant was collected andstoredat−20°C until use. The experimental protocol was approved by the Ethics Committee (reference 21/06/2016/105). 2.3. Antioxidant Activity The antioxidant activity was measured in raw LPH or in the brain using several assays. Whole brain homogenate was used for the FRAP, GSH, and NO assays, and the results were referred to mg of protein. The protein content of each brain was quantified using the Bradford method. Briefly, 5 μL of homogenate brain sample was mixed with Bradford reagent in a final volume of 250 μL. The absorbance at 595 nm was measured using a CLARIOstar Plus microplate reader (BMG Labtech, Ortenberg, Germany). Protein content was extrapolated using a bovine serum albumin (BSA, 0–2 mg mL−1) curve. The hypothalamus was employed for the gene expression analysis. Mol. Nutr. Food Res. 2024,68, 2300503 2300503 (2 of 11) © 2024 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH GmbH 16134133, 2024, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/mnfr.202300503 by Universidad De Sevilla, Wiley Online Library on [21/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.mnf-journal.com 2.3.1. Ferric Reducing Antioxidant Power Assay (FRAP) To quantify the total antioxidant capacity, the ferric reducing antioxidant power (FRAP) assay was performed. 20 μLofLPHat the final concentration of 0.25, 0.5, and 1 mg mL−1or 20 μLbrain homogenate were mixed with 280 μL FRAP reagent. The FRAP reagent was prepared by mixing 1.3 mL of a 10 mM TPTZ solution (Sigma-Aldrich) in 40 mM HCl, 1.3 mL of 20 mM FeCl3 ×6H2O and 13 mL of 0.3 M acetate buffer (pH 3.6). After 30 minofincubationat37°C, the absorbance was recorded at 595 nm with a Synergy HT-multimode microplate reader (BioTek instruments, Winooski, VT, USA). The results were obtained by data extrapolation with a standard curve using 6-hydroxy-2,5,7,8- tetramethylchroman-2-carboxylic acid (Trolox, Sigma). 2.3.2. Total Glutathione Total glutathione (GSH) levels were quantified using the Glutathione Assay Kit (Cayman Chemical, MI, USA), according to the manufacturer’s instructions. Briefly, 50 μL of samples were mixed with 150 μL of the Assay Cocktail (MES buffer, cofactor mixture, enzyme mixture, water, and DTNB) and incubated in the dark on an orbital shaker for 30 min. The absorbance at 405 nm was recorded using the Synergy HT-multimode microplate reader (Biotek Instruments). 2.3.3. Nitric Oxide Quantification Nitric oxide (NO) was quantified in the brain homogenate using the Griess test (Sigma-Aldrich, St. Louis, MO, USA). 50 μL of the Griess reagent was incubated with 50 μL of the sample for 15 min at room temperature in the dark. The absorbance at 540 nm was measured using the Synergy HT-multimode microplate reader (Biotek Instruments). 2.3.4. Relative Expression of Nitric Oxide Synthase mRNA The gene expression of neuronal and inducible NO synthase (nNOS and iNOS, respectively) was evaluated by RT-quantitative PCR (RT-qPCR). Total mRNA was obtained from the frozen hypothalamus using TRIsure (Meridian Bioscience, Cincinnati, OH, USA) standard protocol according to the manufacturer’s instructions. cDNA from 3 μg of RNA was synthesized using the Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostic). qPCR was performed using the LightCycler 480 SYBR Green I Master kit and the Lightcycler 480 thermocycler (Roche), at 55°C (iNOS) or 58°C (nNOS) for 40 cycles (80 ng cDNA per well). The hypoxanthine phosphoribosyl transferase (hprt) gene was used as a housekeeping gene and the relative expression levels of mRNA were calculated using the 2−ΔΔCt method. The primer sequences were: hprt (NM_013556): Forward (Fwd) 5ʹ-TGTTGGATATGCCCTTGACTA-3ʹ, Reverse (Rev) 5ʹ-TGCGCTCATCTTAGGCT-3ʹ;nnos (NM_008712): Fwd 5ʹ-TATTCCATCAGCTCCTCTCCA-3ʹ,Rev5ʹ-GGATGTCAAA TTGTCGCTGTT-3ʹ;inos (NM_001313921): Fwd 5ʹ-ACGGA CGAGACGGATAG-3ʹ,Rev5ʹ-GGGCTTCAAGATAGGGA-3ʹ. 2.3.5. Antioxidant Activities Assays Enzymatic activity of superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione reductase (GR, Randox Laboratories Ltd, Crumlin, UK) and catalase (CAT) (Cayman Chemical, MI, USA) were performed in the brain homogenate according to the manufacturer’s instructions. 2.3.6. 8-Hydroxy-2ʹ-Deoxyguanosine (8-OHdG) Assay The 8-hydroxy-2ʹ-deoxyguanosine (8-OHdG) levels were quantified by the 8-OHdG Check (high sensitive) ELISA (IBL International GmbH, Hamburg, Germany) following the kit instructions. 2.4. Statistical Analysis Data are represented as mean ±standard error of the mean (SEM). Statistical analysis was carried out using one-way analysis of variance (ANOVA) followed by Dunn’s post hoc test. A pvalue ≤0.05 was considered statistically significant. In addition, the size effect was analyzed using Cohen’s test, and a d-value ≥ 0.80 was considered as “large effect size”. Moreover, a nonparametric Spearman’s correlation was applied. Data were analyzed with GraphPad Prism v.8 (GraphPad Software, San Diego, CA, USA) and Jeffreys’s Amazing Statistics Program (JASP v. 0.16.3, Amsterdam, Netherlands). 3. Results 3.1. LPH Characterization LPH was contained by 80.37 ±0.09% of proteins with molecular weights less than 1 kDa, as can be observed in the molecular weight profile (Figure 1A). Furthermore, LPH contained a low content of fiber (2.88 ± 0.08%), lipids (0.58 ±0.00%), soluble sugar (0.02 ±0.00%), and polyphenols (0.06 ±0.00%). Furthermore, 8.62 ±0.09% of the LPH was ash and 7.97 ±0.19% was moisture, as reported in Table 1. The LPH peptide analysis allowed us to identify 278 sequences belonging to the taxonomy L. angustifolius of which 101 sequences (36.33%) showed at least a motif related to antioxidant effects (Table 2). The hydrophobicity of the antioxidant peptides ranged from −0.73 to 0.34. In particular, 15.84% of the sequences had a hydrophobicity of <−0.40, 26.73% were between −0.20 and −0.40, 37.62% had −0.01 to −0.19 and 19.80% had a hydrophobicity ranging −0.34 to 0.00. Furthermore, these 101 LPH sequences contained >6 (1.98%), 5–6 (8.91%), 3–4 (33.6%), and 1–2 (45.54%) aromatic rings. Only 9.90% of the sequences had no aromatic rings (Figure 1B). In this way, 234 out of 1425 (16.42%) amino acids that were part of the antioxidant sequences had at least an aromatic ring. Finally, 39.16% of the amino acids containing in the antioxidant sequences were nonpolar amino acids (39.16%), although the most representative amino acid was glutamic acid (E, 14.11%) (Figure 1C). Regarding the 41 antioxidant motifs identified in 101 sequences mentioned above, these contained 20.0% Leu, 19.0% His, 8.6% Pro, 7.6% Tyr, 7.6% Trp, and Mol. Nutr. Food Res. 2024,68, 2300503 2300503 (3 of 11) © 2024 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH GmbH 16134133, 2024, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/mnfr.202300503 by Universidad De Sevilla, Wiley Online Library on [21/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.mnf-journal.com Figure 1. Molecular weight distribution of the entire Lupinus angustifolius protein hydrolysate (LPH) (A), percentage of hydrophobicity and number of rings (B), and amino acid composition (C) of the sequences with demonstrated antioxidant motifs. 4.8% Val. In addition, IR (ID 8215), LK (ID 8217), EL (ID 7888), and LLPH (ID 3314) were the motifs more represented taking part of 28, 14, 12, and 9 sequences, respectively (Table 2). Table 1. LPH composition. Components LPH Proteins 80.37% Fiber 2.88% Lipids 0.58% Soluble sugar 0.02% Polyphenols 0.06% Ash 8.62% Moisture 7.97% Data are expressed as percentages on a dry basis. LPH, lupin protein hydrolysate. 3.2. In vitro Antioxidant Activity of LPH To evaluate the antioxidant capacity exerted by LPH, an in vitro FRAP assay test was performed. LPH significantly increased FRAP levels in a dose-dependent manner. Specifically, the 0.25 mg mL−1LPH concentration reached a Trolox equivalent antioxidant capacity (TEAC) equal to 9.54 ±0.76 μM(p<0.0001), while 0.5, and 1.0 mg mL−1reached 14.41 ±6.03 μM(p<0.0001) and 30.40 ±1.51 μM(p<0.0001) TEAC, respectively (Figure 2). 3.3. LPH Does Not Modify the Body Weight of the Mice As shown in Figure 3A, no differences (p>0.05) were observed in the body weight of the animals in the three experimental groups weekly and throughout the experiment. However, a significant increase in the final body weight and in the body weight gain Mol. Nutr. Food Res. 2024,68, 2300503 2300503 (4 of 11) © 2024 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH GmbH 16134133, 2024, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/mnfr.202300503 by Universidad De Sevilla, Wiley Online Library on [21/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.mnf-journal.com Table 2. Bioactive motifs with antioxidative demonstrated activity. ID1Bioactive motifsa) No. sequences Secuencesb) 3300 PHH 1 RHRPHHHEEEEEEEEWSHQ 3301 HLH 1 HLHTDEEIRFA 3305 LH 5 GLHLPSYTNYPQL GLHLPSYTNYPQIIM GLHLPSYTNYPQLIF HLHTDEEIRFA GLHLPSYTNYPQ 3314 LLPH 9 INEGGLLLPH EINEGALLLPHYNSK FTEINEGALLLPHYNSK LIFTEISEGALLLPHYNSK TEINEGALLLPHYNSK TEISEGALLLPHYNSK FIEINEGGLLLPHYNSK FIEINEGALLLPHYNSK LTFTEINEGALLLPHYNSK 3317 HL 7 GLHLPSYTNYPQL HLPSYTNYPQIIM GLHLPSYTNYPQIIM GLHLPSYTNYPQLIF HLHTDEEIRFA GLHLPSYTNYPQ HLPSYTNYPQIIM 3319 HH 6 KGILDKIKDKLPGHHN RHRPHHHEEEEEEEEWSHQ HHEEEEEEEEWSH HHEEEEEEEEWSHQ HHEEEEEEEEWS HHEEEEEEEEW 7862 IKK 1 TVGNVGEKIKKPFESITK 7866 AY 4 KLRHNIGQSTSPDAYNPQAGRL KLRHNIGQSTSPDAYNPQAGRLK TSPDAYNPQAGRL AYNDEDLIRIL 7872 LY 2 RADLYNPNAGRIS IADPTRADLYNPTAGRIS 7886 AH 4 AHIPGVVEL SIRALPLDVVAH ALPLDVVAH WLPPWLDAH 7888 EL 12 AHIPGVVEL RIGPVELPYTL EETVRSIELPGLL GRQQEQQLEGELE KAKNELRDSFKLL IERVLLGDDELQ RVLLGDDELQEN SELSGKRPLFGPDLPQTE VVDEGEGNYELVGIR (Continued) Table 2. (Continued) ID1Bioactive motifsa) No. sequences Secuencesb) IERVLLGDDELQE IERVLLGDDELQEN SVLSPEELLAVR 7898 WY 1 NHPEVVAEEPWYGIE 7918 GHH 1 KGILDKIKDKLPGHHN 7980 HHH 1 RHRPHHHEEEEEEEEWSHQ 7995 LHL 4 GLHLPSYTNYPQL GLHLPSYTNYPQIIM GLHLPSYTNYPQLIF GLHLPSYTNYPQ 8001 LHT 1 HLHTDEEIRFA 8038 PHY 8 EINEGALLLPHYNSK FTEINEGALLLPHYNSK LIFTEISEGALLLPHYNSK TEINEGALLLPHYNSK TEISEGALLLPHYNSK FIEINEGGLLLPHYNSK FIEINEGALLLPHYNSK LTFTEINEGALLLPHYNSK 8045 PWL 1 WLPPWLDAH 8053 PWY 1 NHPEVVAEEPWYGIE 8063 RHN 2 KLRHNIGQSTSPDAYNPQAGRL KLRHNIGQSTSPDAYNPQAGRLK 8065 RHR 3 RTTRHRRAQGEEGEEEEEETSTR RHRRAQGEEGEEEEEETSTR RHRPHHHEEEEEEEEWSHQ 8076 RWL 1 TSLDFPILRWL 8103 VKL 1 ARDIWNIEPVKLP 8107 IKL 3 YEGGIKLPLDVI YEGGIKLPTNVL YEGGIKLPLDVIST 8114 GGE 1 RLINEPVEGGEGNLL 8133 KVI 5 KVISPPTLRPR VKEGLKVISPPTLRPR GLKVISPPTLRPR EVKEGLKVISPPTLRPR EGLKVISPPTLRPR 8134 KD 4 KGILDKIKDKLPGHHN KDPNYVDEEDEEEEDVKGF NMVDPDEKDSTGNLPSRAL RDSFGNDIIKDL 8190 PW 2 NHPEVVAEEPWYGIE WLPPWLDAH 8214 RW 1 TSLDFPILRWL 8215 IR 28 SSNIIRFF NDEDLIRIL VGGKIIREPGPLPGL LIFPGSAEDVERLIR QNPNERIKQIIRVE (Continued) Mol. Nutr. Food Res. 2024,68, 2300503 2300503 (5 of 11) © 2024 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH GmbH 16134133, 2024, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/mnfr.202300503 by Universidad De Sevilla, Wiley Online Library on [21/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.mnf-journal.com Table 2. (Continued) ID1Bioactive motifsa) No. sequences Secuencesb) NQRNFLAGSEDNVIRQL HLHTDEEIRFA VGGKIIREPGPLPGLK SEDNVIRQL LAGSEDNVIRQL SIRALPLDVVAH IIRVEEGLGVISPK SIRAGLQFPVGRIG QIIRVEEGLGVISPK VVDEGEGNYELVGIR QIIRLLL QVEDPQINDDEVLIR LENVIRDAVTY IIREPGPLPGL SDALDKIRFL ADENQRNFLAGSEDNVIRQL IFPGSAEDVERLIR PGSAEDVERLIR LDIVIPTIR QGDIIRVPSGTPVYL IFPGSAEDVERLIRNQ AYNDEDLIRIL LIFPGSAEDVERLIRNQ 8216 LKP 1 YSLKPLVPRLS 8217 LK 14 LEVPTIDLK ERLKQIVRVK KLRHNIGQSTSPDAYNPQAGRLK QNPDERLKQIVRVK VKEGLKVISPPTLRPR GLKVISPPTLRPR EVKEGLKVISPPTLRPR VGGKIIREPGPLPGLK EGLKVISPPTLRPR LKLPEGSNDVLLK KFVVDMPGLK KTNDQATTSPLKQVFRGIPAE YSLKPLVPRLS FVVDMPGLK 8218 KP 2 TVGNVGEKIKKPFESITK YSLKPLVPRLS 8219 TY 1 LENVIRDAVTY 8224 VY 1 QGDIIRVPSGTPVYL 8459 TW 1 SIIEEFPRLVTW 8461 VW 1 FVMDSPDDVWRIS 9879 SVL 1 SVLSPEELLAVR 10000 LPL 5 YEGGIKLPLDVI HFWQVDKGHTLPL SIRALPLDVVAH ALPLDVVAH (Continued) Table 2. (Continued) ID1Bioactive motifsa) No. sequences Secuencesb) YEGGIKLPLDVIST 10003 LQL 1 IMLQLVILP 10051 RY 5 EQEEEYEQGEEEVRRY DQQRQQDEQEEEEEEVRRY QRQQDEQEEEYEQGEEEVRRY DQQRQQDEQEEEYEQGEEEVRRY QDEQEEEYEQGEEEVRRY a) According to BIOPEP-UWM Database.[17]b) 1-letter amino acid code. was observed in the WD group (27.83 ±2.30 g, p=0.015; +8.60 ±1.34 g, p=0.014) and in the WD +LPH group (27.35 ±2.48 g, p=0.047; +8.10 ±1.95 g, p=0.028) compared to the SD group (24.53 ±1.24 g and 5.30 ±1.10 g, respectively) (Figure 3B and C). However, no significant differences were observed in the final body weight (p =0.312) and in the body weight gain (p=0.384) when mice were fed WD and treated with the LPH, compared to the WD group (Figure 3B and C). AsshowninFigure3D,nostatisticalchanges(p=0.966) were observed in the brain weight between groups at the endpoint (12 weeks of treatment; SD: 0.42 ±0.02 g; WD: 0.42 ±0.02 g; WD + LPH: 0.42 ±0.01 g). 3.4. LPH Treatment Increases Total Antioxidant Activity To evaluate the antioxidant activity exerted by LPH on the brain, the FRAP assay was performed in the brain of the animals. As shown in Figure 4A, WD ingestion significantly decreased (p= 0.003) FRAP by 18.31% (0.83 ±0.03 mmol equivalent Trolox per liter per milligram protein) compared to SD-fed mice (1.01 ±0.04 mmol equivalent Trolox per liter per milligram protein). LPH treatment for 12 weeks overcame these effects, showing Figure 2. Evaluation of LPH in vitro antioxidant power by ferric reducing antioxidant power (FRAP) assay. Data were represented as mean ±SEM (n=6). Data were analyzed by One-way ANOVA followed by Dunn’s posthoc test. *, p≤0.05; ****, p≤0.0001. C, control; LPH, lupin protein hydrolysate; TEAC, Trolox equivalent antioxidant capacity. Mol. Nutr. Food Res. 2024,68, 2300503 2300503 (6 of 11) © 2024 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH GmbH 16134133, 2024, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/mnfr.202300503 by Universidad De Sevilla, Wiley Online Library on [21/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.mnf-journal.com Figure 3. Weekly body weight (A), final body weight (B), body weight gain (C), and brain weight (D) of the in vivo experiments carried out in male ApoE−/− mice. Data were represented as mean ±SEM (n=4). Data were analyzed by Two-way ANOVA followed by Sidak’s post-hoc test (A) and One-way ANOVA followed by Dunn’s post-hoc test (B-D).*, p≤0.05; LPH, lupin protein hydrolysates; n.s., not significant. Figure 4. Ferric reducing antioxidant power (FRAP) (A) or glutathione (GSH) content (B) in the brain of male ApoE−/−mice from the three experimental groups. Data were represented as mean ±SEM (n=4). Data were analysed by One-way ANOVA followed by Dunn’s post-hoc test. *, p≤0.05; **, p≤ 0.01; ***, p≤0.001; GSH, glutathione; LPH, lupin protein hydrolysates; n.s., not significant. Mol. Nutr. Food Res. 2024,68, 2300503 2300503 (7 of 11) © 2024 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH GmbH 16134133, 2024, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/mnfr.202300503 by Universidad De Sevilla, Wiley Online Library on [21/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.mnf-journal.com Table 3. Spearman’s correlations between total antioxidant power and GSH. Group FRAP vs GSH rp-value SD 0.886 0.033 WD −0.300 0.683 WD +LPH 0.893 0.012 GSH, glutathione; FRAP, ferric reducing antioxidant power; SD, standard diet; WD, western diet; LPH, lupin protein hydrolysate. The bold value indicates a statistical difference. a significant increase of 12.29% (p=0.037) in brain FRAP (0.93 ±0.02 mmol equivalent Trolox per liter per milligram protein) compared to the WD group. No significant differences (p= 0.165) were observed between the WD +LPH and SD-fed mice groups. 3.5. LPH Treatment Increases GSH Levels in the CNS The cerebral GSH content was evaluated in all experimental groups. Mice fed WD showed significantly lower levels (−7.48%, p=0.041) of cerebral GSH (56.68 ±0.44 μM per mg protein) compared to the SD group (61.26 ±0.59 μM per mg protein). LPH was able not only to counteract this effect (76.21 ±3.22 μM per mg protein, p<0.001), increasing it by 34.46%, but also significantly increased (p=0.01) the GSH levels even compared to the SD group (Figure 4B). The nonparametric Spearman’s correlation was applied to study the possible relationship between total antioxidant power and GSH content. As shown in Table 3, a strong positive correlation was observed between FRAP and GSH in the SD group. However, this was lost in the WD group. Significantly, a strong positive correlation between FRAP and GSH was restored in LPH-treated mice (WD +LPH). Graphs with individual data points for each group are shown in Figure S1 (Supporting Information). 3.6. Nitric Oxide Synthesis Is Downregulated by the LPH in the CNS To verify the possible effect of LPH on brain NO synthesis, the relative expression of the nNOS and iNOS mRNA, and the NO concentration were evaluated. As shown in Figure 5,WD- fed mice showed a significant increase of 65.55% and 63.67% in the gene expression of nNOS (1.80 ±0.56-fold change, p= 0.004) (Figure 5A) and iNOS (1.41 ±0.17-fold change, p=0.008) (Figure 5B), respectively, as well as by 19.50% in the NO production (4.36 ±0.07 O.D. per mg protein, p=0.045) (Figure 5C)compared to SD-fed mice. Treatment with LPH for 12 weeks reduced by 49.66% and 18.84% both iNOS gene expression (0.70 ±0.18- fold change, p=0.002) and NO production (3.49 ±0.18 O.D. per mg protein, p=0.01), respectively, compared to the WD group, without affecting the nNOS gene expression (p=0.192). No differences between WD +LPH and SD groups were observed. 3.7. LPH Treatment Increases the Activity of CAT and GR Enzymes and Decreases the DNA Damage in the CNS Once the increase in the brain of total antioxidant activity was demonstrated in mice treated with LPH, the activity of the main antioxidant enzymes as well as oxidative damage to DNA were evaluated. As shown in Figure 6, treatment with LPH significantly increased the activity of the antioxidant enzymes CAT (+ 31.29%, p=0.011) and GR (+26.11, p=0.043), compared to the WD group. On the other hand, LPH treatment did not modify the activity of the antioxidant enzymes SOD (p=0.122) or GPx (p=0.193). Furthermore, a significant decrease in 8-OHdG levels (-27.99%, p=0.031) was shown in the brain of LPH-treated mice, compared to the WD group (Figure 6). 4. Discussion This study highlights the antioxidant effects of LPH treatment on the brain of WD-fed ApoE−/−mice. Specifically, LPH palliated the oxidizing effects on the brain caused by WD feeding. The effects Figure 5. Neuronal (A) and inducible (B) nitric oxide synthase (nNOS and iNOS) gene expression in hypothalamus, and nitric oxide production (C) in brain of the male ApoE−/−mice from the different experimental groups. Data were represented as mean ±SEM (n=4). Data were analyzed by One-way ANOVA followed by Dunn’s post-hoc test. *, p≤0.05; ** p≤0.01; LPH, lupin protein hydrolysates; n.s., not significant. Mol. Nutr. Food Res. 2024,68, 2300503 2300503 (8 of 11) © 2024 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH GmbH 16134133, 2024, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/mnfr.202300503 by Universidad De Sevilla, Wiley Online Library on [21/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License www.advancedsciencenews.com www.mnf-journal.com Figure 6. Percentage of the antioxidant activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and DNA damage (8-OHdG) in the brain of male ApoE−/−mice. After referring the data to mg of protein, results are expressed as a percentage of the standard diet group. Data were represented as mean ±SEM (n =4). Data were analyzed by One-way ANOVA followed by Dunn’s posthoc test. *, p≤0.05; **, p≤0.01; ***, p≤0.001 versus the control group (standard diet, dashed line). 8-OHdG, 8-hydroxy-2ʹ-deoxyguanosine; LPH, lupin protein hydrolysates. of LPH were not related to body weight gain, final body weight, or daily food intake (Figure S2, Supporting Information), which remain unchanged after LPH treatment. LPH is a mixture of peptides obtained after hydrolysis of a lupin (L. angustifolius) protein isolate using the food-grade endopeptidase Alcalase. The protein content of the hydrolysate was higher than that of the previous lupin flour. Additionally, the use of Alcalase generated a hydrolysate with peptides mainly smaller than 8 kDa in size, with a peak in <1 kDa. This fact is due to Alcalase is an endopeptidase that can cleavage between a wide range of amino acids (Glu, Met, Leu, Tyr, Lys, and Gln), providing a hydrolysate with many peptides of small size and hydrophobic characteristics. In this sense, it is well known that the bioactive effect of peptides is influenced by structural properties, such as molecular weight, peptide size, and amino acid composition.[24] In particular, low-molecular-weight peptides are associated with a powerful antioxidant capacity compared to larger peptides.[25] Furthermore, peptides containing i) hydrophobic amino acids such as Leu or Val in their N-terminal regions, ii) imidazole ringcontaining His, iii) nucleophilic sulfur-containing amino acid residues (Cys and Met), iv) pyrrolidine ring (Trp and Pro), and/or v) aromatic amino acid residues (Phe, Trp, and Tyr) have been shown to possess strong antioxidant properties.[25]In this line, a previous study by our group demonstrated that LPH is rich in the amino acids Leu, Val, Pro, His, Thr, Tyr, and Phe.[9] Other food-derived hydrolysates generated with Alcalase have shown potent antioxidant effects, such as pea, corn, soy, zein, amaranth, and rice bran protein hydrolysates.[26–30]Furthermore, numerous scientific articles have shown that hydrolysates obtained from Alcalase have greater antioxidant effects than hydrolysates generated with other proteases such as protamex, neutrase, flavourzyme, papain, and pepsin.[31,32] The characterization of LPH revealed the presence of 101 L. angustifolius peptides (36.33%) that contained at least a known antioxidant motif. The physicochemical analysis of these sequences showed a high content of imidazole rings (90.1% of sequences with at least 1 ring) and hydrophobic amino acids such as Leu (11.71%) and Val (5.75%). In this way, a high proportion of Leu, Val, and amino acids with imidazole rings have been associated with strong antioxidant capacity through direct electron transfer.[24]Moreover, these considerations are supported by the LPH in vitro FRAP test, in which a dose-dependent increase in antioxidant capacity was observed from a LPH concentration of 0.25 mg mL−1. In addition, Wang et al. have previously demonstrated that His- Tyr, Pro-Tyr, and Leu-Pro-Phe motifs, also present in the LPH (6.84%), exert anti-inflammatory and antioxidant effects on the BV-2 microglial cell line. Specifically, these motifs have been shown to reduce LPS-induced nitrite and ROS production after 24 h of cell treatment.[6,18] In the present study, oxidative stress was induced in ApoE−/− mice by feeding a WD. As expected, WD-fed mice showed reduced cerebral levels of FRAP and GSH, as well as increased levels of nNOS, iNOS, and NO, compared to SD-fed mice. These results agree with previous studies that observed an increase in brain oxidative stress caused by high-fat diet feeding.[33,34]Oxidative stress has been implicated in the progression of several neurodegenerative diseases, including Parkinson’s disease and Alzheimer’s disease.[5] Here, we show that LPH treatment palliates the exacerbated oxidative stress triggered by WD consumption in the brain of animals. In particular, LPH treatment counteracts the decrease in FRAP levels and GSH concentration, as well as restores iNOS gene expression and NO content in the brain of WD-fed animals to those of SD-fed mice. While iNOS is produced by pro-inflammatory stimuli such as cytokines, nNOS is constitutively expressed and involved in several physiological procedures in the CNS. In fact, normal NO levels synthesised by nNOS are involved in neuronal plasticity, regulation of blood flow in the CNS, and neurotransmitter release, among others.[35]LPH does not significantly alter the gene expression of nNOS in WD-fed mice, supporting the idea that decreased NO production in the LPH-treated mice is primarily mediated by decreased iNOS mRNA expression, which in turn is involved in a decrease in pro-inflammatory/pro-oxidant status generated in the CNS of mice fed WD. On the other hand, GSH, the most important antioxidant tripeptide (Glu–Cys–Gly) in biological systems, participates in excessive ROS scavenging. It is also important to note that the increase in GSH content in the brain of LPH-treated mice is highly correlated with the increase in total antioxidant power (FRAP). Thus, LPH treatment restores and increases the GSH content, favoring a better response against oxidative stress. Furthermore, an increase in the activity of the antioxidant enzymes CAT and GR and a decrease in 8-OHdG levels were shown in the brain of LPH-treated mice. Thus, the increase of antioxidant system allows the preservation of the integrity of nucleic acids, reducing the severity of danger of the oxidation of the DNA, and so improving the cell’s survival. In this sense, an increase in the activity of antioxidant enzymes was observed previously in plasma and liver of mice treated with LPH.[10,36] Interestingly, a previous study using the same mouse model has shown an anxiolytic role of LPH treatment.[12]Therefore, we suggest that the antioxidant effects observed in the CNS of LPH- treated mice could be a possible explanation for the previous anxiolytic effects demonstrated. All these results are in agreement with our previous studies that showed antioxidant effects of LPH Mol. Nutr. Food Res. 2024,68, 2300503 2300503 (9 of 11) © 2024 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH GmbH 16134133, 2024, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/mnfr.202300503 by Universidad De Sevilla, Wiley Online Library on [21/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License