Methionine sulfoxide modified antioxidant polypeptide targeting keap1 protein and application thereof

Abstract

The present application belongs to the technical field of bioactive peptides, and particularly relates to a methionine-containing sulfoxide-modified antioxidant polypeptide targeting KEAP1 protein and application. The amino acid sequence is shown as SEQ ID NO. 1: M(ox)LAVHDGFEG. Polypeptide precursor protein traceability analysis shows that MG-10 is separated from a digestion solution obtained after chicken meat is fermented by a complex fermentation agent (Candida utilis, Bacillus subtilis and Lactobacillus casei) and then subjected to gastrointestinal juice digestion. The precursor is a PFKM protein (6-phosphofructokinase) in chicken meat, and the 466-475 amino acid region of the PFKM protein is subjected to methionine oxidation modification in the fermentation process. After entering the gastrointestinal juice digestion stage, the region is subjected to coordinated enzymatic hydrolysis of pepsin and trypsin, and finally MG-10 is released. The present application verifies the mechanism of MG-10 targeting KEAP1 protein, activating the NRF2-ARE pathway and improving DSS-induced colitis through molecular docking, cell experiments and animal experiments, and provides a scientific basis for the application of MG-10 in functional foods and the development of drugs for preventing and treating ulcerative colitis.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptide technology, and particularly relates to an antioxidant polypeptide containing methionine sulfoxide modified to target KEAP1 protein and its application. Background Technology

[0002] Bioactive peptides (BAPs) are short-chain polypeptides composed of 2–20 amino acid residues. They typically exist in an inactive form within the natural protein sequence and require release through processes such as enzymatic hydrolysis, microbial fermentation, or gastrointestinal digestion to exert their physiological regulatory functions. Compared to intact proteins, small peptide fragments have higher bioavailability and targeting capabilities. They can interact with biomolecules in the body through non-covalent mechanisms such as hydrogen bonds, electrostatic interactions, and hydrophobic interactions, thereby regulating various physiological processes. In recent years, bioactive peptides from food sources have become a research hotspot in the fields of functional foods and nutritional supplements due to their natural origin, low toxicity, and multifunctionality.

[0003] Chicken, as one of the most consumed animal proteins globally, is not only rich in protein but also boasts superior antioxidant potential compared to pork, beef, and fish, making it an important resource for bioactive peptides. In recent years, research on irritable bowel syndrome (IBS) has increasingly focused on oxidative stress, viewing it as a crucial "catalyst" driving disease development and progression. Oxidative stress is essentially an imbalance between the body's oxidation and antioxidant systems, leading to the accumulation of large amounts of reactive oxygen species (ROS) and subsequent cell damage. In IBS, the intestinal mucosa is chronically in a state of low-grade inflammation and immune activation due to factors such as infection, psychological stress, or diet, a process that can trigger a significant oxidative stress response. Excessive free radicals directly attack intestinal neurons and glial cells, exacerbating their abnormal excitability and thus interfering with the gut-brain axis function. Simultaneously, free radicals also damage the mitochondrial function of intestinal epithelial cells, disrupting tight junctions between cells, leading to a decrease in the integrity of the intestinal barrier and the so-called "leaky gut" phenomenon. This disruption of the intestinal barrier further disrupts the gut microbiota balance and continuously activates the mucosal immune system, exacerbating brain-gut axis signaling disorders. Therefore, oxidative stress is likely a common pathway and core hub connecting psychological stress, infection, immune abnormalities, and neurological dysfunction, providing a more integrated new perspective for understanding the systemic pathophysiological mechanisms of IBS and laying a theoretical foundation for developing antioxidant-targeted therapeutic strategies. NRF2 is a central regulator of cellular antioxidant responses, anchored in the cytoplasm and rapidly degraded in a resting state; when subjected to oxidative stress, NRF2 is activated and enters the nucleus, initiating the expression of a series of endogenous antioxidant genes. Therefore, activating the NRF2 pathway through drugs or natural active substances holds promise for systematically enhancing the antioxidant defense capacity of intestinal cells, neutralizing excess free radicals, protecting intestinal barrier function, and providing a new direction for fundamentally intervening in the pathological process of IBS.

[0004] Oxidative stress results from an imbalance between the body's oxidative and antioxidant systems, playing a central role in various cellular and tissue injuries. The NRF2-KEAP1-ARE pathway is a core endogenous defense system for maintaining cellular homeostasis and resisting oxidative damage. The KEAP1 protein binds to NRF2 through its Kelch domain, mediating the ubiquitination and degradation of NRF2. When oxidative modification triggers a conformational change in KEAP1, NRF2 dissociates, stabilizes, and translocates into the nucleus, initiating the expression of downstream antioxidant genes such as HO-1 and NQO1. Therefore, screening for natural antioxidant peptides targeting KEAP1 has become an important strategy for preventing and treating oxidative stress-related diseases.

[0005] In its previous work, the team used a compound microbial agent composed of *Candida utilis*, *Bacillus subtilis*, and *Lactobacillus casei* to ferment chicken meat (Patent No. ZL 2022 1 0730043.X). Building upon this, the present invention further identified a novel antioxidant peptide, MG-10, modified with methionine sulfoxide, from the in vitro simulated digestive fluid of the fermented chicken meat. The amino acid sequence of MG-10 is M(ox)LAVHDGFEG. Summary of the Invention

[0006] The purpose of this invention is to provide an antioxidant polypeptide containing methionine sulfoxide modified to target the KEAP1 protein and its application.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: An antioxidant polypeptide containing methionine sulfoxide modified to target KEAP1 protein, the polypeptide being MG-10, the amino acid sequence of which is shown in SEQ ID NO.1: M(ox)LAVHDGFEG.

[0008] Furthermore, the polypeptide MG-10 is derived from the amino acid region of chicken PFKM protein (6-phosphofructokinase) at positions 466-475, and is produced by methionine oxidation modification during fermentation and subsequent synergistic enzymatic cleavage by pepsin and trypsin.

[0009] Furthermore, the fermentation process uses a compound microbial agent composed of Candida utilis, Bacillus subtilis, and Lactobacillus casei.

[0010] Another object of the present invention is to provide the use of an antioxidant polypeptide in the preparation of a drug for treating DSS-induced ulcerative colitis.

[0011] The advantages of this invention are as follows: Our team previously used a compound microbial agent composed of *Candida utilis*, *Bacillus subtilis*, and *Lactobacillus casei* to ferment chicken meat (Patent No. ZL 2022 1 0730043.X). Building upon this, this invention further identified a novel antioxidant peptide, MG-10, modified with methionine sulfoxide, from the in vitro simulated digestive fluid of the fermented chicken meat. Its amino acid sequence is M(ox)LAVHDGFEG. For in vitro cell experiments, the human colon cancer Caco-2 cell line was used. This cell line can spontaneously differentiate into a polar monolayer with intestinal epithelial cell characteristics under culture conditions. It is widely used in studies of nutrient absorption and transport, as well as intestinal oxidative stress mechanisms, and is an ideal model for verifying the activation of the NRF2 pathway by bioactive peptides at the intestinal epithelial level. Animal experiments used a mouse model of acute colitis induced by dextran sulfate sodium (DSS). This model induces an innate immune response by disrupting the integrity of the intestinal epithelial barrier. Its clinical and pathological features (weight loss, diarrhea, bloody stools, shortened colon, and inflammatory cell infiltration) are highly similar to those of human ulcerative colitis. It is currently the most commonly used experimental model internationally recognized for studying inflammatory bowel diseases, and its pathogenesis is closely related to oxidative stress and the cascading reaction of inflammatory factors. This invention systematically verified the mechanism by which MG-10 targets the KEAP1 protein, activates the NRF2-ARE pathway, and improves DSS-induced colitis through molecular docking, cell experiments, and animal experiments. This provides a scientific basis for the application of MG-10 in the development of functional foods and drugs for the prevention and treatment of ulcerative colitis. Attached Figure Description

[0012] Figure 1 This is a roadmap of the research and development process of this invention. The invention first uses a compound fermentation agent (Lactobacillus casei, Bacillus subtilis, and Candida utilis) to ferment chicken breast. The fermented and unfermented chicken breasts were then sequentially digested in vitro using simulated pepsin digestion for 2 hours and trypsin digestion for 2 hours, respectively. Peptidomics analysis revealed 1750 specific peptides in the in vitro digestion fluid of the fermented chicken. Subsequently, these peptides were molecularly docked with Keap1 protein, and three candidate peptides were selected. Further kinetic simulations showed that peptide MG-10 exhibited the strongest binding affinity to Keap1. Precursor protein tracking analysis confirmed that MG-10 originates from phosphofructokinase (PFKM), whose N-terminal methionine undergoes oxidative modification during fermentation and is released under the synergistic enzymatic hydrolysis of pepsin and trypsin. In subsequent biological function verification, MG-10 competitively binds to Keap1 protein, thereby promoting nuclear translocation of NRF2 and upregulating the expression level of downstream HO-1 protein.

[0013] Figure 2This is a three-dimensional structural diagram of MG-10 (M(ox) LAVHDGFEG) ​​of the present invention. In the diagram: Primary sequence: Met(O)-Leu-Ala-Val-His-Asp-Gly-Phe-Glu-Gly; oxidized methionine-leucyl-alanyl-valine-histyl-aspartyl-glycyl-phenylalanyl-glutamyl-glycine.

[0014] Figure 3 This section describes the in vitro digestibility characteristics of fermented chicken meat. A is a schematic diagram of the in vitro digestion process; B shows the in vitro protein digestibility, where different values ​​of the same lowercase bar graph (a, b, c, d, and e) indicate a significant difference (P < 0.05), while identical lowercase letters indicate no significant difference; C shows the SDS-PAGE electrophoresis pattern of the in vitro digestion fluid. G0, G2, and G2+I2 represent 0 h of gastric digestion, 2 h of gastric digestion, and 2 h of gastric digestion followed by 2 h of intestinal digestion, respectively. FU and C represent the fermentation group, the non-fermentation group, and the blank control group, respectively.

[0015] Figure 4 The results of the peptidomics analysis are shown in the figure, where (A) is the total number of proteins identified in each group; (B) is the molecular weight distribution of the identified proteins; (C) is the total number of peptides identified in each group; (D) is the molecular weight distribution of the identified peptides; (E) is the Venn diagram of the differentially expressed proteins; and (F) is the Venn diagram of the differentially expressed peptides.

[0016] Figure 5 The results of molecular docking and molecular dynamics simulations are shown in the following figures: (A) The top 10 conformations in molecular docking scores; (B–D) Binding patterns of Keap1 with three polypeptides (M(ox)LAVHDGFEG, FVSDHF, and NGPGPWGFR), with the left figure being an overall three-dimensional view and the right figure being a local two-dimensional interaction diagram. Green dashed lines represent hydrogen bonds, and red dashed lines represent salt bridges; (E) Binding free energy and energy components predicted based on the MM / GBSA (Molecular Mechanics / Generalized Born Surface Area) method. Data were derived from three independent simulations and are expressed as mean ± standard deviation. Different lowercase letters in the same column indicate significant differences (P < 0.05), while the same letter indicates no significant differences (P > 0.05). Data are presented as the time-varying trajectories of various properties during the (F–K) molecular dynamics simulations, including (F) root mean square deviation (RMSD) of peptide ligands, (G) RMSD of the complex, (H) root mean square fluctuation (RMSF) of protein residues, (I) radius of gyration (Rg) of the complex, (J) number of hydrogen bonds in the complex, and (K) solvent-accessible surface area (SASA) of the complex.

[0017] Figure 6Activation of NRF2 pathway-related protein expression in Caco-2 cells by MG-10. Effects of different concentrations of MG-10 on Caco-2 cell viability at 24 h (A) and 48 h (B). (C) Representative Western blot bands of HO-1, Keap1, and cytoplasmic NRF2 in whole-cell lysates, with GAPDH as the internal control. (D) Representative Western blot bands of nuclear NRF2 in nuclear fractions, with Lamin B1 as the internal control. (EG) Quantitative analysis of relative protein expression levels of HO-1 (E), Keap1 (F), and cytoplasmic NRF2 (G) after normalization with GAPDH as the internal control. (H) Quantitative analysis of nuclear NRF2 protein expression after normalization with Lamin B1 as the internal control. Quantitative data are expressed as mean ± standard deviation (SD), n=3 independent experiments. Different lowercase letters (a, b, c) on the bar chart indicate significant differences between groups (P < 0.05), while the same letter indicates no significant difference (P > 0.05). The final concentration of MG-10 was 6.0 μmol / L; the final concentration of SFN (sulforaphane, an NRF2 positive agonist) was 10 μmol / L; and the final concentration of ML385 (an NRF2 selective inhibitor) was 5 μmol / L. The treatment time was 24 h.

[0018] Figure 7 MG-10 competitively binds to Keap1 and promotes NRF2 nuclear translocation. (A) Effect of MG-10 on the Fluc / Rluc ratio. Concentrations used: MG-10 6.0 μmol / L, SFN 10 μmol / L, ML3 85 μmol / L. (B) Pull-down assay to verify the direct interaction between MG-10 and Keap1. Lane 1: Blank control; Lane 2: Bio-MG-10 only; Lane 3: Bio-MG-10 with excess unlabeled MG-10 (competitive); Lane 4: Scramble-Bio (non-specific control). (C) Representative confocal immunofluorescence images showing the subcellular localization of NRF2. NRF2 is green fluorescent, and the nuclei are counterstained with DAPI (blue). Scale bar: 10 μm. All data are expressed as mean ± SD (n=3). Different lowercase letters on the column indicate statistically significant differences between groups (P<0.05), while the same letter indicates no statistically significant difference (P>0.05). Abbreviations: CON: Control; SFN: Sulforaphane (NRF2 agonist); ML385: NRF2 inhibitor; DAPI: 4',6-diamidinyl-2-phenylindole; NRF2: Nuclear factor E2-related factor 2; Fluc / Rluc: Firefly luciferase / Reneiss luciferase ratio.

[0019] Figure 8This study investigated the effect of MG-10 on the expression of NRF2-ARE pathway proteins in the colonic tissue of DSS-induced colitis mice. Normal control group (NC): administered an equal volume of 0.5% CMC-Na solution (0.1 mL / 10 g body weight) daily by gavage, with free access to distilled water; DSS model group (DSS): administered an equal volume of 0.5% CMC-Na solution (0.1 mL / 10 g body weight) daily by gavage, with free access to 3% DSS solution; MG-10 medium-dose monotherapy group (MG-10-M): administered MG-10 100 mg / kg body weight daily by gavage, with free access to distilled water; DSS+MG-10 medium-dose group (DSS+MG-10-M): administered MG-10 100 mg / kg body weight daily by gavage, with free access to 3% DSS solution. Different lowercase letters (a, b, c) in the bar chart indicate significant differences between groups (P<0.05), while the same letter indicates no significant difference (P>0.05). Detailed Implementation Example

[0020] An antioxidant polypeptide containing methionine sulfoxide modified to target KEAP1 protein, the polypeptide being MG-10, the amino acid sequence of which is shown in SEQ ID NO.1: M(ox)LAVHDGFEG.

[0021] Furthermore, the polypeptide MG-10 is derived from the amino acid region of chicken PFKM protein (6-phosphofructokinase) at positions 466-475, and is produced by methionine oxidation modification during fermentation and subsequent synergistic enzymatic cleavage by pepsin and trypsin.

[0022] Furthermore, the fermentation process uses a compound microbial agent composed of Candida utilis, Bacillus subtilis, and Lactobacillus casei.

[0023] Another object of the present invention is to provide the use of an antioxidant polypeptide in the preparation of a drug for treating DSS-induced ulcerative colitis.

[0024] Verification Experiment 1. Materials and Methods 1.1 Experimental Materials Candida utilis, Enterococcus faecalis, and Lactobacillus casei were all obtained from the China General Microbiological Culture Collection Center.

[0025] 1.2 Preparation of bacterial strains Lactobacillus casei and Enterococcus faecalis were inoculated into sterilized MRS medium and incubated statically at 37°C for 2 days. Candida utilis was inoculated into sterilized YPD medium and incubated with shaking at 30°C and 180 r / min for 2 days. The count results of the three microorganisms after incubation are expressed as CFU / mL.

[0026] 1.3 Preparation of Fermented Chicken Breast Cut chicken breast into strips measuring 1cm x 2cm x 4cm (10.00 ± 0.32 g). Marinate the chicken strips for 30 minutes. The concentration of the compound fermentation agent is Lactobacillus casei 1.0 × 10⁻⁶. 9 CFU / mL, Enterococcus faecalis 1×10 8 CFU / mL and Candida utilis 1.0 × 10⁻⁶ 8 The concentration of CFU / mL was mixed in a 1:1:1 ratio. Each chicken breast was inoculated with 0.2 mL of the compound fermentation agent, fermented at 30℃ for 40 h, dried at 65℃ for 2 h, turned over, and dried at 95℃ for 2 h. The chicken breasts in the non-fermented group were marinated using the same method, but without the addition of any fermentation agent after marinating, and then dried directly.

[0027] 1.4 In vitro digestion test 1.4.1 In vitro digestion experimental procedure In vitro digestion experiments were conducted using the INFOOGS 2.0 static simulation method. The 1.25×SGF concentrated stock solution contained 2.57 g / L KCl, 0.61 g / L KH₂PO₄, 10.50 g / L NaHCO₃, 13.80 g / L NaCl, 0.12 g / L MgCl₂·6H₂O, and 0.24 g / L (NH₄)₂CO₃; the 1.25×SIF concentrated stock solution contained 2.54 g / L KCl, 0.54 g / L KH₂PO₄, 35.70 g / L NaHCO₃, 11.23 g / L NaCl, and 0.34 g / L MgCl₂·6H₂O. Without pre-adding to the above concentrated stock solution, add it to the digestion system immediately before use on the day of the digestion experiment to achieve final concentrations of CaCl2·2H2O in SGF and SIF of 0.15 mmol / L and 0.60 mmol / L, respectively. Weigh fermented and unfermented chicken jerky samples separately, and calculate the corresponding sample weight (approximately 0.18 g for fermented group and approximately 0.24 g for unfermented group) based on the measured protein content (55.86% for fermented group and 42.26% for unfermented group), so that the protein content of each sample is approximately 100 mg. Weigh 12 portions of each fermented and unfermented chicken jerky sample, place them in 2 mL grinding tubes, add 1 mL of 1.25×SGF concentrate and 3 stainless steel grinding beads with a diameter of 3 mm, and homogenize using a tissue homogenizer (Seville BioSWE-FP) at -20℃, repeating the cycle 3 times with 30s grinding and 10s intervals. All homogenates from each group were transferred to 50 mL centrifuge tubes. 7 mL of 1.25×SGF concentrate was added, and the pH was adjusted to 3.0 using 1 mol / L HCl. Pepsin (P6887, Sigma-Aldrich) solution was added to achieve a final pepsin activity of 2,000 U / mL. The volume was then brought to 10 mL with ultrapure water. Three tubes from each of the fermented and non-fermented groups were centrifuged and immediately placed in an ice-water bath for 5 min to terminate the reaction. These tubes were labeled F-SGF0h and UF-SGF0h. The remaining nine centrifuge tubes from each of the fermented and non-fermented groups were cultured at 37℃ and 200 r / min for 2 h with shaking. Three tubes from each group were randomly selected, and the reaction was terminated using the same method. Anhydrous ethanol was added, and these tubes were labeled F-SGF2h and UF-SGF2h, respectively.After 2 hours of gastric digestion, all the gastric digestion fluid from the remaining 6 centrifuge tubes was transferred to 100 mL Erlenmeyer flasks. 8 mL of 1.25×SIF concentrate was added, and the pH was adjusted to 7.0 using 1 mol / L NaOH. Trypsin (T0303, Sigma-Aldrich) solution was added to achieve a final trypsin activity of 100 U / mL. The volume was brought to 20 mL with ultrapure water. Three Erlenmeyer flasks from each of the fermented and non-fermented groups were immediately placed in an ice-water bath for 5 minutes to terminate the reaction, and labeled as F-G2+I0h and UF-G2+I0h. The remaining three Erlenmeyer flasks were incubated at 37℃ with shaking at 200 rpm for 2 hours. After digestion, the reaction was terminated by placing them in an ice-water bath for 5 minutes, and labeled as F-SIF2h and UF-SIF2h, respectively. The blank control group was replaced with an equal volume of sterile water to eliminate interference from proteases and the procedure. All other procedures were identical to the sample group, with the corresponding time points labeled CG-0h, CG-2h, and C-GI-2h. A schematic diagram of the experimental procedure is shown below. Figure 3 A. The above digestion experiments were each independently repeated twice, and the resulting samples were used for quantitative determination of protein digestibility and SDS-PAGE electrophoresis analysis, respectively.

[0028] 1.4.2 Determination of protein digestibility Samples at each time point were centrifuged at 10,000g for 20 min at 4℃, the supernatant was discarded, the precipitate was collected, 5 mL of protein extraction buffer was added to resuspend the sample, and after thorough mixing, it was centrifuged at 4,000g for 5 min at 4℃. The supernatant was collected, and the protein content was determined using a BCA protein quantification kit. Protein digestibility was calculated using the following formula: Protein digestibility (%) = (100 mg - W0 - W1 × 5 mL) / 100 mg × 100% In the formula, W0 and W1 are the protein concentrations (mg / mL) in the protein extraction supernatants of the experimental group and the blank sample group, respectively. Each treatment group has 3 parallel samples.

[0029] 1.4.3 SDS-PAGE Analysis Samples at each time point were immediately incubated with 3 volumes of anhydrous ethanol at 4°C overnight (12 h), centrifuged at 10,000g for 20 min at 4°C, and the protein precipitate was collected. The precipitate was dissolved in PBS (pH 7.4) and the protein concentration was adjusted to 1.0 mg / mL. The protein solution was then mixed with 5× loading buffer at a volume ratio of 4:1 and denatured at 100°C for 5 min. 15 μL of the sample was loaded into each well, along with 6 μL of pre-stained protein marker (G2087, Servicebio). SDS-PAGE electrophoresis was performed using a 4%–20% pre-fabricated gradient gel at 4°C, first at 80 V for 30 min, then at 100 V for 80 min. After electrophoresis, the sample was stained with Coomassie Brilliant Blue R-250, and images were acquired using a gel imaging system (GE Healthcare). Each treatment group had three replicates.

[0030] 1.4.4 Evaluation of the antioxidant properties of in vitro digestive fluids Six groups of samples were collected from the gastric juice digestion and gastric juice-to-intestinal juice digestion experiments: control (0 h), control gastric juice (2 h), control intestinal juice (2 h), fermentation (0 h), fermented gastric juice (2 h), and fermented intestinal juice (2 h). These samples were centrifuged at 4000 r / min and 4℃ for 5 min, respectively. The precipitate was removed, and the supernatant was collected to determine its in vitro antioxidant properties. The PTIO free radical scavenging assay, ABTS free radical scavenging assay, DPPH free radical scavenging assay, and FRAP determination were performed according to previous studies.

[0031] PTIO• + (2-phenyl-4,4,5,5-tetramethylimidazolin-3-oxy-1-oxygen, radicalcation) Scavenging experiment: 30 mg of PTIO solid was dissolved in 200 mL of distilled water to prepare the PTIO working solution. 1.6 mL of the working solution was added to a reaction tube, followed by different volumes of sample (0.3, 0.6, 0.9, 1.2, and 1.5 mL), and the total reaction volume was brought up to 3.6 mL with distilled water. The reaction was incubated at 37℃ for 2 h, and the absorbance value A was measured at 557 nm. Preliminary experiments confirmed that when the sample volume was 0.6 mL, the absorbance value no longer decreased with increasing volume. The maximum PTIO scavenging rate (%) = (A0 - A) / A0 × 100%, where A0 represents the absorbance value without sample, and A represents the absorbance value with 0.6 mL of sample added.

[0032] ABTS• +(2,2-Azinobis-(3-ethylbenzothiazoline-6-sulphonate) radicalization assay: 7 mM ABTS stock solution was mixed with 2.45 mM potassium persulfate at a volume ratio of 1:1 and incubated at room temperature in the dark for 12 h. The mixture was then diluted with anhydrous ethanol at pH 7.4 until the absorbance was 0.70 ± 0.02, which was the ABTS+ working solution. Different volumes of sample solution (0.035, 0.07, 0.105, and 0.14 mL) were added to reaction tubes and mixed with 3.2 mL of the working solution. The total reaction volume was then increased to 3.6 mL with ethanol. The reaction was carried out at room temperature for 6 min, and the absorbance value A was measured at 734 nm. Preliminary experiments confirmed that when the sample volume was 0.105 mL, the absorbance value no longer decreased with increasing volume. Maximum ABTS clearance rate (%) = (A0-A) / A0×100%, where A0 represents the value without sample and A represents the value with 0.105 mL of sample added.

[0033] DPPH• + (1,1-Diphenyl-2-picrylhydrazylradical radical cation) scavenging experiment: 0.05-0.1 mM DPPH working solution (absorbance value between 0.6-1.0) was prepared using anhydrous ethanol. Different volumes of sample solution (0.3, 0.6, 0.9, 1.2, and 1.5 mL) were added to reaction tubes and mixed with 3 mL of working solution. The total reaction volume was then increased to 4.5 mL with anhydrous ethanol. The reaction was carried out at room temperature for 30 min, and the absorbance value A was measured at 519 nm. Preliminary experiments determined that when the sample volume was 1.2 mL, the absorbance value no longer decreased with increasing volume. The maximum DPPH scavenging rate = (A0-A) / A0 × 100%, where A0 represents the value without sample, and A represents the value with 1.2 mL of sample added.

[0034] FRAP (Ferric ion reducing antioxidant power) assay: FRAP working solution preparation: Mix 20 mL of 0.3 M pH 3.6 acetate buffer, 2 mL of 10 mmol / L TPTZ solution, and 2 mL of 20 mmol / L FeCl3 solution thoroughly. Add 0.1 mL of sample solution to the reaction tube, then add 2.4 mL of FRAP working solution, mix thoroughly, and incubate at 37°C for 10 min. Read the absorbance at 593 nm. Use 0.1–1.6 mmol / L FeSO4 standard solutions to replace the sample to plot a standard curve. The regression equation is Y = 0.0606x + 0.0241, with a correlation coefficient of R0. 2 =0.995. Substitute the absorbance value into the regression equation to obtain the FRAP value of the sample. 1 FRAP unit = 1 mmol / L FeSO4, that is, the antioxidant capacity of the sample is equivalent to the number of mmol / L FeSO4.

[0035] 1.5 Peptideomics and Virtual Screening Targeting Keap1 Peptides 1.5.1 Identification of digestion products by LC-MS / MS Identification of the incubation broth samples after centrifugation was performed on a Q Exactive mass spectrometer connected to an Easy nLC system. 5 μg peptide samples were loaded into buffer A on a C18 reversed-phase column and separated using buffer B with a linear gradient at a flow rate of 250 nL / min, controlled over 60 min using IntelliFlow technology. Dynamic exclusion lasted for 25 s. Measurement scans were acquired at a resolution of 70,000 m / z 200, and HCD spectroscopy resolution was set to 17,500 m / z 200. MS data were analyzed using MaxQuant software version 1.6.14.0. Data were searched according to Uniprot Sus scrofa, Bos taurus, and plant-based synthetic chicken databases. Enzyme-free cleavage rules were followed, with a mass tolerance of 20 ppm for fragment ions. The overall false discovery rate (FDR) for peptide and protein identification was set to 0.01. Each measurement was repeated five times, and only peptides identified in three measurements were displayed.

[0036] 1.5.2 Molecular docking and molecular dynamics simulation screening The Keap1 protein crystal structure (PDB ID: 2flu) was obtained from the RCSB PDB database. The three-dimensional peptide structure was constructed using PyMol 2.5.5, and energy minimization was performed under the MMFF94 force field. Molecular docking was performed using AutoDock Vina 1.1.2. Before docking, the receptor protein was pretreated using PyMol 2.5.5, including removing water molecules, salt ions, and co-crystallized small molecules, and a docking box was set to encapsulate the entire protein. The ligand and receptor were converted to PDBQT format using ADFRsuite 1.0. The global search exhaustiveness was set to 32 during docking, while other parameters remained at their default values. Based on the docking score, the top three complexes were selected for subsequent molecular dynamics simulations.

[0037] Molecular dynamics simulations were performed using the AMBER 24 software package, and the complex system was described by the ff14SB force field. Hydrogen atoms were added using the LEaP module, and a truncated octahedral TIP3P solvation box was constructed at a distance of 10 Å from the protein, with the addition of Na. + / Cl - Ions neutralize the system charge. Energy optimization was performed sequentially using the steepest descent method (2500 steps) and the conjugate gradient method (2500 steps). Subsequently, the system was heated from 0 K to 298.15 K in 200 ps at a fixed volume, followed by 500 ps NVT ensemble equilibrium and 500 ps NPT ensemble equilibrium. Finally, a 100 ns NPT ensemble production simulation was performed under periodic boundary conditions. The nonbonded cutoff value was set to 10 Å, long-range electrostatic interactions were handled using the Particle Mesh Ewald (PME) method, hydrogen bond constraints were handled using the SHAKE algorithm, and the temperature was controlled by a Langevin thermostat (collision frequency γ = 2 ps). -1 The pressure was maintained at 1 atm, the integration step size was 2 fs, and the trajectory was recorded every 10 ps.

[0038] The free energy was calculated using the MM / GBSA method, based on the MD trajectory over 90–100 ns. The calculation formula is as follows: ΔG bind =ΔG complex −(ΔG receptor +ΔG ligand ) =ΔE internal +ΔE VDW +ΔE elec +ΔG GB +ΔG SA Where: ΔG bind : binding free energy; ΔE internal: internal energy (includesbond, angle, and torsion energies);ΔE vdW : van der Waals energy; ΔE elec Electrostatic energy; ΔG GB : electrostatic contribution to solvation.ΔG SA :non-polar contribution to solvation. ΔG GB and ΔG SA Collectively referred to as solvation free energy. Among them, ΔG GB For polar solvation free energy, ΔG SA This is the nonpolar solvation free energy. For ΔG... GB The calculations were performed using the GB model developed by Nguyen (igb = 2). The nonpolar solvation free energy (ΔGSA) was calculated based on the product of surface tension (γ, empirical constant 0.0072) and solvent-accessible surface area (SA). SA = 0.0072×ΔS ASA (Solvent Accessible Surface Area).

[0039] 1.6 MG-10 precursor protein traceability and computer-aided safety assessment 1.6.1 Identification of precursor proteins of MG-10 peptide Raw mass spectrometry files were retrieved using Peaks Studio 12 software in conjunction with the Gallus gallus UniProt database. To control the false positive rate of mass spectrometry identification, a target-decoy library reverse search strategy was employed. Under this strategy, the false detection rate (FDR) was strictly controlled below 1% (FDR < 0.01), and the FDR was calculated as: FDR = 2 × (number of decoy library matches) / (total number of matches). During MG-10 peptide identification, to determine the origin of the identified natural peptides, the software employed the "parsimony rule": when multiple precursor proteins share the same unique peptide sequence, these precursor proteins are grouped into the same protein group. To eliminate redundant interference from different database entries for the same locus, the software comprehensively sorts the accession numbers of each precursor protein within the group based on peptide count, sequence coverage, and identification score (Score / -10lgP). The accession number with the highest comprehensive ranking within the group is automatically marked as "Top" and assigned the logical flag "TRUE", while all other redundant entries are marked as "FALSE".

[0040] 1.6.2 Prediction of candidate peptides ADMET and TOPKAT Pharmacokinetic and safety risk assessments were conducted on the top three peptides (MG-10, FVSDHF, and NGPGPWGFR) using the ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) and TOPKAT (Computer-Aided Toxicity Prediction) modules in Discovery Studio (BIOVIA, San Diego, CA, USA) software. The ligand 3D structures were constructed using PyMol 2.5.5 and their energy minimized under the MMFF94 force field before being imported into the ADMET descriptor calculation module. Prediction parameters included: intestinal absorption grade, water solubility grade, lipid solubility coefficient (AlogP98), two-dimensional polar surface area (PSA-2D), and blood-brain barrier permeability grade; metabolic and toxicity risk parameters included CYP2D6 inhibition risk, hepatotoxicity risk, and Ames mutagenicity. The reliability of all prediction results was determined based on whether they fell within the model's defined applicability domain; only predictions within the "applicability domain" were adopted. All predictions used the software's default parameters.

[0041] 1.7 Biological validation of MG-10 binding to Keap1 protein 1.7.1 Culture of Caco-2 cells The human colon cancer Caco-2 cell line was purchased from [source missing]. The Caco-2 cell culture medium was high-glucose MEM (containing NEAA) medium containing 20% ​​FBS and 1% penicillin-streptomycin, grown at a concentration of 1.0 × 10⁶ cells / mL. 5 Density inoculation at 25 cm 2 Cells were cultured in square flasks at 37°C in a 5% CO2 incubator. After trypsin digestion and cell counting in the logarithmic growth phase, cells were cultured at 1.0 × 10⁻⁶. 5 The cells were seeded at a density of 1 cell per well in 96-well or 6-well plates and cultured. When the cells reached 60%–70% adhesion, the culture medium was removed, the cells were washed with PBS, and then different induction treatments were performed.

[0042] 1.7.2 Effects of different concentrations and durations of MG-10 on the viability of Caco-2 cells MG-10 was synthesized by Shanghai Qiangyao Biotechnology Co., Ltd., with a purity ≥96.5%. MG-10 was weighed and dissolved in DMSO to prepare a 10 mmol / L stock solution, which was then sterilized by filtration through a 0.22 μm filter. Subsequently, the stock solution was diluted with basal medium to prepare incubation solutions with final concentrations of 0, 2.0, 4.0, 6.0, 8.0, and 10.0 μmol / L. When cell adhesion reached 60%–70%, the medium was removed, and the cells were washed with PBS. Different concentrations of MG-10 incubation solutions were added, and cells were harvested and viable at 24 h and 48 h of stimulation. Cell viability was assessed using the MTT assay: After culture, 10 μL of MTT solution (5 mg / mL) was added to each well, and incubation was performed for 4 h. Afterward, the supernatant was carefully removed, and 150 μL of DMSO was added to each well to dissolve the cells. After shaking on a shaker for 10 min, the absorbance (OD) values ​​at 490 nm and a reference wavelength of 630 nm were measured using a microplate reader. Relative cell viability (%) = (Experimental group A) 490 nm-A 630 nm) / (control group A) 490 nm-A 630 (nm) × 100%. Each group had 8 parallel replicates, and the experiment was independently repeated 3 times.

[0043] 1.7.3 Effects of MG-10 on the expression of NRF2 antioxidant pathway-related proteins in Caco-2 cells Caco-2 cells were grown at a concentration of 5.0 × 10⁻⁶. 5Cells were seeded at a density of cells / well in 6-well plates and cultured overnight at 37°C in a 5% CO2 incubator. When the cell confluence reached 70%-80%, the following treatments were administered for 24 h: control group (CON, solvent only), MG-10 (6.0 µmol / L), SFN (10 µmol / L, NRF2 positive agonist control), or MG-10 combined with ML385 (6.0 µmol / L MG-10 + 5 µmol / L ML385, specific NRF2 inhibitor).

[0044] Total protein was extracted using RIPA lysis buffer for the detection of Keap1 and HO-1. Cytoplasmic and nuclear protein fractions were separated using a nuclear protein extraction kit according to the manufacturer's instructions. The simplified procedure is as follows: Cells were collected, washed twice with pre-chilled PBS, and cytoplasmic extraction buffer was added. The cells were incubated on ice for 10 min. Subsequently, the cells were centrifuged at 12,000 × g for 5 min at 4 °C, and the supernatant was the cytoplasmic fraction. The precipitate was resuspended in nuclear extraction buffer and incubated on ice for 30 min, vortexing every 10 min. The cells were then centrifuged at 12,000 × g for 10 min at 4 °C, and the resulting supernatant was the nuclear fraction. Protein concentration was determined using a BCA protein quantification kit.

[0045] Equal amounts of protein were separated by SDS-PAGE, transferred to a PVDF membrane, and blocked with TBST containing 5% skim milk at room temperature for 2 h. The membrane was incubated with primary antibody overnight at 4°C, followed by incubation with IRDye 680RD goat anti-rabbit secondary antibody at room temperature in the dark for 2 h. Immunoreactive bands were visualized using an Odyssey infrared imaging system and quantified using ImageJ software. GAPDH was used as an internal control for Keap1, HO-1, and cytoplasmic NRF2, while Lamin B1 was used as an internal control for nuclear NRF2. Each experiment was performed in triplicate.

[0046] 1.7.4 Dual-luciferase reporter gene assay To assess the effect of MG-10 on NRF2 transcriptional activity, a dual-luciferase reporter gene system containing antioxidant response elements (AREs) was used. Caco-2 cells were cultured at 5.0 × 10⁶ cells / year. 4Cells were seeded at a density of 1 cell per well in 24-well plates and cultured until confluence reached 70%–75% before transfection. The reporter plasmid pARE-Luc (D2112, Beyotime, Shanghai) and the internal control plasmid pRL-TK (D2760, Beyotime) were co-transfected into cells at a mass ratio of 9:1. The total plasmid amount per well was 800 ng (720 ng pARE-Luc + 80 ng pRL-TK). Lipofectamine™ 3000 (L3000008, Thermo Fisher Scientific, Waltham, MA, USA) was used for transfection. For transfection, 1.5 μL of Lipofectamine™ 3000 and plasmid were diluted separately with 25 μL of Opti-MEM (31985070, Gibco, Grand Island, NY, USA) in each well. After mixing, the mixture was allowed to stand at room temperature for 15 min. Then, 50 μL of the transfection complex was added dropwise to each well (containing 1 mL of antibiotic-free complete medium). Six hours after transfection, the medium was replaced with fresh medium containing the appropriate treatment drug, and the culture was continued for 24 h. The experiment consisted of four groups: control group (CON), MG-10 treatment group (final concentration 6.0 μmol / L), positive control group (sulforaphane, SFN, final concentration 10 μmol / L), and antagonist group (MG-10 + ML385, final concentration 5 μmol / L).

[0047] After treatment, cells were washed twice with pre-cooled PBS, and 100 μL of 1× passive lysis buffer (RG029, Beyotime) was added to each well. Lysis was performed by gentle shaking at room temperature for 20–30 min. The lysis buffer was collected and centrifuged at 12,000 r / min for 3 min at 4 ℃. 30 μL of the supernatant was taken and subjected to luminescence detection according to the instructions of the Dual-Luciferase Reporter Gene Detection Kit (RG029, Beyotime), using blank lysis buffer as a background control. NRF2 transcriptional activity was expressed as the ratio of firefly luciferase activity to Renilla luciferase activity (Fluc / Rluc) after background subtraction. The experiment was independently repeated three times.

[0048] 1.7.5 In vitro pull-down experiment Recombinant full-length human Keap1 protein (unlabeled, expressed in Sf9 insect cells) was purchased from MedChemExpress (HY-P73312; stock solution concentration 50 μg / mL, dissolved in 50 mmol / L Tris-HCl pH 8.0, 500 mmol / L NaCl, 20% glycerol). Biotin-labeled MG-10 (Bio-Ahx-MG-10, sequence: Biotin-(Ahx)-M(O)LAVHDGFEG) ​​and biotin-labeled scrambled peptide (Bio-Scramble, sequence: Biotin-(Ahx)-GELAVMDGFH) were synthesized using a solid-phase peptide synthesis method. A biotinylated protein pull-down kit (magnetic bead method) was purchased from Beyotime Biotechnology.

[0049] Each reaction system had a total volume of 300 μL, containing: 40 μL Keap1 protein (2 μg per tube), 1.5 μL 1 mmol / L biotinylated peptide working solution (final concentration 5 μmol / L), 3 μL protease inhibitor mixture (100×), and 3 μL DTT (100 mmol / L), which was then brought to a final volume of 300 μL with binding buffer. Four groups were set up for the experiment: (1) blank control (without biotinylated peptide); (2) Bio-MG-10 group; (3) competition group (Bio-MG-10 with 50–100 times molar excess of unlabeled MG-10); and (4) Bio-Scramble control group. Each mixture was incubated at 4°C for 1.5–2 h to allow the peptide to fully bind to Keap1. After incubation, 20 μL of the mixture was taken from each tube as the input sample, 5 μL of 5×SDS loading buffer was added, and the mixture was boiled for 5 min before use.

[0050] Streptavidin magnetic beads (50 μL per tube) were washed twice with binding buffer, then blocked with 250 μL of biotin blocking buffer at room temperature for 5–10 min by rotation, followed by washing three times with binding buffer. The remaining 280 μL of each reaction system was transferred to the pretreated magnetic beads and incubated at 4 °C for 45–60 min by rotation to capture the biotinylated peptide-protein complex. After magnetic separation, the supernatant was discarded, and the magnetic beads were washed three times with 500 μL of pre-cooled washing buffer. 50 μL of 1×SDS loading buffer was added to the magnetic bead precipitate, and the mixture was boiled for 10 min. After magnetic separation, the supernatant was collected as the output sample. The input and output samples were subjected to SDS-PAGE and Western blot in parallel, and the enrichment of Keap1 was compared among the groups to assess the binding specificity of MG-10 to Keap1.

[0051] 1.7.6 Immunofluorescence detection of MG-10-induced NRF2 nuclear translocation Caco-2 cells were cultured on glass coverslips in 6-well plates and treated with 6.0 μmol / L MG-10 for 24 h. After the treatment, the supernatant was discarded, and the cells were washed twice with PBS. The cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 10% goat serum at 25°C for 1 h. Then, the anti-NRF2 primary antibody was added and incubated at 4°C for 12 h, followed by three washes with PBS. Thereafter, Cy3-labeled anti-IgG secondary antibody (catalog number not provided) was added and incubated at 25°C for 1 h in the dark. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; G1012, Wuhan Servicebio, Wuhan) at 25°C for 2 min in the dark, and then fluorescence images were captured. Fluorescence was observed using a DM4000B inverted fluorescence microscope, and the fluorescence intensity of Cy3 (NRF2) in each group was compared. The mean fluorescence intensity was calculated by dividing the total fluorescence intensity of the region of interest by the area. The experiment was independently repeated three times.

[0052] 1.8 Animal Experiment 2.8.1 Experimental Animals Specific Pathogen Free (SPF) male C57BL / 6 mice (No. 41098125100723003, aged 6-8 weeks, weighing 18-22 g) were purchased from Henan Huaxing Laboratory Animal Center (Animal Production License No. SCXK (Yu) 2019-0002). The feeding and use scheme of experimental animals in this study was approved by the Animal Care and Use Ethics Committee of Huanghuai University (Approval No. HHU-ACUREC / 2025-1008-1). Feeding conditions: temperature 22±2 °C, relative humidity 50%±10%, 12 h light / 12 h dark cycle, free access to food and water.

[0053] 1.8.2 Grouping of Experimental Animals After adaptive feeding for 7 days, the mice were completely randomly divided into 6 groups with 10 mice in each group, which were respectively: Normal control group (NC) DSS model group (DSS) MG-10 medium dose alone administration group (MG-10-M, 100 mg / kg / d) DSS + MG-10 low dose group (DSS+MG-10-L, 50 mg / kg / d) DSS + MG-10 medium dose group (DSS+MG-10-M, 100 mg / kg / d) DSS + MG-10 high dose group (DSS+MG-10-H, 100 mg / kg / d) 1.8.3 Preparation of MG-10 and Pharmaceutical Formulation Preparation of MG-10 gastric irrigation solution Weigh out MG-10 peptide (purity ≥96.5%) and dissolve it in sterile physiological saline or deionized water. Add 0.5% sodium carboxymethyl cellulose (CMC-Na) as a suspending agent and sonicate until completely dispersed. Prepare low-dose (5 mg / mL), medium-dose (20 mg / mL), and high-dose (40 mg / mL) MG-10 gavage stock solutions, respectively, and store them at 4°C. Bring them to room temperature before use and prepare fresh each time.

[0054] Preparation of DSS solution Weigh DSS powder (MW 36,000–50,000) and dissolve it in sterile ultrapure water to prepare a 3% DSS solution by weight-to-volume ratio (w / v). Stir magnetically at room temperature until completely dissolved and the solution is clear and transparent, then filter through a 0.22 μm filter membrane for sterilization. The DSS solution should be prepared fresh and completely replaced every 2 days.

[0055] 1.8.4 Establishment of a DSS-induced acute colitis model Following previous literature with slight modifications, an acute ulcerative colitis model was established by allowing mice free access to a 3% DSS solution. Except for the normal control group, mice in all other groups had free access to a sterile 3% DSS solution for 7 consecutive days; the normal control group had free access to sterile water throughout the process. During the modeling period, the mice's general condition, weight changes, fecal characteristics, and any instances of bloody stools were observed and recorded daily. The DSS solution was completely changed every 2 days.

[0056] 1.8.5 Drug administration intervention regimen After 7 days of acclimatization feeding, mice were randomly divided into 6 groups of 10 mice each using a completely randomized design: Normal control group (NC): Administered an equal volume of 0.5% CMC-Na solution (0.1 mL / 10 g body weight) by gavage daily, and had free access to distilled water; The DSS model group (DSS) was given an equal volume of 0.5% CMC-Na solution (0.1 mL / 10 g body weight) by gavage daily, and had free access to 3% DSS solution in drinking water. MG-10 medium-dose monotherapy group (MG-10-M): MG-10 100 mg / kg body weight was administered by gavage daily, with free access to distilled water; DSS+MG-10 low-dose group (DSS+MG-10-L): MG-10 50 mg / kg body weight was administered by gavage daily, and 3% DSS solution was allowed to be drunk freely; DSS+MG-10 medium-dose group (DSS+MG-10-M): MG-10 100 mg / kg body weight was administered by gavage daily, and 3% DSS solution was allowed to be drunk freely; The high-dose group of DSS+MG-10 (DSS+MG-10-H): MG-10 200 mg / kg body weight was administered by gavage daily, and 3% DSS solution was allowed to be drunk freely.

[0057] 1.8.6 Sample Collection and Indicator Detection On day 14 of the experiment (the day after day 7 of DSS modeling), all mice were fasted but allowed free access to water overnight (12 h). The following day, they were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg / kg). Blood was collected from the orbital venous plexus, and serum was separated by centrifugation and stored at −80℃ for later use. The entire colon was dissected, its length measured, and then longitudinally dissected. The colon was washed with PBS, flash-frozen in liquid nitrogen, and stored at −80℃. The frozen tissue was weighed, and a 10% homogenate was prepared by adding pre-cooled physiological saline at a ratio of 1:9 (w / v). The supernatant was collected by centrifugation, and the protein concentration was determined by the BCA method for subsequent detection. Serum was also collected.

[0058] (1) Disease Activity Index (DAI) assessment Following the methods described in previous literature, mice were weighed daily at a fixed time (9:00–10:00 AM), and fecal characteristics and bloody stools were observed and recorded. The Disease Activity Index (DAI) was calculated. The scoring criteria are shown in Table 1 below: DAI total score = weight loss score + stool characteristics score + rectal bleeding score (0-12 points).

[0059] (2) Detection of serum inflammatory factors and oxidative stress indicators The supernatant of the serum sample stored at −80℃ was used for testing.

[0060] Inflammatory factor detection: The levels of inflammatory factors in serum and colon tissue homogenate supernatant were detected using enzyme-linked immunosorbent assay (ELISA). The detected indicators included tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-10 (IL-10). All procedures were strictly performed according to the ELISA kit instructions, with three replicates per sample. Serum results are expressed as pg / mL, and colon tissue results are expressed as pg / mg protein.

[0061] Oxidative stress marker detection: The supernatant of colon tissue homogenate was used to detect oxidative stress markers using appropriate kits. Superoxide dismutase (SOD) activity was determined using the xanthine oxidase method, catalase (CAT) activity using the ammonium molybdate method, glutathione peroxidase (GSH-Px) activity using the dithiodinitrobenzoic acid method, and malondialdehyde (MDA) content using the thiobarbituric acid method. All procedures were strictly performed according to the kit instructions, with three replicates per sample. SOD, CAT, and GSH-Px activity results are expressed as U / mg protein, and MDA content results are expressed as nmol / mg protein.

[0062] (3) Western blot analysis of colon tissue Immediately after euthanasia, the abdominal cavity was opened, and the colon (from the distal cecum to the anus) was completely dissected. The mesentery and surrounding adipose tissue were removed, and the colon was laid flat on ice-bath graph paper to measure its length and photograph it. After measurement, the colon was longitudinally dissected and gently rinsed with pre-cooled PBS (pH 7.4) to remove intestinal contents. A 0.5 cm segment of the distal colon was fixed in 4% paraformaldehyde for histopathological analysis. The remaining colon tissue was rapidly aliquoted, flash-frozen in liquid nitrogen, and then transferred to −80°C for storage for subsequent assays.

[0063] Take approximately 30–50 mg of colon tissue stored at −80℃, place it in a pre-chilled mortar, add liquid nitrogen and grind thoroughly into powder. Transfer to a pre-chilled EP tube, add RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors at a mass-to-volume ratio of 1:10 (w / v), and lyse on ice for 30 min, vortexing once every 10 min. After lysis, centrifuge at 12,000 rpm for 15 min at 4℃, collect the supernatant, and determine the protein concentration using a BCA protein quantification kit. Add an appropriate amount of loading buffer, denature in a metal bath at 100℃ for 10 min, aliquot, and store at −80℃ for later use in Western blot detection of NRF2, KEAP1, HO-1, and internal control protein expression levels.

[0064] 1.9 Statistical Analysis Experimental results are expressed as mean ± standard deviation (SD). One-way ANOVA was used to compare means among multiple groups, followed by Duncan's multiple range test for pairwise comparisons. Statistical analysis was performed using SPSS 25.0 software, with p < 0.05 considered statistically significant.

[0065] 2. Results and Analysis 2.1 In vitro digestion data Depend on Figure 3As shown in B, the protein digestibility of chicken breast in the fermented group was significantly higher than that in the control group at three time points: 0 h (gastric juice), 2 h (gastric juice), and 2 h (intestinal juice) (P < 0.05). Looking at the intra-group changes over time, the increase in digestibility in the control group mainly occurred during intestinal digestion, significantly increasing from 10.15% at 2 h (gastric juice) to 52.64% at 2 h (intestinal juice) (P < 0.05). In contrast, the digestibility in the fermented group showed a significant increase during the gastric digestion stage, rising from 23.01% at 0 h (gastric juice) to 70.13% at 2 h (gastric juice) (P < 0.05), but there were no significant changes in digestibility at 2 h (intestinal juice) or 2 h (gastric juice) (P > 0.05).

[0066] 2.2 SDS-PAGE SDS-PAGE protein electrophoresis patterns of the control group, unfermented chicken, and fermented chicken at different digestion time points are shown below. Figure 3 As shown in Figure C. No obvious protein bands were observed in the control group at time points C-G2 and C-G2+I2. In the unfermented group, at time point U-G0, protein bands were distributed within the molecular weight range of 8-195 kDa, showing deep staining and wide distribution; at time point U-G2, band staining was significantly enhanced in the 8-13 kDa range; at time point U-G2+I2, the band intensity in all molecular weight ranges decreased significantly, with only one clear band visible at approximately 20 kDa, and weak staining at other locations. In the fermented group, at time point F-G0, almost no discernible bands were observed in the 8-195 kDa range; at time point F-G2, significant staining appeared in the region below 8 kDa, indicating the release of small peptides / amino acids; at time point F-G2+I2, multiple weak bands appeared in the region below 30 kDa, with a clear band visible at approximately 20 kDa, and this band's position was consistent with the clear band in the unfermented group at time point U-G2+I2.

[0067] 2.3 Antioxidant Experiment of Digestive Juice Table 2 Evaluation of in vitro antioxidant activity in digestive fluids at different time points The results of the antioxidant activity assessment of the digestive fluid at different time points are shown in Table 2. The results showed that at the three time points G0, G2, and G2+I2, the PTIO scavenging rate, ABTS scavenging rate, DPPH scavenging rate, and FRAP value of the fermented group (F) chicken jerky digestive fluid were significantly higher than those of the corresponding unfermented group (U) (P < 0.05). From the intra-group time dynamics, in the unfermented group, compared with U-G0, the four indicators of U-G2 showed no significant changes (P > 0.05), while the PTIO scavenging rate and FRAP value of U-G2+I2 were significantly increased (P < 0.05). Within the fermented group, compared with F-G0, the four indicators of F-G2 were significantly increased (P < 0.05); after further addition of I2 (F-G2+I2), its FRAP value was significantly higher than that of F-G2 (P < 0.05), while the other three indicators (PTIO, ABTS, and DPPH scavenging rates) showed no significant difference compared with F-G2 (P > 0.05).

[0068] 2.4 Peptideomics Peptidomics analysis was performed on samples from 0 h after gastric digestion (U-G0, F-G0) and samples from the end of gastrointestinal digestion (U-GI2, F-GI2) using liquid chromatography-tandem mass spectrometry. The results are shown in [Figure number missing]. Figure 4 At the protein level, the U-G0 group identified 1,668 proteins, while the F-G0 group identified 1,422 proteins during the same period. After continuous digestion in the stomach for 2 hours and then in the intestine for 2 hours, the number of proteins identified in the two groups decreased to 889 (U-GI2 group) and 824 (F-GI2 group), respectively. Figure 4 A), and more than 80% of the identified protein molecular weights are distributed in the 1-100 kDa range ( Figure 4 B). At the peptide level, the U-G0 group identified 8,627 peptides, while the F-G0 group identified 6,695 at the same time; after gastrointestinal digestion, the number decreased to 4,172 (U-GI2 group) and 4,256 (F-GI2 group), respectively. Figure 4 C). From the perspective of peptide molecular weight distribution, the molecular weights of the peptides identified in all samples were mainly concentrated below 3.0 kDa. U-G0h had the highest number of peptides in the 1.5-3.0 kDa range (5,319 peptides), and this range remained dominant after gastrointestinal digestion (U-GI2h) (3,467 peptides). F-G0h had the highest number of peptides in the 0-1.5 kDa range (3,179 peptides), higher than the 2,976 peptides in the same range of U-G0h, and this range remained dominant after gastrointestinal digestion (F-GI2h) (2,846 peptides), also higher than the 2,540 peptides in the same range of U-GI2h. Conversely, in the 1.5-3.0 kDa range, the number of peptides in the fermented group was lower than that in the unfermented group at each digestion stage (…). Figure 4 D). Venn diagram analysis results show that at the protein level ( Figure 4E), a total of 2,122 proteins were identified across the four groups (U-G0h, F-G0h, U-GI2h, and F-GI2h), of which 613 proteins were common to all four groups; at the polypeptide level, a total of 16,100 peptides were identified across the four groups, of which 635 peptides were common to all four groups. Figure 4 F).

[0069] 2.5 Molecular docking and dynamic simulation Molecular docking screening was conducted to evaluate the binding potential of 1750 specific peptides to the Keap1 protein. Figure 5 A shows the top ten conformations ranked by docking scores. Based on the scores, the three highest-scoring peptides, M(ox)LAVHDGFEG(MG-10), NGPGPWGFR, and FVSDHF, were selected for subsequent molecular dynamics simulations. The results are shown in [Figure 1]. Figure 5 Molecular docking results showed that MG-10 forms hydrogen bonds with SER431 and ASN382 residues of the Keap1 protein, and also forms multiple hydrogen bonds and salt bridge interactions with ARG415 and ARG380 residues. Figure 5 B). FVSDHF forms multiple hydrogen bonds with ARG415, ARG483, ARG380, ASN382, and SER363 residues of the Keap1 protein. Figure 5 C). NGPGPWGFR forms multiple hydrogen bonds with ARG380, HIS436, SER602, and GLN530 residues of the Keap1 protein ( Figure 5 D). The binding free energy and energy components calculated by the MM / GBSA method are as follows: Figure 5 As shown in Figure E. Combined with free energy analysis, the absolute value of MG-10 (ΔG_bind = -56.41 kcal / mol) was significantly higher than that of NGPGPWGFR and FVSDHF (P<0.05). Energy decomposition results showed that MG-10 had the largest absolute values ​​of electrostatic interaction energy (ΔE_elec = -229.65 kcal / mol) and desolvation free energy (ΔG_GB = +232.83 kcal / mol); MG-10 and FVSDHF showed no significant difference in the above two energy components (P>0.05), but both were significantly higher than NGPGPWGFR (P<0.05). The 100 ns molecular dynamics simulation results are as follows... Figure 5 As shown in F–K. RMSD analysis ( Figure 5 (F–G) analysis showed that all three peptides and their complexes reached equilibrium after approximately 10 ns, with the FVSDHF ligand and complex exhibiting the smallest fluctuation range, followed by MG-10, and NGPGPWGFR showing the largest fluctuation. RMSF analysis ( Figure 5H) showed that the NGPGPWGFR complex exhibited significant flexibility (peak >0.2 nm) at residues 20–40, while the MG-10 and FVSDHF complexes showed greater overall rigidity. Rg( Figure 5 I) and SASA ( Figure 5 K) analysis showed that all three maintained a compact folded conformation (Rg: 1.76–1.82 nm; SASA: 110–130 nm²). Hydrogen bond analysis ( Figure 5 J) indicates that MG-10 has the highest average number of hydrogen bonds with Keap1. In summary, MG-10 exhibits the strongest binding affinity to Keap1 protein (ΔG_bind = -56.41 kcal / mol), and the complex demonstrates conformational stability and a robust hydrogen bond network in a 100 ns simulation. Therefore, MG-10 was chosen for subsequent biological validation.

[0070] 2.6 ADMET and TOPKAT Predictions The ADMET and TOPKAT prediction results for the three peptides are shown in Table 3. Regarding absorption and physicochemical properties, MG-10's intestinal absorption grade is 3, indicating poor oral absorption, consistent with its high hydrophilicity (AlogP98 = −7.197) and large polar surface area (PSA-2D = 416.07 Ų). Its blood-brain barrier permeability grade is 4, suggesting that MG-10 does not easily cross the blood-brain barrier, reflecting its low risk of exposure to the central nervous system. Its water solubility grade is 5, indicating low water solubility, which may require formulation technology to improve its solubility in practical applications. Regarding metabolic and toxicity risks, the prediction results show that MG-10 has no risk of CYP2D6 inhibition, no risk of hepatotoxicity, and the Ames test result is negative, suggesting it is not mutagenic, preliminarily indicating that MG-10 has good in vitro safety characteristics. In summary, although MG-10 has poor oral absorption, it has low safety risks and has good development potential as a candidate molecule for functional food ingredients, topical skin care active ingredients, or local intestinal administration.

[0071] Table 3. Prediction results of the top three peptides ADMET and TOPKAT Absorption and physicochemical properties Intestinal absorption grade (grades 1–4, 3–4 = poor absorption) 3 3 3 Blood-brain barrier permeability levels (1–4, 4 = lowest) 1 4 4 Water solubility rating (0–6, 6 = lowest) 5 5 5 AlogP98 (lipid-soluble, the larger the negative value, the stronger the hydrophilicity) −5.020 −7.197 −4.879 PSA-2D (Ų, >140 indicates poor permeability) 299.14 416.07 396.31 Metabolic and toxic risks CYP2D6 inhibits risk (FALSE = low risk) FALSE FALSE FALSE Hepatotoxicity risk (FALSE = low risk) FALSE FALSE FALSE Mutagenicity (Ames test) Negative Negative Negative Predicted reliability All predictions are within the applicable domain. Yes Yes Yes Note: ADMET: Absorption, Distribution, Metabolism, Excretion, and Toxicity; TOPKAT: Toxicity Prediction by Komputer Assisted Technology; BBB: Blood-brain barrier; AlogP98: Logarithmic value of the oil-water partition coefficient calculated using the AlogP98 method; PSA-2D: Two-dimensional polar surface area; CYP2D6: Cytochrome P450 2D6 enzyme; Applicability domain: The chemical space defined by the training set of the prediction model, within which predictions are considered reliable. All predictions were performed using Discovery Studio software.

[0072] 2.7 Origin of MG-10 polypeptide precursor protein This study used raw mass spectrometry files in conjunction with the chicken UniProt database for comparative retrieval to trace the precursor protein of the MG-10 polypeptide. Table 4 shows that the four accession numbers (Q90YA3, A0A8V1A2B8, A0A8V1A2C5, and A0A8V1A0E3) all belong to the same proteome (Group 57), corresponding to the PFKM (phosphofructokinase, muscle) gene, encoding ATP-dependent 6-phosphofructokinase (EC2.7.1.11). Sequence alignment revealed that MG-10 (sequence: M(ox)LAVHDGFEG) ​​corresponds to amino acid positions 466–475 of the PFKM sequence, and its unoxidized complete sequence is “MLAVHDGFEG”. Among them, A0A8V1A2C5 was marked as the "Top (TRUE)" representative sequence due to its highest -10LgP score (287.90), total number of peptides (186), number of unique peptides (172), and largest molecular weight (88,315.82 Da). PFKM, with a full length of 814 amino acids, is a key rate-limiting enzyme in the glycolysis pathway and is highly expressed in chicken skeletal muscle. Given that MG-10 was only identified in the F-GI2 group, while the A0A8V1A2C5 protein isoform was present in all four treatment groups, it is suggested that the PFKM protein underwent oxidative modification during the fermentation stage of the compound fermentation agent, oxidizing methionine in the sequence to methionine sulfoxide, which was retained in MG-10. Quantitative analysis showed that the coverage, peak area, and spectral count of A0A8V1A2C5 remained consistent across the four treatment groups: at 0 h (U-G0h, F-G0h), the peak area was significantly higher than after 2 h of gastrointestinal digestion (U-GI2h, F-GI2h), indicating that gastrointestinal digestion had a significant downregulatory effect on this protein. The peak area at 0 h in the non-fermentation group was 6.36 × 10⁻⁶. 5 Both the number of samples and the number of spectra (164) were higher than those in the fermentation group (3.29 × 10⁻⁶). 5The results (112) suggest that fermentation pretreatment led to partial protein degradation or conformational changes. However, after continuous gastrointestinal digestion, there was no significant difference between the fermented and non-fermented groups, indicating that gastrointestinal digestion was the dominant factor in protein degradation. Based on the above results, the release pathway of MG-10 can be inferred as follows: During the fermentation stage, A0A8V1A2C5 is initially hydrolyzed into a large polypeptide library by microbial proteases, while the methionine 466 site is oxidized; subsequently, it enters the in vitro gastrointestinal digestion stage, where it is further degraded under the synergistic action of pepsin and trypsin. Combining the preferential cleavage sites of pepsin and trypsin, it can be inferred that: pepsin preferentially cleaves the C-terminus of leucine (L) at position 476, releasing the C-terminus of MG-10; trypsin preferentially cleaves the C-terminus of arginine (R) at position 465, releasing the N-terminus of MG-10. Through the synergistic action of microbial oxidation and digestive enzymes, the bioactive peptide (M(ox)LAVHDGFEG) ​​MG-10 modified with methionine sulfoxide is released.

[0073] Table 4: Source tracing results of MG-10 polypeptide precursor protein Note: Different capital letters (A, B, C, D) in the same column indicate significant differences (P<0.05), while the same letter indicates no significant differences (P>0.05). U-G0h: Gastric digestion of the unfermented group for 0 h; U-GI2h: Gastrointestinal digestion of the unfermented group completed; F-G0h: Gastric digestion of the fermented group for 0 h; F-GI2h: Gastric digestion of the fermented group for 2 h + intestinal digestion of 2 h.

[0074] 2.8 MG-10 activates the NRF2 antioxidant pathway in Caco-2 cells The relative cell viability of Caco-2 cells after co-incubation with different concentrations of MG-10 for 24 h and 48 h are as follows: Figure 6 A and Figure 6 As shown in Figure B, MG-10 concentrations of 0–10 μmol / L had no significant effect on cell viability; however, when the concentration increased to 10 μmol / L, cell viability decreased to 32.14%, significantly lower than other concentration groups (P<0.05). Under 48 h co-incubation conditions, the cell viability of the 6.0 μmol / L MG-10 treatment group was 83.58%, significantly lower than the control group (P<0.05). Based on these results, subsequent mechanism verification experiments all used 6.0 μmol / L MG-10 treatment for 24 h as the optimal treatment condition.

[0075] Western blot analysis results ( Figure 6E–H results showed that, compared with the control group, MG-10 treatment significantly reduced cytoplasmic NRF2 protein levels while increasing nuclear NRF2 protein levels and simultaneously upregulating HO-1 expression (P<0.05). These changes were similar to those induced by the NRF2 agonist SFN and could be effectively reversed by combined treatment with the specific NRF2 inhibitor ML-385. In summary, these results indicate that MG-10 can promote the translocation of NRF2 from the cytoplasm to the nucleus, thereby activating the NRF2-mediated antioxidant signaling pathway.

[0076] 2.9 MG-10 competitively binds to Keap1 and facilitates NRF2 nuclear translocation The results of the dual-luciferase reporter gene assay showed that ( Figure 7 A) The relative Fluc / Rluc ratios of the MG-10 and SFN groups were significantly higher than those of the CON group (P < 0.05), indicating that both MG-10 and the positive control SFN could enhance the transcriptional activity of downstream target genes of NRF2. However, the Fluc / Rluc ratio of the MG-10 + ML385 group decreased to 0.76 compared with the MG-10 alone group (P < 0.05), suggesting that ML385 can effectively antagonize the transcriptional activation induced by MG-10. Pull-down experiment ( Figure 7 B) shows that Bio-MG-10 can pull down Keap1 protein (lane 2), while the signal is significantly weakened after adding excess unlabeled MG-10 (lane 3); no Keap1 enrichment was detected in the blank control (lane 1) and Bio-Scramble control (lane 4), thus confirming the specific binding between MG-10 and Keap1. Immunofluorescence confocal microscopy observation ( Figure 7 C) shows that in the CON group, the green fluorescence signal of NRF2 was mainly diffusely distributed in the cytoplasm; while in the MG-10 and SFN treatment groups, NRF2 was significantly accumulated in the nucleus, and its co-localization with DAPI-stained nuclei was significantly enhanced. However, in the MG-10 + ML385 group, the nuclear accumulation of NRF2 was weakened, and its fluorescence distribution pattern was similar to that of the CON group.

[0077] 2.10 Animal Experiments: MG-10 Alleviates DSS-Induced Colitis in Mice 2.10.1 Effects of MG-10 on Disease Activity Index (DAI) and Body Weight in DSS-Induced Colitis Mice Table 5 shows the changes in body weight and DAI scores of mice in each group during the experiment. Before modeling, there was no significant difference in body weight among the groups (P > 0.05). From day 3 of DSS modeling, the body weight of mice in the model group continuously decreased, reaching a decrease of 13.25% by day 7, with a DAI score of 9.75. This was significantly different from the normal control group (body weight decrease of 1.23%, DAI score of 0.25) (P < 0.05), indicating successful establishment of the colitis model. Compared with the DSS model group, mice in the medium- and high-dose MG-10 groups showed significantly lower DAI scores from day 5 of modeling (P < 0.05). By day 7, their body weight decreased by 6.82% and 5.36%, respectively, and their DAI scores were 4.50 and 4.25, respectively, both significantly lower than the model group (P < 0.05). There was no statistically significant difference between the medium- and high-dose groups (P > 0.05).

[0078] Table 5. Percentage of body weight loss and DAI score (Mean±SD, n=10) of mice in each group on day 7. NC 1.23±0.54 a 0.25±0.45 a DSS 13.25±2.18 c 9.75±1.28 c MG-10-M 1.18±0.32 a 0.63±0.15 a DSS+MG-10-L 10.41±2.05 c 7.13±1.36 bc DSS+MG-10-M 6.82±1.94 b 4.50±1.27 b DSS+MG-10-H 5.36±1.53 b 4.25±1.04 b Note: Different lowercase letters in the same column indicate statistically significant differences between groups (P < 0.05), while the same letter indicates no statistically significant difference (P > 0.05). The same applies below.

[0079] 2.10.2 Effect of MG-10 on colon length in DSS-induced colitis mice Colon length is an important macroscopic indicator for assessing the severity of colitis. Table 6 shows that compared with the normal control group (8.42 cm), the colon length of mice in the DSS model group was significantly shortened to 4.86 cm (P < 0.05). The colon length in the low-dose MG-10 group was 5.43 cm, with no statistically significant difference compared to the model group (P > 0.05). The colon length in the medium-dose MG-10 group was 7.58 cm, and in the high-dose group it was 7.69 cm, both significantly longer than the model group (P < 0.05), with no statistically significant difference between the medium- and high-dose groups (P > 0.05). Histopathological scores were consistent with the trend in colon length changes. The score in the normal control group was 0.38, while it increased to 7.88 in the DSS model group (P < 0.05). The low-dose MG-10 group scored 6.25 points, which was not significantly different from the model group (P>0.05). The medium- and high-dose MG-10 groups scored 4.38 points and 5.13 points, respectively, both significantly lower than the model group (P<0.05), and there was no significant difference between the medium- and high-dose groups (P>0.05). The medium-dose monotherapy group (0.25 points) did not differ significantly from the normal control group (P>0.05).

[0080] Table 6. Colon length and histopathological scores of mice in each group (Mean±SD, n=10) NC 8.42±0.53 a 0.38±0.52 a DSS 4.86±0.41b 7.88±1.13 c MG-10-M 8.18±0.49a 0.25±0.09 a DSS+MG-10-L 5.43±0.47 b 6.25±1.04 c DSS+MG-10-M 7.58±0.52 a 4.38±0.92 b DSS+MG-10-H 7.69±0.44 a 5.13±0.83 b 2.10.3 Effects of MG-10 on serum oxidative stress markers and inflammatory factors in DSS-induced colitis mice Table 7 shows that, compared with the normal control group, the serum SOD, CAT, and GSH-Px activities of mice in the DSS model group were significantly decreased (P < 0.05), while the MDA content was significantly increased (P < 0.05), indicating that DSS induced a systemic oxidative stress state. There were no significant differences in any indicators between the MG-10 medium-dose monotherapy group and the normal control group (P > 0.05), indicating that MG-10 itself has no adverse effect on the body's redox balance. Compared with the DSS model group, the SOD, CAT, and GSH-Px activities of the DSS+MG-10-M group were significantly increased (P < 0.05), while the MDA content was significantly decreased (P < 0.05), indicating that medium-dose MG-10 can effectively improve the serum oxidative stress level in colitis mice.

[0081] Table 7 Serum oxidative stress indices of mice in each group (Mean±SD, n=10) NC 52.37±4.86 a 21.54±1.03 a 38.46±3.82 a 1.87±0.32 c DSS 22.36±3.15 c 8.42±1.27 c 15.73±2.41 c 6.84±0.76 a MG-10-M 44.83±4.37 ab 17.94±1.85 b 32.75±3.52 ab 2.58±0.41 bc DSS+MG-10-M 38.74±4.26 b 15.23±1.82 b 28.64±3.37 b 3.87±0.52 b Table 8 shows that the serum levels of pro-inflammatory factors TNF-α, IL-1β, and IL-6 in the DSS model group were significantly higher than those in the normal control group (P < 0.05), while the level of anti-inflammatory factor IL-10 was significantly lower (P < 0.05). There were no significant differences in any indicators between the MG-10 medium-dose monotherapy group and the normal control group (P > 0.05). Compared with the DSS model group, the DSS+MG-10-M group showed significantly decreased levels of TNF-α, IL-1β, and IL-6 (P < 0.05), significantly increased IL-10 levels (P < 0.05), and IL-10 levels recovered to a level not significantly different from the normal control group (P > 0.05). These results indicate that medium-dose MG-10 can significantly inhibit DSS-induced systemic inflammatory response and upregulate anti-inflammatory factor levels.

[0082] Table 8. Serum inflammatory factor levels in mice of each group (Mean±SD, n=10) 2.10.4 Effects of MG-10 on NRF2-ARE pathway protein expression in colon tissue of DSS-induced colitis mice Western blot was used to detect the expression levels of NRF2, KEAP1, and the downstream antioxidant protein HO-1 in colon tissue. The results are as follows: Figure 8 As shown in the figure, compared with the normal control group, the expression of KEAP1 protein, NRF2 and its downstream HO-1 protein was significantly reduced in the DSS model group (P < 0.05). Compared with the DSS model group, the DSS+MG-10-M group significantly reversed the abnormal regulation of NRF2-ARE pathway protein expression by DSS.

Claims

1. An antioxidant polypeptide targeting KEAP1 protein and modified with methionine sulfoxide, characterized in that: The polypeptide is MG-10, and its amino acid sequence is shown in SEQ ID NO.1: M(ox)LAVHDGFEG.

2. The antioxidant polypeptide targeting KEAP1 protein modified with methionine sulfoxide as described in claim 1, characterized in that: The polypeptide MG-10 is derived from the amino acid region of PFKM protein (6-phosphofructokinase) in fermented chicken meat, from amino acid positions 466 to 475, through methionine oxidation modification during fermentation and subsequent synergistic enzymatic cleavage by pepsin and trypsin.

3. The antioxidant polypeptide targeting KEAP1 protein modified with methionine sulfoxide as described in claim 2, characterized in that: The fermentation process uses a compound microbial agent composed of Candida utilis, Bacillus subtilis, and Lactobacillus casei.

4. The use of the antioxidant peptide according to any one of claims 1-3 in the preparation of a medicament for treating DSS-induced ulcerative colitis.

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