Grape seed source anti-inflammatory peptide and application thereof

Abstract

The invention discloses a grape seed source anti-inflammatory peptide, and belongs to the technical field of biological medicine. The amino acid sequence of the anti-inflammatory peptide is selected from any one of WGF, SHFGF, EGPFF, WAPR, SFYRAF, HFAFL, PGRF or FDSF. A BPS-induced RAW 264.7 mouse mononuclear macrophage inflammation model is established, and the eight synthetic peptides are found to significantly reduce the content level of NO in inflammatory cells and have an inhibition effect on secretion of inflammatory factors TNF-alpha and IL-6. A qRT-PCR (quantitative reverse transcription-polymerase chain reaction) experiment shows that the synthetic peptide can inhibit expression of mRNA (messenger Ribonucleic Acid) of p65, Tnf alpha, Il6, Il1b and Nos2, and can play an anti-inflammatory role by regulating and controlling an NF-kappa B signal channel. According to the method, the high-activity anti-inflammatory peptide is screened from the wine brewing byproduct grape seeds for the first time, and green and high-value utilization of the grape processing byproduct is realized.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a grape seed-derived anti-inflammatory peptide and application thereof. Background Art

[0002] As a major global agricultural processing industry, the wine industry produces a significant amount of winemaking byproducts annually, primarily grape seeds, skins, and stems. Grape seeds account for approximately 38%-52% of the total dry weight of these byproducts. Grape seeds are rich in high-quality protein, with a crude protein content of up to 8.9%, making them a highly promising plant protein resource. Grape seed protein can be readily and efficiently extracted through biological extraction or synthetic synthesis to produce peptides with specific functions. Due to its small molecular weight, high bioactivity, and specificity, it can precisely target target cells or molecules, minimizing interference with other cells and tissues, making it a potential candidate for disease treatment.

[0003] However, the current development and utilization of grape seeds is still limited to primary feed or waste treatment, resulting in a waste of resources rich in bioactive ingredients. With the advancement of the "Healthy China 2030" strategy and the deepening of the "big food view" and "big health" concepts, people's attention to healthy diets has continued to increase, and the demand for safe and effective anti-inflammatory products has also increased. The development of natural products with anti-inflammatory effects has become a research hotspot in the intersection of food and medicine. Based on this, this study used grape seeds from winemaking by-products as raw materials to extract and sequence protein peptides, and screened out peptides with anti-inflammatory effects through bioinformatics methods such as database prediction, network pharmacology and molecular docking. This invention not only helps to develop new anti-inflammatory products, but also helps to expand the development path of plant protein resources and contribute to the implementation of the Healthy China strategy. Summary of the Invention

[0004] The purpose of the present invention is to provide a grape seed-derived anti-inflammatory peptide, aiming to solve the problem of insufficient quantity of existing anti-inflammatory peptides and realize the green and high-value utilization of grape processing by-products.

[0005] A grape seed-derived anti-inflammatory peptide, whose amino acid sequence is selected from any one of the following: WGF, SHFGF, EGPFF, WAPR, SFYRAF, HFAFL, PGRF or FDSF.

[0006] In the above sequence, W represents tryptophan; G represents glycine; F represents phenylalanine; S represents serine; H represents histidine; E represents glutamic acid; P represents proline; A represents alanine; R represents arginine; Y represents tyrosine; L represents leucine; and D represents aspartic acid.

[0007] The above eight anti-inflammatory peptides were isolated and identified from grape seeds for the first time, providing a new way to make full use of winemaking by-products and also providing new resources for the development of related drugs.

[0008] These anti-inflammatory peptides can be extracted and isolated from grape seeds, or produced using synthetic methods. When using extraction and separation methods, the target peptide can be directly isolated from the extracted grape seed protein peptides, including separation using chromatography methods such as preparative liquid chromatography. Given a clear understanding of the molecular structure of the target peptide, those skilled in the art do not need to overcome technical hurdles when using these extraction and separation methods. When produced using synthetic methods, the small peptides, due to their small molecular weight, offer advantages such as simple operation, short production cycles, and low costs.

[0009] RAW 264.7 cells, derived from Abelson murine leukemia virus-induced tumors in BALB / c mice, are a mouse mononuclear phagocyte leukemia cell line. As a cell line, these cells not only mimic the in vivo process of phagocytosis and clearance of foreign bodies but also respond to external stimuli by secreting ROS and inflammatory mediators (such as TNF-α and IL-6), making them a useful model for inflammatory responses. This study established a BPS (bisphenol S)-induced RAW 264.7 mouse mononuclear phagocyte inflammation model. Eight synthetic peptides were found to have no significant toxic effects on the cells at a concentration of 10.0 µg / mL. They significantly reduced NO levels in inflammatory cells and inhibited the secretion of inflammatory factors, TNF-α and IL-6. qRT-PCR experiments demonstrated that the synthetic peptides inhibited the expression of p65, Tnfα, Il6, Il1b, and Nos2 mRNAs, suggesting that their anti-inflammatory effects may be mediated by regulating the NF-κB signaling pathway.

[0010] The present invention further provides an anti-inflammatory drug comprising the anti-inflammatory peptide and a pharmaceutically acceptable carrier.

[0011] The present invention also provides a method for inhibiting macrophage inflammatory response for non-disease treatment purposes. The anti-inflammatory peptide is used to treat RAW 264.7 cells at a concentration of 10 μg / mL to inhibit BPS-induced cellular inflammatory response, including reducing the NO content level in the cells and inhibiting the secretion of inflammatory factors TNF-α and IL-6.

[0012] The beneficial effects of the present invention are: Inflammatory response is an important pathological mechanism of many diseases (such as arthritis and tumors), and is also a key factor in cosmetic irritation reactions and immune stress in farmed animals. Existing anti-inflammatory drugs (such as non-steroidal anti-inflammatory drugs and glucocorticoids) have problems such as large side effects and high costs.

[0013] This invention, for the first time, has screened highly active anti-inflammatory peptides from grape seeds, a byproduct of winemaking. These peptides have broad application prospects in medicine, functional foods and cosmetics, aquaculture, biomaterials, and other fields. For example, anti-inflammatory peptides can be added to livestock and aquatic feed to inhibit necrotizing enterocolitis caused by overactivation of intestinal macrophages and alleviate the inflammatory stress response of fish in high-density aquaculture. Coating anti-inflammatory peptides on the surface of artificial joints or scaffold materials can inhibit the fusion of foreign macrophages to form granulomas and prolong the life of the implant. Incorporating anti-inflammatory peptides into hydrogel scaffolds can regulate macrophage phenotype and promote angiogenesis. Using anti-inflammatory peptides as anti-inflammatory components in macrophage culture media can maintain immune cell homeostasis. In high-throughput drug screening, they can replace dexamethasone to establish an "inflammation-anti-inflammatory" control group.

[0014] The present invention realizes the green and high-value utilization of grape processing by-products, and the provided anti-inflammatory peptide has the advantages of small molecular weight, low production cost, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 : Molecular docking diagram of peptide PGRF and RELA (p65).

[0016] Figure 2 : Effects of eight synthetic peptides on the viability of RAW 264.7 cells.

[0017] Figure 3 : Effects of eight synthetic peptides on NO secretion in RAW 264.7 cells.

[0018] Figure 4 : Effects of eight synthetic peptides on IL-6 secretion by RAW 264.7 cells.

[0019] Figure 5 : Effects of eight synthetic peptides on TNF-α secretion in RAW 264.7 cells.

[0020] Figure 6 : Effects of synthetic peptides on BPS-induced inflammatory factor p65 mRNA expression in RAW 264.7 cells.

[0021] Figure 7 :Effects of synthetic peptides on BPS-induced inflammatory factor Tnfα mRNA expression in RAW 264.7 cells.

[0022] Figure 8 : Effects of synthetic peptides on BPS-induced inflammatory factor Il6 mRNA expression in RAW 264.7 cells.

[0023] Figure 9 : Effects of synthetic peptides on BPS-induced inflammatory factor Il1b mRNA expression in RAW 264.7 cells.

[0024] Figure 10 : Effects of synthetic peptides on BPS-induced inflammatory factor Nos2 mRNA expression in RAW 264.7 cells.

[0025] The above attached Figure 2-10 The results are expressed as mean ± standard deviation, n = 3, and different letters indicate significant differences among the groups, p < 0.05. DETAILED DESCRIPTION

[0026] The present invention will be described in further detail below with reference to specific examples. It should be noted that the specific examples are intended only to explain the present invention and are not intended to limit the present invention. In addition, the various experimental procedures involved in the examples are conventional in the art unless otherwise stated. For any part not specifically described herein, those skilled in the art can refer to various commonly used reference books, scientific literature, or relevant specifications, manuals, etc. before the filing date of the present invention for implementation.

[0027] Grape seeds are a key byproduct of winemaking and have the potential to be developed as highly effective natural anti-inflammatory drugs. To obtain peptides with potent anti-inflammatory activity, the applicant used wine grape seeds as raw material. First, the protein was extracted using a conventional alkaline-acid precipitation method and then enzymatically hydrolyzed to prepare protein peptides. Next, the applicant used LC-MS / MS-based proteopeptidomics to identify the protein peptide sequences. Bioinformatics methods such as database prediction, network pharmacology, and molecular docking were used to screen for peptides with anti-inflammatory potential. Finally, the anti-inflammatory activity of the selected peptides was verified using the RAW 264.7 cell inflammation model.

[0028] In this study, 9,043 peptides were identified from grape seed protein peptides by LC-MS / MS, of which 59.69% had molecular weights below 1 kDa. Using the Peptide Ranker database, 26 peptides with bioactivity scores >0.9 were identified, and their toxicity and anti-inflammatory activities were further predicted. Network pharmacology analysis of targets and pathways was performed to identify the peptides' inflammatory targets and pathways. Molecular docking was used to simulate the interactions between the peptides and the target, RELA, and peptides with binding affinity to the RELA (p65) protein were identified. Ultimately, eight peptides with significant anti-inflammatory activity were identified: WGF, SHFGF, EGPFF, WAPR, SFYRAF, HFAFL, PGRF, and FDSF. Figure 1 The docking of one of the peptides (PGRF) with the RELA protein was demonstrated, and the docking binding energy was less than -5 kcal / mol, proving that the peptide can spontaneously bind to RELA and the system is stable. It can be used to regulate the NF-κB signaling pathway, and it is speculated that it has good anti-inflammatory activity potential.

[0029] Furthermore, the above eight peptides were synthesized. The synthetic peptides had a significant inhibitory effect on the production of nitric oxide (NO) in inflammatory cells in the RAW 264.7 inflammation model, while reducing the secretion of cellular inflammatory factors tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), and inhibiting the expression of p65, Tnfα, Il6, Il1b and Nos2 mRNA, indicating that they may exert anti-inflammatory activity by regulating the nuclear transcription factor NF-κB (NF-κB) signaling pathway.

[0030] Example 1: Investigating the Effect of Grape Seed Anti-inflammatory Peptide on Cellular Inflammation Levels Based on the RAW 264.7 Macrophage Inflammation Model 1. Materials and Reagents WGF, SHFGF, EGPFF, WAPR, SFYRAF, HFAFL, PGRF, FDSF: synthesized by Hefei Guopi Company, the purity of the synthetic peptides is above 95%.

[0031] Experimental cell line: RAW 264.7 mouse monocyte-macrophage cells were purchased from Shanghai Cell Bank.

[0032] Experimental reagents: Analytical grade reagents such as ammonium bicarbonate, formic acid, acetonitrile, dithiothreitol, and iodoacetamide were all purchased from Sigma-Aldrich, USA; trifluoroacetic acid was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; a nitric oxide (NO) assay kit (A012-1 nitrate reductase method) was purchased from Nanjing Jiancheng Biotechnology Co., Ltd.; a mouse TNF-α rapid ELISA kit and a mouse IL-6 rapid ELISA kit were both purchased from Xinbosheng Biotechnology Co., Ltd.; a total RNA extraction kit was purchased from Beijing Aidelai Biotechnology Co., Ltd.; a cDNA reverse transcription kit and a qPCR kit, SYBR® Premix Ex Taq™, were both purchased from TaKaRa, Japan; and primers used for fluorescence quantitative PCR were purchased from Beijing Qingke Biotechnology Co., Ltd.

[0033] 2. Test methods 2.1 Recovery, culture, and cryopreservation of RAW 264.7 cells (1) Cell recovery: Take out the RAW 264.7 cell cryopreservation tube from the liquid nitrogen tank, quickly place it in a 37℃ water bath, and gently shake to melt the cell cryopreservation solution in the tube as soon as possible. Pipette the completely thawed cell cryopreservation solution into a 4mL centrifuge tube, add an equal amount of preheated DMEM complete medium to dilute, centrifuge at 1000r / min for 3min, discard the supernatant, add 4mL complete medium to resuspend the cells, transfer to a 25T cell culture flask, and place it in a cell constant temperature incubator at 37℃ and 5% CO2 to continue culturing.

[0034] (2) Cell culture and passaging: RAW 264.7 is an adherent cell that has high requirements for inoculation and growth density and is prone to differentiation. When the cells are in good condition, they are usually passaged once every 12-18 hours. When the cells grow to about 80% of the bottom of the bottle, they need to be passaged in time. Discard the original culture medium in the bottle, rinse the cells with preheated PBS, repeat twice, add an appropriate amount of new DMEM complete culture medium, carefully blow down the cells and evenly disperse them in the culture medium, take half of the culture medium containing cells and transfer it to a new 75T culture flask (passage culture at a ratio of 1:2), add complete culture medium, and place it in a cell constant temperature incubator for continued culture.

[0035] (3) Cell freezing: When the RAW 264.7 cells have grown to about 80% and are in good condition, gently blow the cells off the culture flask and transfer them to a 4 mL centrifuge tube. Centrifuge at 1000 r / min for 3 min, discard the supernatant, add 1 mL of preheated cell freezing solution to resuspend the cells, quickly transfer them to an explosion-proof cryotube, place in a -80 °C refrigerator overnight, and then place in a liquid nitrogen tank for long-term storage.

[0036] 2.2 Effects of synthetic peptides on RAW 264.7 cell viability Synthetic peptide solutions of different concentrations (3.125 μg / mL, 6.25 μg / mL, 12.5 μg / mL, and 25 μg / mL) were prepared in DMEM basal medium, and cell viability was determined using the following MTT assay.

[0037] (1) RAW 264.7 cells in good growth condition and in the logarithmic growth phase were blown down from the bottom of the culture flask with preheated PBS to prepare a cell suspension. After counting the cells using a hemocytometer, the cell concentration was adjusted to 1×10 5 The cells were seeded in 96-well cell culture plates with 100 μL per well.

[0038] (2) After the cells adhered for 12 hours, different concentrations of BPS (10 -2 mol / L, 10 -3 mol / L, 10 -4 mol / L, 10 -5 mol / L, 10-6 mol / L, 10 -7 mol / L, 10 -8 mol / L, 10 -9 RAW 264.7 cells were treated with 100 μL of 5-hydroxy-1,4-diol (200 μL) and 100 μL of synthetic peptide solution for 24 hours. After treatment, the culture medium was aspirated. 200 μL of basal medium containing 0.5 mg / mL MTT was then added to each well and incubated for a further 4 hours (protected from light throughout the process).

[0039] (3) After 4 hours, carefully remove the medium containing MTT from each well, add 150 μL DMSO, and shake in the dark for 5 minutes to fully dissolve the purple crystals. Use a microplate reader to measure the absorbance value (OD490) of each well at a wavelength of 490 nm, and calculate the cell survival rate according to the following formula: Cell survival rate = (OD490 of treatment group - OD490 of blank group) / (OD490 of control group - OD490 of blank group) × 100% 2.3 Effects of synthetic peptides on the secretion of NO, TNF-α, and IL-6 by RAW 264.7 cells Take RAW 264.7 macrophages that are in good growth condition and in the logarithmic growth phase and adjust the cell concentration to 1×10 6 10 μg / mL of synthetic peptide was added to 12-well cell culture plates and cultured in a cell incubator at 37°C and 5% CO2 for 12 hours. -7 The cells were cultured with 100 μmol / L BPS for 6 h; the same volume of DMEM basal medium was used as a control. After treatment, the cell culture medium was collected and the NO content (μmol / L) in the cell culture medium was determined using the NO kit.

[0040] According to the determination steps of each ELISA kit, the standard curves of TNF-α and IL-6 were drawn respectively, and the content of each inflammatory factor in the corresponding cell supernatant (pg / mL) was calculated based on the standard curves.

[0041] 2.4 Extraction of total cellular RNA (1) After the drug treatment, discard the cell culture medium, slowly add 1 mL of pre-cooled PBS to rinse the cells twice, aspirate the remaining PBS buffer, add 0.8 mL of Trizol to lyse the cells, repeatedly pipette to mix, transfer to a 1.5 mL enzyme-free centrifuge tube, and lyse at room temperature (25°C) for 5 minutes; (2) Add 0.1 mL of chloroform, cover the sample tube tightly, shake vigorously for 15 seconds, and incubate at room temperature for 5 minutes; (3) Centrifuge at 12,000 rpm for 10 min at 4°C. Carefully aspirate the top aqueous phase and transfer it to a new tube (do not touch the middle layer). Record the volume of the aqueous phase. Add half the volume of the aqueous phase, i.e., 0.5 times the volume of anhydrous ethanol, and mix thoroughly. Transfer the resulting mixed solution to the adsorption column RA and centrifuge at 12,000 rpm for 30 s. Discard the waste liquid and return the adsorption column to the collection tube. (4) Add 500 μL of deproteinized solution RE, centrifuge at 12000 rpm for 30 s, and discard the waste liquid; (5) Add 500 μL of rinse solution RW, centrifuge at 12000 rpm for 30 s, and discard the waste liquid; (6) Repeat adding 500 μL of rinse solution RW, centrifuging at 12000 rpm for 30 s, and discarding the waste solution; (7) Place the adsorption column RA back into the empty collection tube and centrifuge at 12,000 rpm for 2 min to remove as much rinse solution as possible to prevent residual ethanol in the rinse solution from inhibiting downstream reactions; (8) Remove the adsorption column RA and place it in a new RNase-free centrifuge tube. Add 20 μL of RNase-free water to the middle of the adsorption membrane, leave it at room temperature for 2 minutes, and centrifuge it at 12,000 rpm for 1 minute. Add the resulting solution back to the centrifugal adsorption column and centrifuge it for 1 minute. (9) Use a micro-spectrophotometer to measure the RNA concentration and OD260 / OD280, OD260 / OD230 values ​​to determine whether the RNA is contaminated with salt and protein. Aliquot the RNA and store it in a -80℃ refrigerator.

[0042] 2.5 Reverse transcription and cDNA synthesis (1) Dilute the obtained RNA to a concentration of approximately 200 ng / μL.

[0043] (2) Prepare the reaction system for removing gDNA: 2 μL 5 × gDNA Eraser Buffer, 1 μL gDNA Eraser Buffer, 7 μL RNA, mix well, and incubate at 42°C for 2 min to remove genomic DNA.

[0044] (3) Prepare the reverse transcription system: 1 μL PrimeScript RT Enzyme MixⅠ, 1 μL RT Primer Mix, 4 μL 5 × PrimeScript Buffer, 4 μL RNase Free dd H2O, and 10 μL of the reaction solution in step 2. Mix well and incubate at 37°C for 15 min, then incubate at 85°C for 5 s for reverse transcription. The obtained cDNA was aliquoted and stored at -80°C.

[0045] 2.6 qRT-PCR determination of related gene expression Primer sequences for all genes were obtained from the qPrimerDB-qPCR Primer Database (https: / / biodb.swu.edu.cn / qprimerdb / ) and synthesized by Wuhan Qingke Biotechnology Co., Ltd. Detailed primer information is shown in Table 1. Quantitative amplification was performed according to the SYBR Premix EX Tap II instructions. The reaction system consisted of 5 μL 2× qPCR Mix, 3.7 μL ddH2O, 0.4 μL forward primer (10 μmol / L), 0.4 μL reverse primer (10 μmol / L), and 0.5 μL cDNA template. The reaction program was as follows: 95°C pre-denaturation for 30 s, 95°C denaturation for 5 s, 57°C annealing for 30 s, and 72°C extension for 30 s, for 40 cycles. Fluorescence signals were collected during the extension phase. The melting curve was performed in the temperature range of 57°C to 95°C, with a 1°C increase every 15 s. The relative expression of the target gene was calculated using the 2-∆∆CT method. Mactin was used as an internal reference gene.

[0046] Table 1 qRT-PCR primer sequences

[0047] 2.7 Data processing and statistical analysis All experiments were repeated three or more times, and the results are expressed as mean ± standard deviation (mean ± SD). Data were analyzed using IBM SPSS Statistic Version 25.0. Data from different groups were analyzed using one-way ANOVA, with significance analyzed using the t-test. Graphs were generated using GraphPad Prism 9.5. Significance was determined at p < 0.05.

[0048] 3. Test results 3.1 Cytotoxicity assay The MTT assay was used to determine the effects of different concentrations of eight synthetic peptides on the survival rate of RAW 264.7 cells. Figure 2As shown, within the concentration range of 3.125-12.5 μg / mL, SHFGF, EGPFF, WAPR, HFAFL, and FDSF had no significant effect on cell viability. WGF significantly reduced macrophage viability at a concentration of 6.25 μg / mL (p<0.05), and SFYRAF significantly reduced cell viability at concentrations of 3.125 μg / mL, 6.25 μg / mL, and 25 μg / mL. PGRF exhibited significant toxicity to cells at concentrations above 3.125 μg / mL, with cell viability reaching only 65.21±2.61% at a concentration of 25 μg / mL. Based on the above data, a synthetic peptide concentration of 10.0 μg / mL will be used in subsequent cell experiments to ensure no significant toxicity to macrophages.

[0049] 3.2 Effects of synthetic peptides on NO secretion by RAW 264.7 cells To evaluate the inhibitory effect of 8 kinds of synthetic peptides on the inflammatory response of mouse mononuclear macrophage RAW 264.7 cells, the inhibitory effect of 10 μg / mL synthetic peptides on the inflammatory response of mouse mononuclear macrophage RAW 264.7 cells was determined. -7 mol / L BPS stimulated NO secretion in mouse RAW 264.7 cells. The results showed that ( Figure 3 ), 10 -7 mol / L BPS significantly increased NO secretion in macrophages (p<0.01), demonstrating the successful establishment of an inflammatory model. All eight synthetic peptides significantly inhibited NO secretion in inflammatory cells (p<0.05).

[0050] 3.3 Effects of synthetic peptides on the secretion of inflammatory factors by RAW 264.7 cells like Figure 4 As shown, in 10 -7 In a mouse RAW 264.7 macrophage inflammation model stimulated by 1 mol / L BPS, short-term BPS exposure significantly increased the levels of inflammatory cytokines IL-6 and TNF-α in macrophages (p<0.001). Compared with the model group, IL-6 secretion was reduced in the WGF, SHFGF, EGPFF, WAPR, SFYRAF, HFAFL, PGRF, and FDSF-treated groups. IL-6 secretion in the WGF, SHFGF, EGPFF, WAPR, and SFYRAF groups was significantly lower than that in the model group (p<0.001). IL-6 levels in the HFAFL, PGRF, and FDSF groups decreased to levels not significantly different from those in the control group (p>0.05).

[0051] like Figure 5As shown, the TNF-α content in normal cells is 384.44±1.97pg / mL. Under BPS stimulation, the TNF-α content in the model group cells was 2168.26±17.31pg / mL, which was significantly higher than that in the control group (p<0.0001). In addition, the TNF-α content in cells treated with synthetic peptides was significantly reduced (p<0.0001). These results indicate that the eight synthetic peptides have a good inhibitory effect on the secretion of inflammatory factors IL-6 and TNF-α, and all show good anti-inflammatory activity.

[0052] 3.4 Effects of synthetic peptides on the expression of genes related to the NF-κB signaling pathway in RAW 264.7 cells The nuclear transcription factor NF-κB plays a key regulatory role in the inflammatory response. p65, as a key subunit of the NF-κB complex, binds to the NF-κB inhibitory protein IκB under normal physiological conditions and is retained in the cytoplasm. When inflammation occurs, the inflammatory signal activates the IKK complex, phosphorylates and degrades IκB, and releases the p65 / p50 heterodimer. At this time, p65 enters the cell nucleus and binds to DNA to initiate the transcription of pro-inflammatory genes (Tnfα, Il6, Nos2). Under inflammatory stimulation, IκB is phosphorylated and degraded, releasing p65, enabling it to transfer to the cell nucleus and activate the expression of inflammation-related genes (Assis et al 2024). In addition, the phosphorylation level of p65 will also increase, further enhancing its transcriptional activity. Figure 6 As shown in the results, under the stimulation of BPS, the expression of p65 mRNA in mouse macrophages increased significantly, indicating that BPS has a significant effect on the inflammation-induced macrophages (p<0.01). The eight synthetic peptides can inhibit the expression of p65 mRNA at a concentration of 10 μg / mL, thereby regulating cellular inflammation. Among them, WGF and EGPFF have a very significant effect on reducing the expression of p65 mRNA in inflammatory cells (p<0.0001), and their expression levels are 59.05% and 59.13% of that in the model group, respectively.

[0053] TNF-α is an important pro-inflammatory cytokine, whose gene expression is regulated by the NF-κB signaling pathway. After activation, NF-κB is transferred to the cell nucleus and binds to the promoter region of the Tnfα gene, promoting its transcription and translation (Narayananand Sonika 2010, Darendelioglu et al 2025). At the same time, under the stimulation of inflammation, inflammatory signals will activate macrophages, T cells, etc., promoting the transcription of the Tnfα gene. TNF-α can bind to the tumor necrosis factor receptor (TNFR) through autocrine or paracrine pathways, further activating the NF-κB and MAPK pathways, forming a positive feedback loop to amplify the inflammatory effect (Akanda and Park 2017). Figure 7 As shown, in BPS-induced mouse macrophages, Tnfα mRNA expression was significantly increased. Compared with the model group, Tnfα mRNA expression in the EGPFF and SFYRAF groups was significantly decreased (p<0.0001), by 39.40% and 45.69%, respectively. Expression in the WGF, SHFGF, WAPR, HFAFL, and FDSF groups was also significantly decreased (p<0.05), while no significant change was observed in the PGRF group. These results suggest that the synthetic peptides WGF, SHFGF, EGPFF, WAPR, SFYRAF, HFAFL, and FDSF can exert anti-inflammatory effects by inhibiting Tnfα mRNA expression.

[0054] The expression of Il6 mRNA is also regulated by the NF-κB signaling pathway (Suda et al 2025). Under inflammatory stimulation, the activation of NF-κB will lead to increased transcription of the IL-6 gene. In addition, the expression of Il6 mRNA is also affected by other JAK-STAT signaling pathways (Yang et al 2022) and is closely related to the activation and proliferation of inflammatory cells. Therefore, the expression of Il6 mRNA can be used to judge the generation of cellular inflammation and the regulation of the anti-inflammatory effect of synthetic peptides. Figure 8 All eight synthetic peptides significantly inhibited BPS-induced upregulation of Il6 mRNA in macrophages (p<0.0001), with expression levels reaching 74.92%, 76.80%, 70.22%, 72.10%, 66.77%, 57.99%, 70.53%, and 67.71% of those in the model group, respectively. This indicates that at a concentration of 10 μg / mL, all eight synthetic peptides significantly inhibited Il6 mRNA expression in the BPS-induced RAW 264.7 cell inflammation model, with HFAFL having the most significant effect.

[0055] Il1b mRNA expression is primarily regulated by activation of the NLRP3 inflammasome (Liu et al. 2016). Under inflammatory stimuli, the NLRP3 inflammasome is activated, leading to the cleavage and secretion of the inactive pro-IL-1β precursor, pro-IL-1β. Activation of NF-κB, acting as a "priming signal" for inflammatory responses, also promotes Il1b mRNA expression (Dissanayake et al. 2021). Regarding IL-1β in RAW 264.7 cells, short-term exposure to BPS for 6 hours significantly increased Il1b mRNA expression (p < 0.0001), with Il1b mRNA levels in the model group significantly higher than in the control group (p < 0.01). After treatment with WGF, SHFGF, EGPFF, WAPR, SFYRAF, HFAFL, and FDSF, the expression levels of Il1b mRNA were all reduced compared with the model group, which were 85.61%, 79.65%, 61.75%, 55.47%, 85.96%, 60.00%, and 82.11%, respectively. PGRF had no significant effect on the expression level of Il1b mRNA (p>0.05) ( Figure 9 ).

[0056] The expression of Nos2 mRNA is regulated by the NF-κB signaling pathway (Tam et al 2025). Under inflammatory stimulation, the activation of NF-κB promotes the transcription and translation of the Nos2 gene, and NF-κB can synergistically activate its transcription with signal transducer and activator of transcription 1 (STAT1) (Amani et al 2024). STAT1 can be activated through interferon γ (IFN-γ) signaling, resulting in increased gene expression, which further catalyzes the production of a large amount of NO. NO has multiple biological functions in inflammatory responses, including regulating vascular tension and immune cell activity. When there is excessive NO in cells, it will induce inflammation (Seleem et al 2025). Therefore, when inflammation occurs, the expression of Nos2 mRNA in cells will increase accordingly. Figure 10 As shown in the results, compared with the model group, the expression level of Nos2 mRNA in mouse macrophages in the synthetic peptide WGF, SHFGF, EGPFF, WAPR, SFYRAF, HFAFL and FDSF treatment groups was significantly decreased (p<0.05), indicating that they played an effective inhibitory effect on BPS-induced cellular inflammation.

[0057] The above results show that the anti-inflammatory peptides screened from grape seeds can significantly inhibit the expression of major targets in the NF-κB signaling pathway and its downstream genes, indicating that it may alleviate BPS-induced inflammatory response in RAW264.7 mouse mononuclear macrophages by inhibiting the NF-κB signaling pathway.

[0058] References 1.Akanda MR, Park BY. Involvement of MAPK / NF-κB signal transductionpathways: Camellia japonica mitigates inflammation and gastric ulcer. BiomedPharmacother, 2017, 95: 1139-1146. 2.Amani S, Alinejad S, Asadi N, Yousefi E, Khademvatan S, Howarth GS. Anti-Leishmania major activity of Calotropis procera extract by increasingROS production and upregulating TNF-α, IFN-γ and iNOS mRNA expression under in vitro conditions. Trop Med Health, 2024, 52(1): 16-16. 3.Assis SISd, Amendola LS, Okamoto MM, Ferreira GdS, Iborra RT, Santos DR, Santana MdFM, Santana KG, Giannella MLC, Barbeiro DF, Soriano FG, Machado UF, Passarelli M. The prolonged activation of the p65 subunit of the NF-Kappa-B nuclear factor sustains the persistent effect of advancedglycation end products on inflammatory sensitization in macrophages. Int JMol Sci, 2024, 25(5). 4.Darendelioglu E, Caglayan C, Küçükler S, Bayav İ, Kandemir FM, AynaA, Sağ S. 18β-glycyrrhetinic acid Mitigates bisphenol A-induced liver andrenal damage: Inhibition of TNF-α / NF-κB / p38-MAPK, JAK1 / STAT1 pathways,oxidative stress and apoptosis. Food Chem Toxicol, 2025, 196: 115218-115218. 5.Dissanayake WC, Oh JK, Sorrenson B, Shepherd PR. Glucose regulatesexpression of pro-inflammatory genes, IL-1β and IL-12, through a mechanisminvolving hexosamine biosynthesis pathway-dependent regulation of α-Ecatenin. Biosci Rep, 2021, 41(7). 6.Liu T, Zhou Y, Li P, Duan JX, Liu YP, Sun GY, Wan L, Dong L, FangX, Jiang JX, Guan CX. Blocking triggering receptor expressed on myeloidcells-1 attenuates lipopolysaccharide-induced acute lung injury viainhibiting NLRP3 inflammasome activation. Sci Rep, 2016, 6(1): 39473. 7.Narayanan P, Sonika P. Tumor necrosis factor-α signaling inmacrophages. Crit Rev Eukaryot Gene Expr, 2010, 20(2): 87-103. 8.Seleem MA, Salem OM, Basha E, Ibrahim HA, Elshamy AM, Azzam AR,Ismail R, Homouda AA, Elkordy A, Faheem H. The protective effects ofSaxagliptin and Cilostazol in an experimental model of Cyclophosphamide-Induced nephrotoxicity in rats: targeting iNOS / NF-kB and Nrf-2 / HO-1 pathways.J Biochem Mol Toxicol, 2025, 39(3): e70196. 9.Suda Y, Ikuta K, Hayashi S, Wada K, Anjiki K, Kamenaga T, TsubosakaM, Kuroda Y, Nakano N, Maeda T, Tsumiyama K, Matsumoto T, Kuroda R, MatsubaraT. Comparison of anti-inflammatory and anti-angiogenic effects of JAKinhibitors in IL-6 and TNFα-stimulated fibroblast-like synoviocytes derivedfrom patients with RA. Sci Rep, 2025, 15(1): 9736-9736. 10.Tam FF, Dumlao JM, Lee AH, Choy JC. Endogenous production ofnitric oxide by iNOS in human cells restricts inflammatory activation andcholesterol / fatty acid biosynthesis. Free Radic Biol Med, 2025, 231: 1-10. 11.Yang ML, Wu S, Cai WS, Ming XP, Zhou YH, Chen X. Hypoxia-inducedMIF induces dysregulation of lipid metabolism in Hep2 laryngocarcinomathrough the IL-6 / JAK-STAT pathway. Lipids Health Dis, 2022, 21(1): 82-82.

Claims

1. A grape seed-derived anti-inflammatory peptide, characterized in that: The amino acid sequence of the anti-inflammatory peptide is selected from any one of the following: WGF, SHFGF, EGPFF, WAPR, SFYRAF, HFAFL, PGRF or FDSF.

2. Use of the anti-inflammatory peptide according to claim 1 in the preparation of anti-inflammatory drugs.

3. An anti-inflammatory drug comprising the anti-inflammatory peptide according to claim 1 and a pharmaceutically acceptable carrier.

4. A method for inhibiting macrophage inflammatory response, characterized in that: Treating RAW 264.7 cells with the anti-inflammatory peptide described in claim 1 at a concentration of 10 μg / mL can inhibit BPS-induced cellular inflammatory response, including reducing the level of NO in the cells and inhibiting the secretion of inflammatory factors TNF-α and IL-6.

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