Active peptide for preventing or treating alcoholic liver injury and application thereof

Abstract

The application belongs to the technical field of biotechnology, and discloses an active peptide for preventing or treating alcoholic liver injury and application thereof.Three sea cucumber gonad-derived polypeptides capable of preventing or treating alcoholic liver injury are obtained by enzymolysis combined with network pharmacology, and the amino acid sequences of the polypeptides are shown as SEQ.ID.NO.1-3.A polypeptide sequence shown as SEQ.ID.NO.1 has not been reported yet.Experiments prove that the three polypeptides all have the potential of reducing alcoholic liver cell injury and can play a protective role by inhibiting enzyme leakage;the three polypeptides can all reduce the content of MDA, increase the activity of SOD and the content of GSH, and can all reduce the damage of alcohol to HepG2 cells by reducing the content of inflammatory factors, thus showing a potential anti-inflammatory effect.Therefore, the three polypeptides obtained by the application have the effect of relieving alcoholic liver injury, and can be applied to the preparation of drugs for preventing or treating alcoholic liver injury.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an active peptide for the prevention or treatment of alcoholic liver injury and its application. Background Technology

[0002] The liver, as a vital metabolic and detoxification organ, undertakes crucial physiological functions such as alcohol metabolism and toxin removal, and its functional integrity is directly related to the body's health. Alcohol abuse is one of the leading causes of liver disease worldwide, and alcoholic liver disease (ALD) has become a global public health challenge. According to the World Health Organization (WHO), approximately 3 million people die globally each year from harmful alcohol use, with ALD being one of the leading causes of death. In China, the prevalence of ALD is rising significantly, and the patient population is gradually becoming younger. ALD has a broad spectrum, progressing from early simple steatosis to alcoholic hepatitis, liver fibrosis, cirrhosis, and ultimately even hepatocellular carcinoma. This process not only seriously threatens patients' lives and health but also places a heavy economic burden on the social healthcare system.

[0003] Currently, clinical treatment of ALD mainly relies on drug intervention, lifestyle modifications, and liver transplantation. However, these existing prevention and treatment methods still have certain limitations. While chemically synthesized drugs can improve some clinical symptoms in the short term, long-term use can easily lead to adverse reactions such as infection, gastrointestinal reactions, metabolic disorders, and secondary damage to liver and kidney function, making safety difficult to guarantee. Lifestyle interventions, mainly involving abstinence from alcohol and dietary control, are fundamental prevention and treatment measures, but problems such as poor patient compliance and high relapse rates make it difficult to achieve stable and widespread prevention and treatment effects. Liver transplantation remains an effective treatment for end-stage ALD, but the shortage of donors, high treatment costs, and postoperative immune rejection greatly limit its clinical application and widespread use. Against this backdrop, natural products with high safety and good bioactivity have gradually become a research hotspot in the field of liver protection. Natural products are widely available, have few toxic side effects, and good biocompatibility, making them an important resource for screening novel hepatoprotective active substances. Existing research has confirmed that various natural products can alleviate alcohol-induced liver tissue damage and slow down disease progression by regulating multiple cell death signaling pathways, demonstrating good research value and application prospects in the prevention and treatment of ALD. Summary of the Invention

[0004] This invention provides an active peptide for the prevention or treatment of alcoholic liver injury and its application. The peptide was prepared and screened from sea cucumber gonadal tissue and exhibits good efficacy in preventing or treating alcoholic liver injury.

[0005] The specific technical solution is as follows:

[0006] One objective of this invention is to provide an active peptide for the prevention or treatment of alcoholic liver injury, characterized in that the amino acid sequence is as shown in SEQ.ID.NO.1.

[0007] Specifically, the aforementioned active peptides for preventing or treating alcoholic liver injury are derived from sea cucumber gonads.

[0008] The preparation method of bioactive peptides for preventing or treating alcoholic liver injury is as follows: In this invention, sea cucumber gonad hydrolysate is obtained by enzymatic hydrolysis with protease. After purification, the peptides obtained by hydrolysis are sequenced and target prediction is performed. Then, targets related to alcoholic liver injury are screened from the hydrolysate. KEGG pathway analysis and virtual screening for antioxidant and anti-inflammatory effects are performed on the obtained targets. The peptides obtained by screening are subjected to toxicity evaluation and activity experiments to verify their activity. Finally, three bioactive peptides for preventing or treating alcoholic liver injury are obtained, and their amino acid sequences are shown in SEQ.ID.NO.1~3, respectively.

[0009] Among them, the peptide sequence with amino acid sequence as shown in SEQ.ID.NO.1 has not been reported, while the peptide sequences with amino acid sequence as shown in SEQ.ID.NO.2 and SEQ.ID.NO.3 have been reported, but their application in the prevention or treatment of alcoholic liver injury has not been disclosed.

[0010] Among them, SEQ.ID.NO.1 is GPAGPQGP.

[0011] Among them, SEQ.ID.NO.2 is PSNLGTGLR.

[0012] Among them, SEQ.ID.NO.3 is VPYPR.

[0013] The enzymatic hydrolysis involves first adding pepsin for hydrolysis, and then adding flavor protease for hydrolysis.

[0014] The purification process involves obtaining a component with a molecular weight of 200-3000 Da through membrane filtration.

[0015] A second objective of this invention is to provide the application of the above-mentioned active peptides for preventing or treating alcoholic liver injury in the preparation of products for preventing or treating alcoholic liver injury, wherein the active peptides are selected from at least one of the peptides with amino acid sequences as shown in SEQ.ID.NO.1~3.

[0016] Specifically, the aforementioned active peptides for preventing or treating alcoholic liver injury are used in the preparation of drugs for preventing or treating alcoholic liver injury.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] This invention obtained three sea cucumber gonad-derived peptides through enzymatic hydrolysis combined with network pharmacology, among which the amino acid sequence of the peptide shown in SEQ.ID.NO.1 has not been reported before. Experiments confirmed that all three peptides have the potential to alleviate alcoholic hepatocellular damage and may exert a protective effect by inhibiting enzyme leakage; all three peptides can decrease MDA content while increasing SOD activity and GSH content, and all can alleviate alcohol-induced damage to HepG2 cells by reducing inflammatory cytokine levels, exhibiting potential anti-inflammatory effects. Therefore, the three peptides obtained in this invention possess a mitigating effect on alcoholic liver injury and can be applied to the preparation of drugs for the prevention or treatment of alcoholic liver injury. Attached Figure Description

[0019] Figure 1 This illustrates the effect of ethanol concentration on cell viability in Example 1.

[0020] Figure 2 The effect of the three peptides in Example 1 on the viability of HepG2 cells;

[0021] Figure 3 The effect of the three peptides in Example 1 on the viability of alcohol-damaged HepG2 cells;

[0022] Figure 4 The effect of the three peptides in Example 1 on the intracellular AST level in alcohol-damaged HepG2 cells;

[0023] Figure 5 The effect of the three peptides in Example 1 on the intracellular ALT level in alcohol-damaged HepG2 cells;

[0024] Figure 6 The effect of the three peptides in Example 1 on MDA activity in alcohol-damaged HepG2 cells;

[0025] Figure 7 The effect of the three peptides in Example 1 on SOD activity in alcohol-damaged HepG2 cells;

[0026] Figure 8 The effect of the three peptides in Example 1 on GSH activity in alcohol-damaged HepG2 cells;

[0027] Figure 9 The effect of the three peptides in Example 1 on TNF-α in alcohol-damaged HepG2 cells;

[0028] Figure 10 The effect of the three peptides in Example 1 on intracellular IL-6 in alcohol-damaged HepG2 cells;

[0029] Figure 11 The effect of the three peptides in Example 1 on IL-1β in alcohol-damaged HepG2 cells;

[0030] Figure 12 This is an intersection analysis of the three peptide targets and alcoholic liver disease targets in Example 2;

[0031] Figure 13 This refers to the effect of the three peptides in Example 2 on the relative expression level of the TNF gene;

[0032] Figure 14 The effect of the three peptides on the relative expression level of the PI3K gene in Example 2;

[0033] Figure 15 This illustrates the effect of the three peptides in Example 2 on the relative expression level of the AKT1 gene.

[0034] Figure 16 The effect of the three peptides on the relative expression level of the NF-κB gene in Example 2;

[0035] Figure 17 The effect of the three peptides on the relative expression level of the PTGS2 gene in Example 2;

[0036] Figure 18 The effect of the three peptides on the relative expression level of the PPARG gene in Example 2;

[0037] Figure 19 This describes the effect of the three peptides in Example 2 on the relative expression level of the SOD2 gene.

[0038] Figures 1-11 , Figures 13-19 In Chinese: different lowercase letters indicate significant differences (P < 0.05). Detailed Implementation

[0039] The embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and examples. The following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0040] In the specific implementation: the pepsin used was purchased from Sangon Biotech; the flavor protease used was purchased from Solarbio.

[0041] Example 1

[0042] The steps for preparing bioactive peptides to prevent or treat alcoholic liver injury are as follows:

[0043] S1. Preparation of sea cucumber gonadal peptides:

[0044] (1) Raw material pretreatment: After homogenizing the sea cucumber gonadal tissue (mixed male and female gonads), heat it in a boiling water bath for 15 minutes to obtain a homogenate, and let it cool before use.

[0045] (2) Raw material enzymatic hydrolysis: Add 2000 U / g of pepsin based on its mass to the homogenate obtained in step (1), adjust the pH to 2.5, and enzymatically hydrolyze at 37℃ for 4 h; then add 1200 U / g of flavor protease based on the mass of the homogenate, adjust the pH to 7.0, and enzymatically hydrolyze at 50℃ for 2 h; boil at 100℃ to inactivate the enzyme; then centrifuge at 5000 r / min for 15 min, take the supernatant, and obtain the enzymatic hydrolysate.

[0046] (3) Enzyme hydrolysate purification: The enzyme hydrolysate obtained in step (2) is subjected to nanofiltration and ultrafiltration fractionation. First, a 200 Da nanofiltration membrane is used to remove salt and free amino acids, and then a 3000 Da spiral wound membrane is selected for ultrafiltration. The 200~3000 Da fraction is freeze-dried to obtain sea cucumber gonadal peptide powder, which is stored at -20℃ for later use.

[0047] S2. Perform peptide sequence analysis: Desalt the sea cucumber gonadal peptide powder obtained in step S1 using a C18 StageTip column, and perform peptide sequence analysis using LC-MS / MS. The complete peptide sequence was obtained by comparison analysis with the database. The mass spectrometry database search software was MaxQuant 2.4.14.0, and the sample database used was the uniprot protein database.

[0048] S3. Screening for bioactive peptides for the prevention or treatment of alcoholic liver injury using network pharmacology methods:

[0049] Based on the "SMILES" functional module in the BIOPEP-UWM platform (https: / / biochemia.uwm.edu.pl / biopep / finding_smiles.php), peptide sequences were converted into corresponding SMILES structures. Subsequently, the potential targets were predicted using the SwissTarget Prediction online tool (http: / / www.swisstargetprediction.ch / ). All prediction results were integrated and duplicates removed to obtain the final set of potential targets related to the peptide sequences.

[0050] Using "Alcoholic Liver Disease" as the search term, searches were conducted in three databases: GeneCards (https: / / www.genecards.org / ), OMIM (https: / / omim.org / ), and TDD (https: / / db.idrblab.net / ttd / ). The target information obtained from each database was aggregated, and duplicates were removed to establish a dataset of target targets related to ALD.

[0051] We used the Venny online tool (http: / / www.liuxiaoyuyuan.cn / ) to perform an intersection analysis on the predicted targets of sea cucumber gonadal peptides and ALD-related targets to obtain the set of targets shared by both.

[0052] Drug-disease common targets were uploaded to the DAVID database (https: / / david.ncifcrf.gov / ). KEGG pathway analysis was conducted on the relevant targets of sea cucumber gonadal active components for the treatment of ALD. Peptide sequences were sorted using the enriched "TNF signaling pathway," and virtual screening for antioxidant and anti-inflammatory effects was performed. The top-ranked peptide sequences were selected for chemical synthesis and subsequent experimental screening.

[0053] S4. Evaluate the toxic effects and activity screening of sea cucumber gonadal peptides on HepG2 cells:

[0054] The effect of the peptide sequences obtained in step S3 on cell viability was tested to evaluate toxicity. HepG2 cells were diluted to 2.5 × 10⁻⁶. 5 HepG2 cells were seeded at a concentration of 100 μL / well in 96-well cell culture plates and cultured for 24 h. Sea cucumber gonadotropic peptides were prepared into solutions of 50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, 600 μg / mL, and 800 μg / mL using culture medium. Different concentrations of sea cucumber gonadotropic peptides were added to the corresponding wells to act on HepG2 cells, serving as the experimental group. Culture medium was added to the cells in the normal group, with blank culture medium serving as the control group. After culturing for another 24 h, the supernatant was aspirated, and the CCK-8 assay was used to determine whether the sea cucumber gonadotropic peptides within this concentration range were toxic to HepG2 cells, and the relative cell proliferation rate was calculated. Non-toxic peptide sequences were selected for subsequent experiments. The relative cell proliferation rate was calculated using the following formula:

[0055] %.

[0056] Cellular experiments were used to screen the activity of the selected peptide sequences. An alcoholic hepatocyte injury model was established using ethanol induction. The method for establishing the alcoholic hepatocyte injury model was as follows: when the HepG2 cell density reached above 80%, the cells were digested with trypsin and resuspended, and the density was adjusted to 2.5 × 10⁻⁶ cells / year. 5Cells were seeded at a rate of 100 μL per well in a 96-well plate and cultured for 24 h. The supernatant was discarded. Complete culture medium was added to the normal group, while the alcohol group was added to culture medium containing different final concentrations of ethanol (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9 mol / L). Cultured for another 24 h, the supernatant was then removed, and culture medium containing 10% CCK-8 reagent was added to each well. The cells were incubated in the dark for 1 h, and the absorbance was measured at 450 nm. Blank culture medium was used as a control. Cell viability was calculated based on the absorbance values ​​to screen for suitable alcohol concentrations for cell modeling. Cell viability was calculated using the following formula:

[0057] %.

[0058] Test results are available Figure 1 The results showed that cell viability gradually decreased with increasing ethanol concentration. A 30%–50% decrease in cell viability was used as the criterion for successful modeling. After treatment with 0.6 mol / L ethanol, the cell viability was 60.54%, a decrease of 39.45% compared to the normal group, indicating successful cell modeling. Subsequent experiments all used 0.6 mol / L ethanol to construct the damage model.

[0059] To verify the effect of sea cucumber gonadotropic peptide on alcoholic hepatocyte injury, HepG2 cells were seeded into 6-well plates at a volume of 2 mL per well and incubated for 24 h. After incubation, the supernatant was aspirated, and the culture medium for the normal and model groups was replaced. The experimental groups were treated with 100 μg / mL, 200 μg / mL, and 400 μg / mL sea cucumber gonadotropic peptide, respectively, and cultured for another 24 h. The culture medium was then replaced again, with the normal group receiving fresh medium, the model group receiving culture medium with a final ethanol concentration of 0.6 mol / L, and the experimental groups receiving culture medium containing both the corresponding concentrations of sea cucumber gonadotropic peptide and a final ethanol concentration of 0.6 mol / L. Cells in each group were cultured for another 24 h, and the supernatant and cells were collected from each group for subsequent activity assays and calculation of relative cell proliferation rate. The relative cell proliferation rate was calculated using the following formula:

[0060] %;or

[0061] %

[0062] Cells were lysed using an ultrasonic cell disruptor and then subjected to ice bath lysis. Alanine aminotransferase (ALT) / glutamic acid (GPT / ALT) activity assay kits (Solepro) and aspartate aminotransferase (GOT / AST) activity assay kits (Solepro) were used to measure AST and ALT liver function indicators according to the manufacturer's instructions. Superoxide dismutase (SOD) activity assay kits (Solepro), reduced glutathione (GSH) content assay kits (Solepro), and malondialdehyde (MDA) content assay kits (Solepro) were used to measure the antioxidant activities of SOD, GSH, and MDA according to the manufacturer's instructions. Tumor necrosis factor α ELISA kits (Solepro), interleukin 1β ELISA kits (Solepro), and interleukin 6 ELISA kits (Solepro) were used to measure the release levels of inflammatory factors TNF-α, IL-1β, and IL-6 in the cell culture medium according to the manufacturer's instructions. Three peptides were finally screened, and their peptide sequences are shown in SEQ.ID.NO.1~3, respectively. Among them, SEQ.ID.NO.1 is GPAGPQGP, SEQ.ID.NO.2 is PSNLGTGLR, and SEQ.ID.NO.3 is VPYPR.

[0063] The effects of the three peptides on HepG2 cell viability are shown in the figure. Figure 2 The results showed that, compared with the normal group, the three different concentrations of peptides did not significantly affect cell viability, indicating that sea cucumber gonadal peptides had no toxic effect on HepG2 cells within the concentration range of 50–800 μg / mL. Among them, the peptide (VPYPR) with the amino acid sequence shown in SEQ.ID.NO.3 inhibited cell proliferation to some extent at 800 μg / mL, which may be due to the change in external drug concentration affecting extracellular osmotic pressure, thereby affecting cell proliferation. Therefore, based on the significant differences between the groups, three drug concentrations of 100–400 μg / mL were ultimately selected for subsequent experiments.

[0064] The effects of the three peptides on the viability of alcohol-damaged HepG2 cells are as follows: Figure 3 The results showed that the cell survival rate in the alcohol model group was significantly lower than that in the normal group (P < 0.05). Compared with the alcohol model group, the cell survival rate of each peptide treatment group was improved in a concentration-dependent manner. Among them, at 400 μg / mL, peptide SEQ.ID.NO.1 (GPAGPQGP) had the most significant restorative effect on damaged cell viability, followed by peptides SEQ.ID.NO.2 (PSNLGTGLR) and SEQ.ID.NO.3 (VPYPR). There were statistically significant differences in cell survival rate between each peptide treatment group and the alcohol model group, indicating that within the range of 100–400 μg / mL, all three peptides had a protective effect against alcohol-induced HepG2 cell damage in a dose-dependent manner.

[0065] The effects of the three peptides on intracellular AST and ALT levels in alcohol-damaged HepG2 cells are shown in the following figures. Figure 4 , Figure 5 The results showed that compared with the normal group, the activities of AST and ALT in the alcohol model group were significantly increased, reaching 30.08 U / mg port and 10.51 U / mg port, respectively. After treatment with sea cucumber gonadal peptides, the activities of AST and ALT were significantly decreased, and significantly lower than those in the alcohol model group (P<0.05). Among them, peptide SEQ.ID.NO.1 (GPAGPQGP) showed the best effect, significantly reducing AST and ALT activities to 13.44 U / mg port and 5.51 U / mg port, respectively, at 400 μg / mL. The results indicate that the three sea cucumber gonadal peptides have the potential to alleviate alcoholic hepatocellular damage and may exert a protective effect by inhibiting enzyme leakage.

[0066] The effects of the three peptides on the activities of MDA, SOD, and GSH in alcohol-damaged HepG2 cells are as follows: Figure 6 , Figure 7 , Figure 8 The results showed that all three peptides decreased MDA content while increasing SOD activity and GSH content. At a concentration of 400 μg / mL, compared with the alcohol model group, the MDA content of the peptides SEQ.ID.NO.1 (GPAGPQGP), SEQ.ID.NO.2 (PSNLGTGLR), and SEQ.ID.NO.3 (VPYPR) treatment groups decreased by approximately 30.40%, 16.65%, and 22.55%, respectively; the SOD activity of the three peptide treatment groups increased by approximately 125%, 50.41%, and 47.99%, respectively, compared with the alcohol model group; and the GSH content also recovered to its highest value, exceeding the alcohol model group by approximately 86.52%, 53.05%, and 67.95%, respectively. In terms of increasing SOD activity and restoring GSH content, the PSNLGTGLR and VPYPR treatment groups at the same concentration were slightly less effective than the GPAGPQGP treatment group.

[0067] The effects of the three peptides on intracellular TNF-α, IL-6, and IL-1β in alcohol-damaged HepG2 cells are as follows: Figure 9 , Figure 10 , Figure 11The results showed that the levels of TNF-α, IL-1β, and IL-6 in the alcohol model group were significantly higher than those in the normal group (P<0.05), indicating that alcohol intake led to the massive release of pro-inflammatory factors, thereby triggering an inflammatory response. Compared with the alcohol model group, all three peptides significantly reduced the inflammatory response. Among them, the downregulation of IL-6 was the most prominent, with a reduction of more than 40% in some groups and a maximum of nearly 63%. They also showed a strong inhibitory effect on TNF-α, with a reduction of more than 25% in most cases, demonstrating strong anti-inflammatory potential. All three peptides could alleviate the damage of alcohol to HepG2 cells by reducing the levels of inflammatory factors, showing potential anti-inflammatory effects.

[0068] Example 2

[0069] Target analysis was performed on the three peptides obtained in Example 1.

[0070] Potential targets for the three sea cucumber gonadal peptides obtained in Example 1 were initially screened from the Swiss Target Prediction database. By integrating the GeneCards, OMIM, and TDD databases, a total of 3162 ALD-related targets were collected. Figure 12 As shown, Venn diagram analysis was performed on the potential targets corresponding to the three peptide sequences and the ALD target to obtain the intersection targets, which were then used as potential targets for the action of sea cucumber gonadal peptides on ALD for further analysis. KEGG signaling pathway analysis showed that the TNF signaling pathway was one of the pathways with the highest enrichment significance among the three peptide sequences.

[0071] The common targets of the three peptide sequences obtained in Example 1 and ALD (alcoholic liver disease)-related targets were imported into the STRING database (https: / / string-db.org / ), with the species set to "human," to obtain the interaction data between targets, which were then downloaded as .TSV format files. A protein-protein interaction network was constructed using Cytoscape software, and the top ten core targets in the network were further screened through topology analysis. The PPI network analysis results showed that, using degree value as the screening criterion for core targets, the gene targets corresponding to each peptide were ranked, and the top 10 core gene targets for each peptide were finally screened out as follows: the core targets corresponding to peptide SEQ.ID.NO.1 are TNF, AGTR1, PLG, CTSB, MMP9, PTGS2, PPARG, CASP, ACE, and DPP4; the core targets corresponding to peptide SEQ.ID.NO.2 are AGTR1, CTSL, PTGS2, PLG, FURIN, ACE, CTSD, CAPNT1, F2, and DPP4; and the core targets corresponding to peptide SEQ.ID.NO.3 are AKT1, SRC, STAT3, AGTR1, PLG, ACE, HDAC1, BDKRB1, PTGS2, and CTSB. These screened gene targets are presumed to be the core regulatory targets for the therapeutic effect of sea cucumber gonadal peptides on alcoholic liver injury. This result further confirms that sea cucumber gonadal peptides can achieve a mitigating effect on alcoholic liver injury by regulating the expression of multiple gene targets.

[0072] The gene expression levels of the three peptides in Example 1 after treatment and culture in an alcoholic liver injury cell model were detected using qPCR. RNA was extracted from the treated cells according to the instructions of the total RNA extraction kit, and the RNA was reverse transcribed into cDNA using a reverse transcription kit. The quality and concentration of cDNA were then analyzed by qPCR, and the relative mRNA expression levels of each target gene were calculated. The primer sequences and sequence numbers of each target gene are shown in Table 1.

[0073] Table 1 Primer Sequences

[0074]

[0075] The effect of the three peptides on the relative expression level of the TNF gene is shown in the figure. Figure 13The results showed that TNF mRNA expression in the alcohol model group was significantly increased compared to the normal group, approximately three times that of the normal group. After treatment with peptides GPAGPQGP, PSNLGTGLR, and VPYPR, TNF mRNA expression gradually decreased with increasing concentration (P<0.05), with the 400 μg / mL peptide SEQ.ID.NO.1 showing the most significant inhibitory effect, superior to the treatment groups SEQ.ID.NO.2 and SEQ.ID.NO.3. These results indicate that the three sea cucumber gonadal peptides can effectively inhibit TNF mRNA expression and have a potential regulatory role in alcohol-induced inflammatory responses.

[0076] The effects of the three peptides on the relative expression level of the PI3K gene are shown in the figure. Figure 14 The effect of the three peptides on the relative expression level of the AKT1 gene is shown in [the table below]. Figure 15 The results showed that at a concentration of 400 μg / mL, the peptide GPAGPQGP exhibited the most significant inhibitory effect on PI3K mRNA, decreasing by 50.53% compared to the alcohol model group; the PSNLGTGLR and VPYPR treatment groups showed decreases of 42.83% and 41.51%, respectively, with similar inhibitory effects. Regarding AKT1 mRNA expression, the peptide VPYPR showed the strongest inhibitory effect at 400 μg / mL, significantly decreasing by 65.78% compared to the alcohol model group; the GPAGPQGP and PSNLGTGLR treatment groups showed decreases of 50.80% and 33.18%, respectively. These results indicate that sea cucumber gonadal peptides can regulate the activation of their downstream AKT signaling pathway by modulating PI3K expression.

[0077] The effects of the three peptides on the relative expression level of the NF-κB gene are shown in the figure. Figure 16 The effect of the three peptides on the relative expression level of the PTGS2 gene is shown in [the table below]. Figure 17 The results showed that the expression level of NF-κB mRNA in the model group was significantly higher than that in the normal group (P<0.05), indicating that oxidative stress and inflammatory signals induced by alcohol metabolism had induced the activation of NF-κB and PTGS2. After treatment with sea cucumber gonadal peptides, the expression levels of NF-κB and PTGS2 mRNA gradually decreased with increasing concentration (P<0.05). At 400 μg / mL, the expression levels of NF-κB and PTGS2 mRNA in the peptide GPAGPQGP were close to the levels in the normal group (P<0.05), representing a decrease of 60.51% and 48.49% respectively compared to the alcohol model group. This effect was significantly better than that in the PSNLGTGLR and VPYPR treatment groups, showing a concentration-dependent characteristic.

[0078] The effects of the three peptides on the relative expression level of the PPARG gene are shown in the figure. Figure 18 The effects of the three peptides on the relative expression levels of the SOD2 gene are shown in [the table below]. Figure 19The results showed that sea cucumber gonadotropic peptides could reverse alcohol-induced inhibition of PPARG expression and upregulate SOD2 mRNA levels, suggesting that its protective effect may be achieved by activating PPARG to regulate SOD2. PPARG, as a key member of the nuclear receptor superfamily, not only participates in the regulation of lipid metabolism and insulin sensitivity but also regulates the antioxidant defense system through direct or indirect mechanisms. After treatment with sea cucumber gonadotropic peptides, PPARG expression recovered in a dose-dependent manner, SOD2 expression was significantly upregulated, and NF-κB activation was inhibited. This result suggests that the peptide may activate PPARG to directly upregulate SOD2 to enhance antioxidant capacity, and indirectly protect SOD2 from inhibition by inflammatory factors by antagonizing NF-κB activation and inflammatory gene expression. Furthermore, PPARG activation can also improve mitochondrial function, providing a stable microenvironment for SOD2 to exert its role in scavenging superoxide anions.

[0079] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of an active peptide for preventing or treating alcoholic liver injury in the preparation of products for preventing or treating alcoholic liver injury, characterized in that, The active peptide is selected from at least one of the peptides whose amino acid sequences are shown in SEQ ID NO. 1~3.

2. The application according to claim 1, characterized in that, It is used in the preparation of drugs for the prevention or treatment of alcoholic liver injury.

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