Application of a buckwheat antioxidant peptide as a PPARα agonist

Abstract

This invention provides an application of tartary buckwheat antioxidant peptide as a PPARα agonist. The tartary buckwheat antioxidant peptide (SEQ ID NO:1) is used to prepare a PPARα agonist, and this tartary buckwheat antioxidant peptide is used to prepare a drug for improving metabolic dysfunction-related fatty liver disease (MASLD). The tartary buckwheat antioxidant peptide of this invention provides a new drug candidate for improving MASLD and has broad clinical application prospects.

Description

Technical Field

[0001] This invention belongs to the field of buckwheat antioxidant peptide technology, specifically relating to the application of a buckwheat antioxidant peptide as a PPARα agonist. Background Technology

[0002] Metabolic dysfunction-associated fatty liver disease (MASLD) is one of the most common chronic liver diseases worldwide. Its pathological features include excessive fat accumulation in hepatocytes, metabolic disorders, inflammatory responses, and liver fibrosis. In severe cases, it can develop into fatty hepatitis, cirrhosis, or even liver cancer.

[0003] Peroxisome proliferator-activated receptor alpha (PPARα), a core regulator of lipid metabolism, regulates fatty acid oxidation, anti-inflammation, and anti-fibrosis in hepatocytes by activating the expression of downstream fatty acid oxidation genes. Therefore, PPARα is a key target for the treatment of MASLD, and developing PPARα agonists for use as drugs or functional foods for MASLD treatment is of great significance. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to address the shortcomings of the prior art by providing a buckwheat antioxidant peptide as a PPARα agonist. This buckwheat antioxidant peptide provides a new candidate drug for improving MASLD and has broad clinical application prospects.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: the application of a buckwheat antioxidant peptide as a PPARα agonist, wherein the buckwheat antioxidant peptide is used to prepare a PPARα agonist, and the buckwheat antioxidant peptide is used to prepare a drug for improving metabolic dysfunction-associated fatty liver disease (MASLD); the amino acid sequence of the buckwheat antioxidant peptide is shown in SEQ ID NO:1.

[0006] Preferably, the tartary buckwheat antioxidant peptide is used to specifically activate PPARα and upregulate the expression of downstream fatty acid oxidation-related genes of PPARα.

[0007] Preferably, the downstream fatty acid oxidation-related genes of PPARα include CPT1a, Resat, Cyp4a, and Cyp7a.

[0008] Preferably, the tartary buckwheat antioxidant peptides are used to reduce lipid accumulation and inhibit the process of liver fibrosis.

[0009] Preferably, the PPARα agonist is a pharmaceutically acceptable carrier and excipient.

[0010] Preferably, the drug contains one or more pharmaceutically acceptable carriers comprising an amino acid sequence of tartary buckwheat antioxidant peptides.

[0011] Preferably, the tartary buckwheat antioxidant peptides are used as the active ingredient to prepare any dosage form, such as tablets, capsules, powders, mixtures, pills, sprays, granules, oral liquids, or injections.

[0012] Compared with the prior art, the present invention has the following advantages: The tartary buckwheat antioxidant peptide (SEQ ID NO:1) of the present invention has PPARα-specific activating activity. Its application as a PPARα agonist fills the technical gap of natural tartary buckwheat polypeptide as a PPARα agonist, enriches the types of PPARα agonists, and can be used to prepare drugs to improve metabolic dysfunction-associated fatty liver disease (MASLD).

[0013] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0014] Figure 1 This is a diagram showing the activation of PPARα by the buckwheat antioxidant peptide AFYRW in Example 2 of the present invention.

[0015] Figure 2 This figure shows the expression levels of the tartary buckwheat antioxidant peptide AFYRW upregulating the expression levels of downstream fatty acid oxidation-related genes CPT1a, Resat, Cyp4a, and Cyp7a of PPARα in the MASLD mouse model of Example 3 of this invention. ** in the figure indicates a significant difference of p < 0.01, and *** indicates a significant difference of p < 0.001.

[0016] Figure 3 In the MASLD mouse model of Example 3 of this invention, the buckwheat antioxidant peptide AFYRW reduced lipid accumulation and lowered blood lipids. A is a mouse liver image, B is a mouse liver Oil Red O staining image, C is a mouse liver H&E staining image, and D is a graph showing the levels of free fatty acids, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol in mouse serum. In the figure, * indicates a significant difference of p < 0.05, ** indicates a significant difference of p < 0.01, and *** indicates a significant difference of p < 0.001.

[0017] Figure 4 The figures show the inhibition of inflammation and liver fibrosis by the tartary buckwheat antioxidant peptide AFYRW in the MASLD mouse model of Example 3 of this invention. A shows the expression level of IL-6 protein in mouse liver, B shows the Oil Red O staining of mouse liver, and C shows the H&E staining of mouse liver. ** in the figures indicate significant differences (p < 0.01).

[0018] Figure 5This diagram illustrates the activation of PPARα and upregulation of downstream genes CPT1a, Resat, Cyp4a, and Cyp7a by the tartary buckwheat antioxidant peptide AFYRW in the lipid accumulation cell model of Example 4 of this invention. A shows the PPARα protein expression level in mouse liver, B shows the luciferase gene reporter assay results, and C shows the expression levels of downstream fatty acid oxidation-related genes CPT1a, Resat, Cyp4a, and Cyp7a. In the figures, * indicates a significant difference of p < 0.05, ** indicates a significant difference of p < 0.01, and *** indicates a significant difference of p < 0.001.

[0019] Figure 6 This image shows the inhibition of lipid accumulation by the tartary buckwheat antioxidant peptide AFYRW in the lipid accumulation cell model of Example 4 of this invention. A is an Oil Red O staining image of cells, B is a graph showing the levels of free fatty acids, triglycerides, and total cholesterol in cells, and C is a graph showing the expression level of IL-6 protein in cells. In the figure, * indicates a significant difference with p < 0.05, and ** indicates a significant difference with p < 0.01. Detailed Implementation

[0020] Example 1

[0021] The preparation method of the buckwheat antioxidant peptide (SEQ ID NO:1) in this invention is described in Chinese Patent CN109336953B. The buckwheat antioxidant peptide in this invention is the buckwheat antioxidant peptide P3 prepared in Chinese Patent CN109336953B. In this invention, the buckwheat antioxidant peptide is named buckwheat antioxidant peptide AFYRW.

[0022] The application of the buckwheat antioxidant peptide AFYRW as a PPARα agonist in this embodiment is described in Examples 2 and 3, which are in vivo experiments, and Example 4 is an in vitro experiment.

[0023] Tartary buckwheat antioxidant peptides are used to prepare PPARα agonists, and tartary buckwheat antioxidant peptide AFYRW is used to prepare drugs to improve metabolic dysfunction-associated fatty liver disease (MASLD).

[0024] The tartary buckwheat antioxidant peptide AFYRW is used to specifically activate PPARα and upregulate the expression of downstream fatty acid oxidation-related genes of PPARα.

[0025] The downstream fatty acid oxidation-related genes of PPARα include CPT1a, Resat, Cyp4a, and Cyp7a.

[0026] The tartary buckwheat antioxidant peptide AFYRW is used to reduce lipid accumulation and inhibit the process of liver fibrosis.

[0027] The PPARα agonist is a pharmaceutically acceptable carrier and excipient.

[0028] The drug contains one or more pharmaceutically acceptable carriers comprising an amino acid sequence containing tartary buckwheat antioxidant peptides.

[0029] Using the aforementioned buckwheat antioxidant peptides as active ingredients, the preparation can be made into any of the following dosage forms: tablets, capsules, powders, mixtures, pills, sprays, granules, oral liquids, or injections. Example 2

[0030] This example demonstrates how the tartary buckwheat antioxidant peptide AFYRW stimulates the expression of PPARα.

[0031] Male C57BL / 6 mice (6-8 weeks old) were purchased from the Animal Experiment Center of Guizhou Medical University. All animals were housed in a specific pathogen-free (SPF) environment at a temperature of 22±2℃ and a light / dark cycle of 12 hours.

[0032] The experiment was divided into two groups: NC: Control group mice, fed standard diet for 20 weeks; NC+AFYRW: Mice in the AFYRW treatment group were fed a standard diet and injected intraperitoneally with AFYRW solution (80 mg / kg / d) for 20 weeks.

[0033] After the reaction, all mice were anesthetized and sacrificed, and liver tissue was collected to extract tissue proteins for immunoblotting experiments. Figure 1 As shown, the expression level of PPARα in the liver tissue of mice treated with AFYRW (NC+AFYRW) was significantly higher than that in the control group (NC), indicating that AFYRW effectively stimulated the expression of PPARα. Example 3

[0034] This embodiment demonstrates how the tartary buckwheat antioxidant peptide AFYRW stimulates the expression of downstream genes of PPARα in the liver of MASLD mice and inhibits lipid accumulation and blood lipid levels in the liver of MASLD mice.

[0035] Male C57BL / 6 mice (6-8 weeks old) were purchased from the Animal Experiment Center of Guizhou Medical University. All animals were housed in a specific pathogen-free (SPF) environment at a temperature of 22±2℃ and a light / dark cycle of 12 hours.

[0036] The experiment was divided into four groups: NC: Control group mice, fed standard diet for 20 weeks; NC+AFYRW: Mice in the AFYRW treatment group were fed a standard diet and injected intraperitoneally with AFYRW solution (80 mg / kg / d) for 20 weeks. MASLD: Mice in the metabolic dysfunction-associated fatty liver disease group were fed a high-fat diet (78.5% standard diet, 21% lard and 0.5% cholesterol) for 20 weeks. MASLD+AFYRW: Mice in the AFYRW treatment group were fed a high-fat diet (78.5% standard diet, 21% lard and 0.5% cholesterol) and were intraperitoneally injected with AFYRW solution (80 mg / kg / d) for 20 weeks.

[0037] After the reaction was completed, all mice were anesthetized and sacrificed, and their liver tissue and serum were collected for subsequent experiments.

[0038] like Figure 2 As shown, the expression levels of CPT1a, Rest, Cyp4a, and Cyp7a genes in the liver tissue of mice treated with AFYRW (NC+AFYRW) were significantly higher than those in the control group (NC); moreover, the expression levels of CPT1a, Rest, Cyp4a, and Cyp7a genes in the liver tissue of mice treated with AFYRW (MASLD+AFYRW) were also significantly higher than those in mice with metabolic dysfunction-related fatty liver disease (MASLD). These results indicate that the tartary buckwheat antioxidant peptide AFYRW activates the expression of downstream lipid oxidation-related genes CPT1a, Rest, Cyp4a, and Cyp7a associated with PPARα.

[0039] like Figure 3 As shown in (A), the livers of the control group (NC), the AFYRW treatment group (NC+AFYRW), and the AFYRW therapy group (MASLD+AFYRW) were all dark red, while the livers of the metabolic dysfunction-associated fatty liver disease group (MASLD) were light red. Figure 3 As shown in (BC), Oil Red O staining and H&E staining results of mouse liver tissue showed that in the MASLD group of mice with metabolic dysfunction-associated fatty liver disease, the cytoplasm appeared red, and a large number of fat vacuoles appeared in the liver lobule structure (arrows in the MASLD group in Figure C point to lipid droplets); while in the AFYRW treatment group of mice (MASLD+AFYRW), the cytoplasm of the liver tissue appeared blue, and the number of fat vacuoles was reduced. Figure 3As shown in (D), serum free fatty acids and low-density lipoprotein cholesterol levels were significantly upregulated in mice with metabolic dysfunction-associated fatty liver disease (MASLD) compared to the control group (NC), while AFYRW (MASLD+AFYRW) significantly reduced serum free fatty acids and low-density lipoprotein cholesterol levels. Serum high-density lipoprotein cholesterol levels were significantly downregulated in MASLD mice compared to the control group (NC), while AFYRW (MASLD+AFYRW) significantly restored serum high-density lipoprotein cholesterol levels. These results indicate that the tartary buckwheat antioxidant peptide AFYRW significantly reduced hepatic lipid accumulation and blood lipid levels in MASLD mice.

[0040] like Figure 4 As shown in (A), the level of the liver inflammatory factor IL-6 in mice with metabolic dysfunction-associated fatty liver disease (MASLD) was significantly upregulated compared with the control group (NC), while AFYRW (MASLD+AFYRW) significantly reduced IL-6 levels. Figure 4 As shown in (BC), MASSON staining and α-SMA immunohistochemical staining results revealed significantly increased α-SMA expression in the central hepatic vein of mice with metabolic dysfunction-associated fatty liver disease (MASLD) (α-SMA is a marker of myofibroblasts), indicating that some hepatocytes in the central vein of MASLD have transformed into myofibroblasts, marking the occurrence of local fibrosis. AFYRW treatment (MASLD+AFYRW) effectively reduced α-SMA protein expression in the central hepatic vein, reduced myofibroblast formation, and alleviated the malignant progression of MASLD. These results indicate that the tartary buckwheat antioxidant peptide AFYRW significantly inhibited liver inflammation and fibrosis in MASLD mice. Example 4

[0041] This example illustrates the effect of the tartary buckwheat antioxidant peptide AFYRW on a lipid-accumulating cell model.

[0042] A lipid-accumulating cell model was constructed by co-culturing HepG2 cells with a mixture of 0.25 mmol / L palmitic acid and oleic acid for 12 h.

[0043] The experiment was divided into three groups: Control: Control group cells; M: Lipid-accumulated cell model group, HepG2 cells were co-cultured with a mixture of 0.25 mmol / L palmitic acid and oleic acid for 12 h; M+AFYRW: Cells in the AFYRW treatment group were co-cultured with HepG2 cells for 12 h with a mixture of 0.25 mmol / L palmitic acid and oleic acid, while AFYRW (40 μg / mL) was added for treatment.

[0044] like Figure 5 As shown in (A), PPARα expression in the AFYRW treatment group (M+AFYRW) cells was significantly upregulated compared to the lipid accumulation cell model group (M). Luciferase gene reporter assays also showed that PPARα activation was more pronounced in the AFYRW treatment group cells (M+AFYRW), such as... Figure 5 (B). Furthermore, AFYRW significantly upregulated the expression of downstream fatty acid oxidation-related genes CPT1a, Resat, Cyp4a, and Cyp7a of PPARα, such as... Figure 5 (C).

[0045] like Figure 6 As shown in (A), Oil Red O staining results indicated that, compared to the control group (Control), the lipid accumulation cell model group (M) had a large number of red lipid droplets distributed in the cytoplasm, while AFYRW (M+AFYRW) reduced intracellular lipid droplets and lipid accumulation. Furthermore, as... Figure 6 As shown in (B), AFYRW (M+AFYRW) significantly reduced the levels of free fatty acids, triglycerides, and total cholesterol in the lipid-accumulated cell model group (M). Simultaneously, AFYRW (M+AFYRW) significantly reduced the expression level of IL-6 in the lipid-accumulated cell model group (M), such as... Figure 6 (C).

[0046] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. The application of a buckwheat antioxidant peptide as a PPARα agonist, characterized in that, The tartary buckwheat antioxidant peptide is used to prepare a PPARα agonist, and the tartary buckwheat antioxidant peptide is used to prepare a drug to improve fatty liver disease related to metabolic dysfunction; the amino acid sequence of the tartary buckwheat antioxidant peptide is shown in SEQ ID NO:

1.

2. The application of the buckwheat antioxidant peptide according to claim 1 as a PPARα agonist, characterized in that, The tartary buckwheat antioxidant peptides are used to specifically activate PPARα and upregulate the expression of downstream fatty acid oxidation-related genes of PPARα.

3. The application of the buckwheat antioxidant peptide according to claim 2 as a PPARα agonist, characterized in that, The downstream fatty acid oxidation-related genes of PPARα include CPT1a, Resat, Cyp4a, and Cyp7a.

4. The application of the buckwheat antioxidant peptide according to claim 2 as a PPARα agonist, characterized in that, The tartary buckwheat antioxidant peptides are used to reduce lipid accumulation and inhibit the process of liver fibrosis.

5. The application of the buckwheat antioxidant peptide according to claim 1 as a PPARα agonist, characterized in that, The PPARα agonist is a pharmaceutically acceptable carrier and excipient.

6. The application of the buckwheat antioxidant peptide according to claim 1 as a PPARα agonist, characterized in that, The drug contains one or more pharmaceutically acceptable carriers comprising an amino acid sequence containing tartary buckwheat antioxidant peptides.

7. The application of a tartary buckwheat antioxidant peptide according to any one of claims 1-6 as a PPARα agonist, characterized in that, Using the aforementioned buckwheat antioxidant peptides as active ingredients, the preparation can be made into any of the following dosage forms: tablets, capsules, powders, mixtures, pills, sprays, granules, oral liquids, or injections.

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