Application of tartary buckwheat antioxidant peptide as OGT inhibitor

Abstract

The invention provides application of a tartary buckwheat antioxidant peptide as an OGT inhibitor, the tartary buckwheat antioxidant peptide has an amino acid sequence as shown in SEQ ID NO: 1, and the tartary buckwheat antioxidant peptide is used for preparing an N-acetylglucosamine transferase inhibitor and preparing a medicine for improving insulin resistance. The tartary buckwheat antioxidant peptide disclosed by the invention can be used as an N-acetylglucosamine transferase (O-GlcNAc transferase, OGT) and a coded sugar chain O-linked N-acetylglucosamine (O-GlcNA) inhibitor for treating insulin resistance, and is used for preparing a medicine for improving the insulin resistance.

Description

Technical Field

[0001] The invention belongs to the technical field of application of antioxidant active peptides, and particularly relates to application of tartary buckwheat antioxidant peptide as an OGT inhibitor. Background Art

[0002] Insulin resistance is specifically manifested as the insensitivity of target organs or target cells to the effects of insulin, resulting in a reduced response of the body to insulin. In addition, insulin resistance is closely related to obesity, metabolic syndrome, cardiovascular disease and cancer. Therefore, improving insulin resistance is critical to the treatment of related diseases.

[0003] N-acetylglucosamine transferase (O-GlcNAc transferase, OGT) and its encoded sugar chain O-linked N-acetylglucosamine (O-GlcNA) are closely related to diabetes and cardiovascular diseases. Among them, insulin resistance can be improved by inhibiting the expression of OGT and O-GlcNAc. Therefore, it is very necessary to develop new OGT and O-GlcNAc inhibitors and use them as drugs for the treatment of insulin resistance and related diseases. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide an application of a buckwheat antioxidant peptide as an OGT inhibitor in view of the above-mentioned deficiencies in the prior art. The antioxidant active peptide can be used to prepare drugs for improving insulin resistance and has broad clinical application prospects.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is: an application of a tartary buckwheat antioxidant peptide as an OGT inhibitor, the tartary buckwheat antioxidant peptide is used to prepare an N-acetylglucosamine transferase inhibitor, and is used to prepare a drug for improving insulin resistance; the amino acid sequence of the tartary buckwheat antioxidant peptide is shown in SEQ ID NO: 1.

[0006] Preferably, the N-acetylglucosamine transferase inhibitor is a pharmaceutically acceptable carrier and excipient.

[0007] Preferably, the drug for improving insulin resistance contains one or more pharmaceutically acceptable salts or carriers comprising the amino acid sequence of tartary buckwheat antioxidant peptide.

[0008] Preferably, the tartary buckwheat antioxidant peptide is an active ingredient, and conventional adjuvants are added according to conventional processes to prepare any dosage form of clinically acceptable tablets, capsules, powders, mixtures, pills, sprays, granules, capsules, oral liquids, and injections.

[0009] Compared with the prior art, the present invention has the following advantages:

[0010] The tartary buckwheat antioxidant peptide (SEQ ID NO: 1) of the present invention can significantly improve insulin resistance by inhibiting OGT, and can be used as an OGT inhibitor for preparing a drug for improving insulin resistance.

[0011] The present invention is further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is a graph showing the effect of the bitter buckwheat antioxidant peptide AFYRW on the OGT gene expression level (A), OGT protein expression level (B) and O-GlcNAc glycosylation modification expression level (C) in the insulin resistance cell model of Example 2 of the present invention.

[0013] Figure 2 This is a diagram showing the ability of the tartary buckwheat antioxidant peptide AFYRW to improve glucose uptake in the insulin resistance cell model of Example 2 of the present invention.

[0014] Figure 3 In the insulin resistance cell model of Example 2 of the present invention, the bitter buckwheat antioxidant peptide AFYRW improves glucose uptake by inhibiting OGT. (A) Effects of OGT agonist (Thiamet G) and AFYRW on the expression levels of OGT and O-GlcNAc in insulin resistance cells. (B) Effects of OGT agonist (Thiamet G) and AFYRW on the glucose uptake capacity of insulin resistance cells.

[0015] Figure 4 This is a graph showing that the tartary buckwheat antioxidant peptide AFYRW inhibits the expression levels of OGT and O-GlcNAc in the liver of insulin-resistant mice in the insulin-resistant mouse model of Example 3 of the present invention.

[0016] Figure 5 This is a graph showing that the tartary buckwheat antioxidant peptide AFYRW improves the insulin level and insulin resistance index of insulin-resistant mice in the insulin-resistant mouse model of Example 3 of the present invention. DETAILED DESCRIPTION

[0017] Example 1

[0018] The preparation method of the tartary buckwheat antioxidant peptide (SEQ ID NO: 1) in the present invention is shown in Chinese patent CN109336953B. The tartary buckwheat antioxidant peptide in the present invention is the tartary buckwheat antioxidant peptide P3 prepared in Chinese patent CN109336953B. In the present invention, the tartary buckwheat antioxidant peptide is named as tartary buckwheat antioxidant peptide AFYRW.

[0019] Example 2 and Example 3 are applications of the bitter buckwheat antioxidant peptide AFYRW as N-acetylglucosamine transferase (O-GlcNAc transferase, OGT) and improving insulin resistance. Example 2 is an in vitro experiment, and Example 3 is an in vivo experiment.

[0020] The buckwheat antioxidant peptide AFYRW has inhibitory activity against N-acetylglucosamine transferase (O-GlcNAc transferase, OGT) and its encoded sugar chain O-linked N-acetylglucosamine (O-GlcNA), and can be used as an OGT inhibitor.

[0021] The bitter buckwheat antioxidant peptide AFYRW is used to prepare N-acetylglucosamine transferase (O-GlcNActransferase, OGT) and its encoded sugar chain O-linked N-acetylglucosamine (O-GlcNA) inhibitor, and the OGT inhibitor is a pharmaceutically acceptable carrier and excipient.

[0022] The tartary buckwheat antioxidant peptide AFYRW is used to prepare a drug for improving insulin resistance;

[0023] The medicine for improving insulin resistance contains one or more pharmaceutically acceptable salts or carriers containing the amino acid sequence of the tartary buckwheat antioxidant peptide.

[0024] The tartary buckwheat antioxidant peptide AFYRW is an active ingredient, and conventional adjuvants are added according to conventional processes to prepare any dosage form of clinically acceptable tablets, capsules, powders, mixtures, pills, sprays, granules, capsules, oral solutions and injections.

[0025] Example 2

[0026] Test A:

[0027] 1. Buckwheat antioxidant peptide AFYRW inhibits the expression of OGT and O-GlcNAc in insulin resistance cell model.

[0028] First, palmitic acid (0.25mmol / L) was co-cultured with a hepatocyte cell line (HepG2) for 24 hours, and then insulin (100nmol / L) was added to stimulate the cells for 20 minutes to construct an insulin resistance cell model.

[0029] The experiment was divided into four groups:

[0030] Control: control group;

[0031] IR-HepG2: insulin resistance cell model group;

[0032] IR-HepG2+AFYRW (40 μg / mL): insulin-resistant cells + buckwheat antioxidant peptide AFYRW (24 hours, 40 μg / mL) treatment group;

[0033] IR-HepG2+AFYRW (160μg / mL): insulin-resistant cells + buckwheat antioxidant peptide AFYRW (24 hours, 160μg / mL) treatment group.

[0034] After the reaction, cell proteins and mRNA were extracted for protein immunoblotting and real-time fluorescence quantitative PCR to detect the effect of tartary buckwheat antioxidant peptide AFYRW on the expression of OGT and O-GlcNAc glycosylation modification in insulin-resistant cells. Figure 1 As shown in A-1B, the gene and protein expression levels of OGT were upregulated in the insulin resistance cell model group (IR-HepG2), while the bitter buckwheat antioxidant peptide AFYRW could significantly inhibit the gene and protein expression levels of OGT in the insulin resistance cell model (IR-HepG2+AFYRW). Figure 1 As shown in C, the O-GlcNAc glycosylation modification level was upregulated in the insulin resistance cell model group (IR-HepG2), while AFYRW could significantly inhibit the expression level of O-GlcNAc glycosylation modification in the insulin resistance cell model (IR-HepG2+AFYRW).

[0035] 2. Buckwheat antioxidant peptide AFYRW improves the glucose uptake ability of insulin resistance cell model.

[0036] First, palmitic acid (0.25mmol / L) was co-cultured with a hepatocyte cell line (HepG2) for 24 hours, and then insulin (100nmol / L) was added to stimulate the cells for 20 minutes to construct an insulin resistance cell model.

[0037] The experiment was divided into four groups:

[0038] Control: control group;

[0039] IR-HepG2: insulin resistance cell model group;

[0040] IR-HepG2+AFYRW (40μg / mL): insulin-resistant cells + buckwheat antioxidant peptide AFYRW (24 hours, 40μg / mL) treatment group

[0041] IR-HepG2+AFYRW (160μg / mL): insulin-resistant cells + buckwheat antioxidant peptide AFYRW (24 hours, 160μg / mL) treatment group.

[0042] After the reaction, the insulin-resistant cells were co-cultured with insulin (100 nmol / L) for 20 min, and then the residual glucose in the cell culture supernatant was measured. The glucose uptake of the cells was equal to the initial glucose content (25 mmol / L) minus the residual glucose. Figure 2 As shown, the glucose uptake ability of the insulin resistance cell model group (IR-HepG2) was significantly inhibited, while the buckwheat antioxidant peptide AFYRW could significantly improve the glucose uptake ability of the insulin resistance cell model (IR-HepG2+AFYRW), indicating that the buckwheat antioxidant peptide AFYRW can improve insulin resistance by increasing glucose uptake.

[0043] 3. Buckwheat antioxidant peptide AFYRW improves the glucose uptake ability of insulin resistance cell model by inhibiting OGT.

[0044] First, palmitic acid (0.25mmol / L) was co-cultured with a hepatocyte cell line (HepG2) for 24 hours, and then insulin (100nmol / L) was added to stimulate the cells for 20 minutes to construct an insulin resistance cell model.

[0045] The experiment was divided into five groups:

[0046] Control: control group;

[0047] IR-HepG2: insulin resistance cell model group;

[0048] IR-HepG2+AFYRW (160 μg / mL): insulin-resistant cells + buckwheat antioxidant peptide AFYRW (24 hours, 160 μg / mL) treatment group;

[0049] IR-HepG2+Thiamet G: insulin-resistant cells+OGT agonist group (2 hours, 100 μM);

[0050] IR-HepG2+AFYRW+Thiamt G: insulin-resistant cells+buckwheat antioxidant peptide AFYRW (24 hours, 160 μg / mL)+OGT agonist group (2 hours, 100 μM).

[0051] like Figure 3 As shown in A, OGT agonist (Thiamet G) and palmitic acid significantly upregulated the expression of OGT and O-GlcNAc in insulin-resistant cells, while AFYRW could significantly inhibit the upregulation of OGT and O-GlcNAc induced by OGT agonist (Thiamet G) or palmitic acid. These results confirm that AFYRW is an inhibitor of OGT and O-GlcNAc expression.

[0052] In addition, if Figure 3 As shown in B, Thiamet G and palmitic acid both impaired the glucose uptake capacity of cells after upregulating OGT and O-GlcNAc, while AFYRW could completely improve the glucose uptake capacity impaired by Thiamet G or palmitic acid.

[0053] Example 3

[0054] Test A:

[0055] 1. Buckwheat antioxidant peptide AFYRW inhibits the expression of OGT and O-GlcNAc in the liver of insulin-resistant mice.

[0056] Male C57BL / 6 mice (6-8 weeks old) were purchased from the Animal Experiment Center of Guizhou Medical University. All mice were housed in a specific pathogen-free (SPF) environment at a temperature of 22 ± 2 °C and a light / dark cycle of 12 h.

[0057] The experiment was divided into three groups:

[0058] Control: The mice in the control group were fed with a normal diet.

[0059] HFD: The insulin resistance mouse model group (HFD) was fed with a high-fat diet containing 21% lard, 10% sugar, and 0.5% cholesterol for 5 consecutive weeks.

[0060] HFD+AFYRW: The insulin resistance mouse model + AFYRW treatment group (HFD+AFYRW) was intraperitoneally injected with buckwheat antioxidant peptide AFYRW (20 mg / kg) every day 1 week in advance, and then fed a high-fat diet and injected with buckwheat antioxidant peptide AFYRW (20 mg / kg) every day for 5 consecutive weeks.

[0061] Finally, the HFD group and HFD+AFYRW group were injected with streptozotocin (55 mg / kg) once a day for 5 consecutive days, and the control group (Control) was injected with citrate buffer.

[0062] After the reaction, all mice were anesthetized and killed, and liver tissue was taken to extract tissue proteins for immunoblotting experiments. Figure 4 As shown in the results, the expression levels of OGT and O-GlcNAc in the liver tissue of the insulin resistance mouse model group (HFD) were significantly higher than those in the normal control group (Control), while the buckwheat antioxidant peptide AFYRW could significantly inhibit the expression of OGT and O-GlcNAc in insulin resistance mice (HFD+AFYRW), reversing them to normal expression levels.

[0063] 2. Buckwheat antioxidant peptide AFYRW improves insulin levels and insulin resistance index in insulin-resistant mice.

[0064] Male C57BL / 6 mice (6-8 weeks old) were obtained 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 °C and a 12-h light / dark cycle.

[0065] The experiment was divided into four groups:

[0066] Control: The mice in the control group were fed with a normal diet.

[0067] HFD: The insulin resistance mouse model group (HFD) was fed with a high-fat diet containing 21% lard, 10% sugar, and 0.5% cholesterol for 5 consecutive weeks.

[0068] HFD+AFYRW: The insulin resistance mouse model + AFYRW treatment group (HFD+AFYRW) was intraperitoneally injected with buckwheat antioxidant peptide AFYRW (20 mg / kg) every day 1 week in advance, and then fed a high-fat diet and injected with buckwheat antioxidant peptide AFYRW (20 mg / kg) every day for 5 consecutive weeks.

[0069] HFD+Met: Insulin resistance mouse model + metformin treatment group (HFD+Met, 0.5 g / kg) served as the positive drug control group.

[0070] Finally, the HFD, HFD+AFYRW, and HFD+Met groups were injected with streptozotocin (55 mg / kg) once a day for 5 days, and the control group was injected with citrate buffer.

[0071] After the reaction, all mice were anesthetized and killed, and serum samples were taken to detect insulin levels. Figure 5 As shown in the data, the fasting insulin level (FINS) and insulin resistance index (HOMA-IR) of the insulin resistance mouse model group (HFD) were significantly increased, while AFYRW or the positive drug metformin (HFD+AFYRW, HFD+Met) could reduce their FINS and HOMA-IR.

[0072] In summary, the present invention improves insulin resistance by inhibiting OGT expression through the bitter buckwheat antioxidant peptide AFYRW. Therefore, the bitter buckwheat antioxidant peptide AFYRW can be used to prepare drugs that inhibit OGT and improve insulin resistance. The drug contains a pharmaceutically acceptable carrier and excipient with an amino acid sequence of SEQ ID NO: 1, which is used to treat insulin resistance. The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent change made to the above embodiments according to the essence of the invention technology still falls within the protection scope of the technical solution of the present invention.

Claims

1. A use of a tartary buckwheat antioxidant peptide as an OGT inhibitor, characterized in that: The tartary buckwheat antioxidant peptide is used for preparing an N-acetylglucosamine transferase inhibitor and a drug for improving insulin resistance; the amino acid sequence of the tartary buckwheat antioxidant peptide is shown in SEQ ID NO:

1.

2. The use of a tartary buckwheat antioxidant peptide as an OGT inhibitor according to claim 1, characterized in that: The N-acetylglucosamine transferase inhibitor is a pharmaceutically acceptable carrier and excipient.

3. The use of a tartary buckwheat antioxidant peptide as an OGT inhibitor according to claim 1, characterized in that: The drug for improving insulin resistance contains one or more pharmaceutically acceptable salts or carriers containing the amino acid sequence of tartary buckwheat antioxidant peptide.

4. The use of a tartary buckwheat antioxidant peptide as an OGT inhibitor according to claim 1, characterized in that: The tartary buckwheat antioxidant peptide is an active ingredient, and is prepared into any dosage form of clinically acceptable tablets, capsules, powders, mixtures, pills, sprays, granules, capsules, oral liquids, and injections by conventional processes and adding conventional adjuvants.

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