Application of a buckwheat antioxidant peptide as a PARP1 inhibitor

Abstract

This invention provides an application of a tartary buckwheat antioxidant peptide as a PARP1 inhibitor. This tartary buckwheat antioxidant peptide (SEQ ID NO:1) is used to prepare a poly(ADP-ribose) polymerase 1 inhibitor, and is used to prepare a drug for preventing and / or improving diabetic retinopathy. The tartary buckwheat antioxidant peptide of this invention exerts multiple biological functions by inhibiting PARP1 / PAR: on the one hand, it reduces NAD... + On the one hand, it depletes nutrients, and on the other hand, it protects vascular endothelial function by inhibiting angiogenesis. This buckwheat antioxidant peptide can be used as a drug to prevent and / or improve diabetic retinopathy.

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 PARP1 inhibitor. Background Technology

[0002] Diabetic retinopathy (DR) is one of the most common microvascular complications of diabetes. The pathological mechanisms of DR involve hyperglycemia-induced oxidative stress, inflammatory responses, and pathological angiogenesis. Currently, the main clinical drugs for DR are anti-VEGF antibodies, which require frequent intravitreal injections, resulting in poor patient compliance and inadequate response in some patients. Therefore, developing novel therapeutic agents that can be administered orally or via eye drops is of significant clinical importance.

[0003] PARP1 is a core molecule for cellular sensing of oxidative stress. Under the hyperglycemic conditions of diabetes, excessively activated PARP1 consumes large amounts of NAD+. + This leads to mitochondrial dysfunction and induces pathological angiogenesis by promoting VEGF expression. Therefore, targeting PARP1 is a potential new strategy for treating DR. Summary of the Invention

[0004] The technical problem to be solved by this invention is to address the shortcomings of the prior art by providing a buckwheat antioxidant peptide as a PARP1 inhibitor. This buckwheat antioxidant peptide exerts multiple biological functions by inhibiting PARP1 / PAR: on the one hand, it reduces NAD... + On the one hand, it depletes nutrients, and on the other hand, it protects vascular endothelial function by inhibiting angiogenesis. This buckwheat antioxidant peptide can be used as a drug to prevent and / or improve diabetic retinopathy.

[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 PARP1 inhibitor, wherein the buckwheat antioxidant peptide is used to prepare a polyadenosine diphosphate ribose polymerase 1 (PARP1) inhibitor, and the buckwheat antioxidant peptide is used to prepare a drug for preventing and / or improving diabetic retinopathy; the amino acid sequence of the buckwheat antioxidant peptide is shown in SEQ ID NO:1.

[0006] Preferably, the buckwheat antioxidant peptides are used to reduce PAR levels.

[0007] Preferably, the tartary buckwheat antioxidant peptide is used to inhibit NAD. + Consumption.

[0008] Preferably, the tartary buckwheat antioxidant peptide is used to inhibit angiogenesis.

[0009] Preferably, the drug is one or more pharmaceutically acceptable oral or ophthalmic preparations containing the amino acid sequence of tartary buckwheat antioxidant peptides.

[0010] Preferably, the oral preparation is any clinically acceptable dosage form, such as tablets, capsules, powders, mixtures, pills, sprays, granules, or oral liquids; the ophthalmic preparation is any dosage form, such as eye drops, ophthalmic gels, or intravitreal injections.

[0011] Compared with the prior art, the present invention has the following advantages: The buckwheat antioxidant peptide (SEQ ID NO:1) of the present invention reduces angiogenesis by inhibiting PARP1 and its product PAR, and can be used as a PARP1 inhibitor for the preparation of drugs for the treatment and prevention of diabetic retinopathy.

[0012] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0013] Figure 1 In Embodiment 2 of the present invention, NAD is enhanced by inhibiting PARP1. + Levels. (A) Small interfering RNA (siPARP1) inhibits the expression levels of PARP1 and PAR in cells. (B) Small interfering RNA (siPARP1) reduces NAD in cells by inhibiting PARP1. + Consumption, thereby increasing NAD + Level. * in the figure indicates a significant difference with p < 0.05.

[0014] Figure 2 The graph shows the effect of the tartary buckwheat antioxidant peptide AFYRW on PARP1, PAR expression levels (A), and PARP1 enzyme activity (B) in Example 2 of this invention.

[0015] Figure 3 In the insulin-resistant cell model of Example 2 of this invention, the tartary buckwheat antioxidant peptide AFYRW affected the expression levels of PARP1, PAR (A), and NAD. + Effect of content (B) plot. *** in the plot indicates a significant difference of p < 0.001.

[0016] Figure 4 In the oxidative stress cell model of Example 3 of this invention, the tartary buckwheat antioxidant peptide AFYRW affected the expression levels of PARP1, PAR (A), and NAD. + Effect of content (B) plot. In the figure, * indicates a significant difference with p < 0.05, and ** indicates a significant difference with p < 0.01.

[0017] Figure 5This figure shows the effects of the tartary buckwheat antioxidant peptide AFYRW on in vitro angiogenesis (AB), VEGF mRNA expression level (C), and VEGF protein level (DE) in the oxidative stress cell model of Example 3 of this invention. * 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. Detailed Implementation

[0018] Example 1

[0019] 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.

[0020] The application of buckwheat antioxidant peptides as PARP1 inhibitors in the present embodiment, wherein the buckwheat antioxidant peptides are used to prepare polyadenosine diphosphate ribose polymerase 1 (PARP1) inhibitors, and wherein the buckwheat antioxidant peptides are used to prepare drugs for preventing and / or improving diabetic retinopathy.

[0021] The tartary buckwheat antioxidant peptide is used to reduce PAR levels. As a PARP1 inhibitor, the tartary buckwheat antioxidant peptide achieves its function by reducing PAR levels.

[0022] The tartary buckwheat antioxidant peptides are used to inhibit NAD. + Buckwheat antioxidant peptides, as PARP1 inhibitors, work by inhibiting NAD+. + It consumes resources to achieve its function.

[0023] The tartary buckwheat antioxidant peptide is used to inhibit angiogenesis and is used to prepare drugs that inhibit angiogenesis.

[0024] Examples 2 and 3 demonstrate that the tartary buckwheat antioxidant peptide AFYRW acts as a poly(ADP-ribose) polymerase 1 (PARP1) inhibitor, reducing NAD+. + Applications that consume and inhibit angiogenesis.

[0025] The drug is one or more pharmaceutically acceptable oral or ophthalmic preparations containing the amino acid sequence of tartary buckwheat antioxidant peptides; the oral preparation is any clinically acceptable dosage form of tablets, capsules, powders, mixtures, pills, sprays, granules, or oral liquids; the ophthalmic preparation is any dosage form of eye drops, ophthalmic gels, or intravitreal injections. Example 2

[0026] I. Inhibiting PARP1 levels via siRNA to reduce NAD in cells + Consumption.

[0027] Small interfering RNA (siRNA) was used to co-culture hepatocyte lines for 48-72 hours, followed by detection of PARP1 and PAR levels, as well as NAD+ levels. + content.

[0028] The experiment was divided into two groups: siControl: Control group; siPARP1: siRNA knockdown of PARP1 expression group.

[0029] like Figure 1 As shown in (A), compared with the siControl group, the expression levels of PARP1 and its catalytic product PAR were decreased in the siPARP1 group. Further NAD detection... + The levels of NAD in the siPARP1 group were found to be higher than those in the siPARP1 group. + The level was significantly higher than that of the siControl group. Figure 1 (B). The above results indicate that inhibiting PARP1 expression can effectively reduce PAR production, thereby inhibiting NAD. + Consumption, increase intracellular NAD + level.

[0030] II. The inhibitory effect of AFYRW, an antioxidant peptide from tartary buckwheat, on PARP1 and PAR in cells.

[0031] First, hepatocytes were co-cultured with palmitic acid (0.25 mmol / L) for 24 hours, and then insulin (100 nmol / L) was added to stimulate the cells for 20 minutes to construct an insulin-resistant cell model.

[0032] The experiment was divided into three groups: Control group; IR: Insulin-resistant cell model group; IR + AFYRW: Insulin-resistant cells + tartary buckwheat antioxidant peptide AFYRW (160µg / mL) treatment group.

[0033] After the reaction, cellular proteins were extracted, and the PARP1 enzyme activity in the cells was measured. For example... Figure 2 As shown in (A), PARP1 and PAR expression were upregulated in the insulin-resistant cell model group (IR), while the tartary buckwheat antioxidant peptide AFYRW could inhibit the expression levels of PARP1 and PAR in the insulin-resistant cell model (IR+AFYRW). Figure 2 As shown in (B), the tartary buckwheat antioxidant peptide AFYRW inhibits the activity of PARP1 enzyme in cells.

[0034] III. The tartary buckwheat antioxidant peptide AFYRW inhibits the expression of PARP1 and PAR in insulin-resistant cells.

[0035] First, hepatocytes were co-cultured with palmitic acid (0.25 mmol / L) for 24 hours, and then insulin (100 nmol / L) was added to stimulate the cells for 20 minutes to construct an insulin-resistant cell model.

[0036] The experiment was divided into four groups: Control group; IR: Insulin-resistant cell model group; IR+AFYRW: Insulin-resistant cells + tartary buckwheat antioxidant peptide AFYRW (160µg / mL) treatment group; IR+AFYRW+siPARP1: Insulin-resistant cells + tartary buckwheat antioxidant peptide AFYRW (160µg / mL) + siPARP1 knockdown group; like Figure 3 As shown in (A), compared with insulin-resistant cells (IR), AFYRW inhibited the expression of PARP1 and PAR (IR+AFYRW); moreover, AFYRW and siPARP1 worked together (IR+AFYRW+siPARP1), and the inhibitory effect on PARP1 and PAR expression in insulin-resistant cells was more obvious.

[0037] like Figure 3 As shown in (B), AFYRW (IR+AFYRW) reduces NAD in insulin-resistant cells (IR). + Consumption, thereby increasing NAD + The content; moreover, AFYRW and siPARP1 work together (IR+AFYRW+siPARP1) to reduce NAD in insulin-resistant cells (IR). + The increase in NAD+ levels was more pronounced. These results confirm that AFYRW is an inhibitor of PARP1 and PAR expression, and increases NAD+ levels by inhibiting PARP1 and PAR. + content. Example 3

[0038] I. Buckwheat antioxidant peptide AFYRW inhibits PAR expression in cells under oxidative stress.

[0039] A cell model of oxidative stress was constructed by co-culturing human umbilical vein endothelial cells with H2O2 (100 μmol / L) for 2 hours.

[0040] The experiment was divided into four groups: Control group; H2O2: Oxidative stress cell model group; H2O2+AFYRW (10μg / mL): Oxidative stress cells + tartary buckwheat antioxidant peptide AFYRW (24 hours, 10μg / mL) treatment group; H2O2+AFYRW (60μg / mL): Oxidative stress cells + tartary buckwheat antioxidant peptide AFYRW (24 hours, 60μg / mL) treatment group.

[0041] After the reaction, the expression levels of PARP1, PAR, and NAD in the cells of each group were measured. + content.

[0042] like Figure 4 As shown in (A), the expression level of PAR in oxidative stress cells (H2O2) was higher than that in the normal control group (Control), while the tartary buckwheat antioxidant peptide AFYRW could inhibit the expression level of PAR in oxidative stress cells (H2O2+AFYRW). Figure 4 As shown in (B), NAD in oxidative stress cells (H2O2) + The content was lower than that of the normal control group (Control), while the tartary buckwheat antioxidant peptide AFYRW can increase NAD in oxidative stress cells (H2O2+AFYRW). + content.

[0043] II. Buckwheat antioxidant peptide AFYRW inhibits angiogenesis in cells under oxidative stress.

[0044] Human umbilical vein endothelial cells were co-cultured with H2O2 (100 μmol / L) for 2 hours to construct an oxidative stress anti-cell model.

[0045] The experiment was divided into four groups: Control group; H2O2: Oxidative stress cell model group; H2O2+AFYRW (10μg / mL): Oxidative stress cells + tartary buckwheat antioxidant peptide AFYRW (24 hours, 10μg / mL) treatment group; H2O2+AFYRW (60μg / mL): Oxidative stress cells + tartary buckwheat antioxidant peptide AFYRW (24 hours, 60μg / mL) treatment group.

[0046] After the reaction, the tube formation ability and VEGF expression level in each group of cells were measured.

[0047] like Figure 5 As shown in (AB), oxidative stress cells (H2O2) form more tubular structures, while AFYRW (H2O2+AFYRW) can reduce tubular formation and inhibit angiogenesis. Figure 5As shown in C-5E, the mRNA and protein expression levels of VEGF are upregulated in oxidative stress cells (H2O2), while AFYRW (H2O2+AFYRW) can inhibit VEGF expression.

[0048] In summary, this invention reduces NAD by inhibiting the expression of PARP1 and its catalytic product PAR through the tartary buckwheat antioxidant peptide AFYRW. + It consumes and inhibits angiogenesis. Therefore, the tartary buckwheat antioxidant peptide AFYRW can be used to prepare PARP1 inhibitors for the preparation of drugs or functional foods for the treatment and prevention of diabetic retinopathy. The drugs contain pharmaceutically acceptable carriers and excipients with the amino acid sequence SEQ ID NO:1.

[0049] 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 PARP1 inhibitor, characterized in that, The tartary buckwheat antioxidant peptide is used to prepare a poly(ADP-ribose) polymerase 1 inhibitor, and the tartary buckwheat antioxidant peptide is used to prepare a drug for preventing and / or improving diabetic retinopathy; 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 PARP1 inhibitor, characterized in that, The tartary buckwheat antioxidant peptides are used to reduce PAR levels.

3. The application of the buckwheat antioxidant peptide according to claim 1 as a PARP1 inhibitor, characterized in that, The tartary buckwheat antioxidant peptides are used to inhibit NAD. + Consumption.

4. The application of the buckwheat antioxidant peptide according to claim 1 as a PARP1 inhibitor, characterized in that, The tartary buckwheat antioxidant peptides are used to inhibit angiogenesis.

5. The application of the tartary buckwheat antioxidant peptide according to any one of claims 1-4 as a PARP1 inhibitor, characterized in that, The drug is one or more pharmaceutically acceptable oral or ophthalmic preparations containing the amino acid sequence of tartary buckwheat antioxidant peptides.

6. The application of the buckwheat antioxidant peptide according to claim 5 as a PARP1 inhibitor, characterized in that, The oral preparation is any clinically acceptable dosage form, including tablets, capsules, powders, mixtures, pills, sprays, granules, and oral liquids; the ophthalmic preparation is any dosage form, including eye drops, ophthalmic gels, and intravitreal injections.

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