Application of beta-hydroxybutyric acid in preparation of medicine for treating glycogenic liver diseases

Abstract

The invention discloses application of beta-hydroxybutyric acid in preparation of a medicine for treating glycogenic liver diseases. The glycogenic liver diseases are usually found in young type 1 diabetes patients with poor blood glucose control, and the symptoms are liver swelling, transaminase increase, hepatic glycogen increase and the like. According to the application, the beta-hydroxybutyric acid is found to be capable of remarkably improving the hepatic complications (namely the glycogenic liver diseases) of the type 1 diabetes for the first time, and a new medication and a new treatment strategy are provided for treating the glycogenic liver diseases.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceutical technology, and particularly to the application of β-hydroxybutyric acid in the preparation of drugs for treating glycogenotic hepatopathy. Background Art

[0002] Type 1 diabetes (T1D) is a chronic metabolic disease, mainly manifested by the destruction of pancreatic islet β cells, resulting in insufficient or complete lack of insulin secretion. T1D accounts for about 10% of global diabetes cases and is the most common type of diabetes in children and adolescents. T1D patients face the dual challenges of difficult blood glucose control and serious complications, including cardiovascular diseases, liver diseases, and kidney diseases, etc., which not only seriously affect the quality of life of patients but also shorten the life expectancy of patients.

[0003] Before the discovery of insulin in 1921, the only treatment option for T1D patients was a low-carbohydrate diet. By reducing carbohydrate intake, the catabolism of fatty acids in the body produces ketone bodies (KB) as an alternative energy source for blood glucose. Ketone bodies include β-hydroxybutyric acid, acetoacetic acid, and acetone, among which β-hydroxybutyric acid (3HB) is the most abundant and stable ketone body. Although ketone bodies play beneficial roles as signaling molecules in various physiological and pathological processes such as metabolic regulation and neuroprotection, currently, the specific effects of ketone bodies on complications in T1D, especially their effects on the liver, are still unclear. Summary of the Invention

[0004] The purpose of the present invention is to provide the application of β-hydroxybutyric acid in the preparation of drugs for treating glycogenotic hepatopathy in view of the deficiencies in the prior art.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is:

[0006] Provide the application of β-hydroxybutyric acid in the preparation of drugs for treating glycogenotic hepatopathy.

[0007] Furthermore, the only active ingredient of the drug for treating glycogenotic hepatopathy is β-hydroxybutyric acid.

[0008] Even further, the drug for treating glycogenotic hepatopathy further includes pharmaceutically acceptable excipients.

[0009] By adopting the above technical solution, compared with the prior art, the present invention has the following technical effects:

[0010] Glycogenotic hepatopathy is usually seen in young type 1 diabetes patients with poor blood glucose control, manifested as liver enlargement, elevated transaminases, and increased hepatic glycogen, etc. The present invention for the first time discovers that β-hydroxybutyric acid can significantly improve the liver complications (i.e., glycogenotic hepatopathy) of type 1 diabetes, providing a new medication and treatment strategy for treating glycogenotic hepatopathy. Brief Description of the Drawings

[0011] Figure 1 It shows the effect of 3HB on the body weight of type 1 diabetic mice.

[0012] Figure 2 It shows the effect of 3HB on the food intake of type 1 diabetic mice.

[0013] Figure 3 It shows the effect of 3HB on the water intake of type 1 diabetic mice.

[0014] Figure 4 It shows the effect of 3HB on the serum insulin level of type 1 diabetic mice.

[0015] Figure 5 It shows that 3HB significantly improves glycogenotic liver disease in type 1 diabetic mice, alleviates hepatomegaly, reduces liver glycogen content, and inhibits active glycogen synthesis in the liver; among them, (A) representative liver photos obtained by macroscopic examination; (B) liver weights of mice in each group; (C) liver index (the ratio of liver weight to body weight); (D) representative pictures of H&E and periodic acid-Schiff (PAS) staining from liver tissues; scale bar, 50 μm; (E) glycogen levels in liver tissues; (F) serum AST and ALT levels; (G) protein levels of glycogen synthesis metabolism-related genes in the liver; (H-I) relative protein ratios of p-GS / GS (H) and p-GSK3β / GSK3β (I) were determined by densitometric analysis of immunoblotting results.

[0016] Figure 6 It shows that 3HB directly inhibits glycogen synthesis in primary mouse hepatocytes; among them, (A) representative pictures of glycogen staining in primary hepatocytes, scale bar, 100 μm; (B) quantitative analysis of glycogen content in primary hepatocytes (n = 4); (C) expression levels of glycogen synthesis-related proteins in primary hepatocytes; (D) relative protein ratios of p-GS / GS and p-GSK3β / GSK3β were determined by densitometric analysis of immunoblotting results. Detailed implementation manners

[0017] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but it is not intended to limit the present invention. It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other.

[0018] The present invention discovers that β-hydroxybutyric acid (3HB) can significantly improve the liver complications of type 1 diabetes, namely glycogenotic liver disease. The specific experiments are as follows:

[0019] 1. Experimental animals and cells

[0020] Male C57BL / 6J mice at 9 to 10 weeks of age (body weight 22–24 g) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. Primary hepatocytes were isolated from male C57BL / 6J mice at 8–10 weeks of age.

[0021] 2. Animal models and interventions

[0022] After a one-week adaptation period, 24 mice were randomly assigned to a control group (CON, n = 8) and an experimental group (n = 16). All mice were fed a standard normal diet. Mice in the experimental group were intraperitoneally injected with STZ (150 mg / kg, dissolved in sodium citrate buffer, pH = 4.2 - 4.5) to induce type 1 diabetes, and mice in the control group were injected with an equal volume of sodium citrate buffer. Three days after STZ injection, the fasting blood glucose (FBG) of the experimental mice was measured. When FBG > 16.8 mM and accompanied by polydipsia, polyphagia, polyuria, and weight loss, the mice were determined to be type 1 diabetic model mice. All T1D mice were further randomly divided into two groups according to body weight and FBG level: a model group (STZ group, n = 8, orally administered normal drinking water, the same as the control group) and a treatment group (STZ + 3HB-Na, n = 8, orally administered sodium β-hydroxybutyrate solution). The sodium β-hydroxybutyrate solution was prepared by dissolving 4.0 g of sodium β-hydroxybutyrate powder (Solarbio) in 1 L of distilled water. Mice in each group received continuous intervention for 7 weeks. During this period, the food intake and water intake were recorded daily, and the body weight changes were recorded every 3 days.

[0023] 3. Experimental results

[0024] The livers of mice in the STZ group showed typical features of glycogenotic liver disease, while 3HB treatment significantly alleviated this complication ( Figures 1 - 4 ).

[0025] The liver weight and liver index of mice in the STZ group were significantly increased. However, the liver morphology of mice in the 3HB treatment group was significantly improved, and the liver weight and liver index were significantly lower than those in the STZ group ( Figure 5 A - C).

[0026] We performed histopathological analysis on liver tissues. The results of H&E staining showed that the hepatic lobule structure was clear in the liver sections of mice in the normal group, and the eosinophilic cytoplasm was abundant; while the sections of mice in the STZ group showed typical pathological features of glycogenotic liver disease, including significant diffuse hepatocyte changes, pale hepatocytes, sparse cytoplasm, and varying degrees of cell swelling and vacuolation. Notably, these pathological changes were significantly alleviated in the 3HB treatment group. PAS staining showed that the glycogen distribution in the livers of mice in the STZ group increased diffusely, while the glycogen distribution in the livers of mice in the 3HB treatment group decreased significantly ( Figure 5 D).

[0027] Further determination of liver tissue glycogen content showed that the liver glycogen content in the STZ group of mice increased by more than 5-fold compared to the normal control group, and the serum transaminase level increased significantly. In contrast, the liver glycogen accumulation in the 3HB-Na group of mice was significantly improved ( Figure 5 E-F).

[0028] Furthermore, Western blot experiments were conducted to investigate the effect of 3HB on the protein levels related to the liver glycogen metabolism pathway. As shown in Figure 5 G-I), the phosphorylation level of glycogen synthase (GS) in the liver of mice in the STZ group decreased, while the phosphorylation level of glycogen synthase kinase 3β (GSK3β) increased significantly. This indicates that glycogen synthesis in the liver of the STZ group was very active, and 3HB treatment significantly inhibited this active glycogen synthesis metabolism in the liver.

[0029] In addition, the present invention evaluated the effect of different concentrations of glucose or 3HB on the glycogen content of primary hepatocytes by glycogen staining. The results showed that Figure 6 ), 3HB treatment significantly inhibited glycogen synthesis under high glucose (50 mM) conditions. Under low glucose conditions (5.6 mM), relatively less glycogen was synthesized, and the concentration-dependent effect of 3HB was manifested as a decrease in glycogen content. Through immunoblot analysis, it was found that as the glucose concentration increased, the expression level of phosphorylated glycogen synthase (GS) decreased, while the level of phosphorylated glycogen synthase kinase 3β (GSK3β) increased significantly. This indicates that high glucose directly promoted glycogen synthesis independently of insulin. Importantly, as the concentration of 3HB increased, glycogen synthesis in hepatocytes was significantly inhibited, indicating that 3HB has a direct inhibitory effect on glycogen synthesis in hepatocytes. In summary, 3HB has a direct effect on the remission of glycogenotic liver disease in type 1 diabetic mice.

[0030] The above are only the preferred embodiments of the present invention, and do not limit the implementation manner and protection scope of the present invention. For those skilled in the art, it should be realized that all equivalent replacements and obvious changes made by using the content of the present invention's specification and illustrations should be included in the protection scope of the present invention.

Claims

1. Application of β-hydroxybutyric acid in the preparation of drugs for the treatment of glycogen liver disease.

2. The use according to claim 1, characterized in that: The only active ingredient of the drug for treating glycogen liver disease is β-hydroxybutyric acid.

3. The use according to claim 2, characterized in that: The drug for treating glycogen liver disease also includes pharmaceutically acceptable excipients.

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