Application of nattokinase in preparation of medicine for relieving alcoholic liver injury disease

Abstract

The invention belongs to the technical field of microbial fermentation protease, and particularly relates to application of nattokinase in preparation of a medicine for relieving alcoholic liver injury diseases. The nattokinase disclosed by the invention can be used for effectively relieving the increase of the liver-weight ratio of mice caused by alcohol; meanwhile, nattokinase can improve liver fat accumulation induced by alcohol; hepatocyte arrangement orderliness is recovered, and inflammatory cell infiltration is reduced; the antioxidant capacity can be improved, and the condition of liver function damage can be relieved; and the effect of recovering the normal liver blood fat level is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial fermentation protease, and particularly relates to the application of nattokinase in the preparation of drugs for relieving alcoholic liver injury diseases. Background Art

[0002] Alcoholic liver injury (ALD) is a liver disease caused by long-term excessive drinking, and its development process includes three stages: fatty liver, alcoholic hepatitis, and cirrhosis. Acetaldehyde produced by alcohol metabolism in the liver can trigger oxidative stress and inflammatory responses. This process is not only related to the direct toxic effects of alcohol and its metabolites (such as acetaldehyde), but also involves multiple pathophysiological mechanisms such as oxidative stress, inflammatory responses, lipid metabolism disorders, gut microbiota dysbiosis, and immune system dysfunction. Clinical manifestations include symptoms such as fatigue, jaundice, and ascites, and severe cases can progress to liver failure. The progression of ALD has stage characteristics, and each stage may be accompanied by different clinical manifestations and pathological changes. Therefore, early intervention and treatment are crucial for preventing the progression of the disease.

[0003] Nattokinase (NK) is an alkaline serine protease produced by Bacillus subtilis during natto fermentation. It is well-known for its strong fibrinolytic activity and is considered a safe, effective, low-cost, and easily absorbable functional dietary supplement for the treatment of cardiovascular diseases. Compared with existing clinical drugs, it has the advantages of safety, definite efficacy, and convenient oral administration, making NK have broad application prospects in drug research. The present invention further studies nattokinase (NK) and finds that nattokinase can effectively reduce the increase in liver weight ratio caused by alcohol in mice; at the same time, nattokinase can improve alcohol-induced liver fat accumulation; restore the orderly arrangement of hepatocytes and reduce inflammatory cell infiltration; it is also found that it can improve its antioxidant capacity, reduce liver function impairment; and has the effect of restoring normal liver blood lipid levels. The present invention provides the application of nattokinase in the preparation of drugs for relieving alcoholic liver injury diseases. Summary of the Invention

[0004] To achieve the above object, the present invention provides the application of nattokinase in the preparation of drugs for relieving alcoholic liver injury diseases.

[0005] The technical solution adopted by the present invention is:

[0006] The application of nattokinase in the preparation of drugs for relieving alcoholic liver injury diseases.

[0007] Further, in the above application, the manifestation of relieving alcoholic liver injury diseases is that nattokinase restores the body weight level in alcoholic liver injury.

[0008] Further, in the above application, the manifestation of relieving alcoholic liver injury diseases is that nattokinase relieves the increase in liver weight ratio in alcoholic liver injury.

[0009] Further, in the above application, the manifestation of alleviating alcoholic liver injury disease is that nattokinase alleviates liver fat accumulation in alcoholic liver injury.

[0010] Further, in the above application, the manifestation of alleviating alcoholic liver injury disease is that nattokinase restores the orderly arrangement of hepatocytes and reduces inflammatory cell infiltration in alcoholic liver injury.

[0011] Further, in the above application, the manifestation of alleviating alcoholic liver injury disease is that nattokinase improves the antioxidant capacity in alcoholic liver injury.

[0012] Further, in the above application, the manifestation of alleviating alcoholic liver injury disease is that nattokinase alleviates the impairment of liver function in alcoholic liver injury.

[0013] Further, in the above application, the manifestation of alleviating alcoholic liver injury disease is that nattokinase restores the normal liver blood lipid level in alcoholic liver injury.

[0014] Compared with the prior art, the present invention has the following beneficial effects: Compared with the prior art, the present invention firstly discloses that nattokinase can effectively reduce the increase in liver weight of mice caused by alcohol; at the same time, nattokinase can improve alcohol-induced liver fat accumulation; restore the orderly arrangement of hepatocytes and reduce inflammatory cell infiltration; it is also found that it can improve its antioxidant capacity, alleviate the impairment of liver function; and has the effect of restoring the normal liver blood lipid level. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is a graph showing the effect of nattokinase on the body weight of mice (compared with the alcohol group, ***p < 0.001).

[0016] Figure 2 It is a quantitative graph of the pathological score of the liver weight ratio of nattokinase (compared with the alcohol group, **p < 0.01, compared with the positive drug treatment group, *p < 0.5).

[0017] Figure 3 It is a graph showing the effect of nattokinase on the liver morphology of mice. Among them, (A) normal control group; (B) alcohol group;

[0018] (C) positive drug treatment group; (D) nattokinase treatment group.

[0019] Figure 4 It is a graph showing the pathological effect of nattokinase on the liver tissue of mice. Among them, (A) normal control group; (B) alcohol group;

[0020] (C) positive drug treatment group; (D) nattokinase treatment group.

[0021] Figure 5 It is a graph showing the effect of nattokinase on the antioxidant capacity of mice. Among them, (A) Serum MDA level (compared with the alcohol group, ***p < 0.001, compared with the positive drug treatment group, the p value is not significant); (B) Serum SOD level (compared with the alcohol group, *p < 0.5, compared with the positive drug treatment group, the p value is not significant).

[0022] Figure 6 It is a graph showing the effect of nattokinase on liver function impairment in mice. Among them, (A) Serum ALT level (compared with the alcohol group, ***p < 0.001, compared with the positive drug treatment group, the p value is not significant); (B) Serum AST level (compared with the alcohol group, ***p < 0.001, compared with the positive drug treatment group, the p value is not significant).

[0023] Figure 7 It is a graph showing the effect of nattokinase on the blood lipid levels in the liver of mice. Among them, (A) T-CHO value in liver tissue (compared with the alcohol group, ***p < 0.001, compared with the positive drug treatment group, the p value is not significant); (B) TG value in liver tissue (compared with the alcohol group, *p < 0.5, compared with the positive drug treatment group, *p < 0.5); (C) LDL value in liver tissue (compared with the alcohol group, **p < 0.01, compared with the positive drug treatment group, the p value is not significant); (D) HDL value in liver tissue (compared with the alcohol group, *p < 0.5, compared with the positive drug treatment group, the p value is not significant). Detailed implementation method

[0024] For the convenience of further understanding of the present invention, the following examples provide more detailed descriptions of it. However, these examples are only for better understanding of the present invention and not for limiting the scope or implementation principles of the present invention. The implementation methods of the present invention are not limited to the following content.

[0025] Example 1 Nattokinase restores body weight level in alcoholic liver injury

[0026] 1. Main materials and reagents

[0027] Nattokinase was purchased from Shuangjun Biotechnology Co., Ltd. SPF-grade 8-week-old ICR male mice were purchased from Liaoning Changsheng Biotechnology Co., Ltd. The positive drug silybin was purchased from Tasly Sant Pharmaceutical Co., Ltd.

[0028] 2. Animal intervention and grouping: Administration was carried out through the gavage route for each group. The normal control group was gavaged with normal saline; the alcohol group (model group) was given 50% ethanol at a dose of 10 g / kg; the nattokinase treatment group was given alcohol and nattokinase at a specific concentration, and the positive drug treatment group was given a certain concentration of alcohol and silybin. The test substances were given 2 hours after gavage of alcohol in the latter two groups, and the alcohol dose was the same as that of the model group.

[0029] 3. Specimen collection and processing: After the last administration, the mice were subjected to 12-hour fasting (free access to water). Subsequently, the mice were anesthetized by intraperitoneal injection of 3% sodium pentobarbital solution (40 mg / kg). The liver was quickly excised, weighed, and rinsed with pre-cooled normal saline.

[0030] 4. Record the body weight every morning and evening. One-way ANOVA was performed using Graphpad Prism. A significant difference was observed when p < 0.05, a very significant difference when p < 0.01, and an extremely significant difference when p < 0.001.

[0031] As Figure 1 shown, compared with the normal control group, the body weight of the mice in the alcohol group was significantly reduced, and the damage to the digestive system led to a short-term weight loss. There was no significant difference between the nattokinase treatment group and the positive drug treatment group, and the body weight was significantly increased compared with the alcohol group. This indicates that nattokinase can improve the short-term weight loss in mice induced by alcohol.

[0032] Example 2 Nattokinase alleviates the increase in liver weight ratio in alcoholic liver injury

[0033] Animal intervention, grouping, specimen collection, and processing were the same as in Example 1. By separating the liver tissues of the mice, weighing the liver, and calculating the liver index, the effect of alcohol gavage on liver weight and the effect of nattokinase were evaluated by comparing the liver indices of each group of mice. The data processing method was the same as in Example 1.

[0034] Liver weight ratio = liver weight / body weight × 100%

[0035] As Figure 2 shown, compared with the normal control group, the liver index of the mice in the alcohol group showed a significant upward trend, indicating that alcohol-induced liver injury led to an increase in liver weight. The liver index of the nattokinase treatment group was significantly reduced, and the liver index of the positive drug treatment group (silibinin) was also significantly lower than that of the alcohol group, suggesting that nattokinase can effectively reduce the increase in liver weight caused by alcohol.

[0036] Example 3 Nattokinase alleviates liver fat accumulation in alcoholic liver injury

[0037] Animal intervention, grouping, specimen collection, and processing were the same as in Example 1. Observe the gross morphology of the liver to preliminarily evaluate the degree of liver injury.

[0038] The results are as Figure 3 shown. Compared with the normal control group, visible fat granules were accumulated in the alcohol group, while there was no obvious fat granule accumulation in the nattokinase treatment group and the positive drug treatment group. Nattokinase improved the liver fat accumulation caused by alcohol.

[0039] Example 4 Nattokinase restores the orderly arrangement of hepatocytes and reduces inflammatory cell infiltration in alcoholic liver injury

[0040] Animal intervention, grouping, specimen collection and processing were the same as in Example 1. HE staining: Its standard operation process includes key steps such as tissue fixation, dehydration and clearing, paraffin embedding, sectioning, dewaxing and hydration, staining, differentiation and blueing, dehydration and clearing, and mounting. After staining, the cell nuclei were blue, the cytoplasm and collagen fibers were pink, and the pathological changes of liver tissue were observed and photographed under a light microscope.

[0041] The results were as Figure 4 shown that the hepatocytes of mice in the normal control group had intact morphology, were arranged orderly, the cell nuclei were large and round, located in the center of the cells, and the nucleoli and liver structures were clearly visible. In the alcohol group, the hepatocytes showed fatty degeneration, with vacuoles of different sizes distributed in the cytoplasm, accompanied by inflammatory cells, and the hepatic cords were arranged disorderly and the structure was blurred. Compared with the alcohol group, the hepatocyte damage in the nattokinase treatment group was significantly alleviated, only a small amount of lipid droplets were seen, no obvious pathological abnormalities were found, and the arrangement of hepatic cord structures tended to be regular. In the positive drug treatment group, the hepatocytes had no obvious degeneration or necrosis, occasional small lipid vacuoles were seen, the hepatic sinusoids were not dilated, and the hepatic lobule structure was clearly intact. Nattokinase could restore the orderly arrangement of hepatocytes and reduce fatty degeneration in alcoholic liver injury.

[0042] Example 5 Nattokinase improves its antioxidant capacity in alcoholic liver injury

[0043] Animal intervention, grouping, specimen collection and processing were the same as in Example 1. Determination of MDA and SOD values: Whole blood samples were collected by cardiac puncture. The whole blood samples could be left standing at room temperature for 2 hours or placed in a 4°C refrigerator overnight; centrifuged at 3000 rpm for 15 minutes at 4°C, and then centrifuged at 3000 revolutions per minute at 4°C for 15 minutes; carefully aspirate the supernatant, and use a malondialdehyde (MDA) assay kit and a total superoxide dismutase (T-sod) assay kit, and an automatic biochemical analyzer to detect the indicators. The data processing method was the same as in Example 1.

[0044] The results were as Figure 5 shown that the MDA value of mice in the alcohol group was significantly increased compared with that of mice in the normal control group, while the MDA values of the nattokinase treatment group and the positive drug treatment group were significantly decreased compared with the alcohol group. The SOD value of mice in the alcohol group was significantly decreased compared with that of mice in the normal control group, and the SOD values of the nattokinase treatment group and the positive drug treatment group were significantly increased compared with the alcohol group. Nattokinase improves its antioxidant capacity in alcoholic liver injury.

[0045] Example 6 Nattokinase alleviates liver function impairment in alcoholic liver injury

[0046] Animal intervention, grouping, specimen collection and processing were the same as in Example 1. Determination of ALT and AST levels: The alanine aminotransferase (ALT / GPT) colorimetric assay kit and aspartate aminotransferase (AST / GOT) colorimetric assay kit were used for determination, and the enzyme marker was used to detect the indexes. The data processing method was the same as in Example 1.

[0047] The results were as Figure 6 shown. By detecting their biochemical levels, the AST and ALT values of the mice in the alcohol group were significantly higher than those of the mice in the normal control group. The AST and ALT values of the nattokinase treatment group and the positive drug treatment group were significantly lower than those of the alcohol group. Nattokinase alleviated the impairment of liver function in mice with alcoholic liver injury.

[0048] Example 7 Nattokinase restores normal liver blood lipid levels in alcoholic liver injury

[0049] Animal intervention, grouping, specimen collection and processing were the same as in Example 1. The low-density lipoprotein cholesterol (LDL) assay kit, high-density lipoprotein cholesterol (HDL) assay kit, triglyceride (TG) assay kit and total cholesterol (T-CHO) assay kit were used for determination, and the enzyme marker was used to detect the indexes. The data processing method was the same as in Example 1.

[0050] The results were as Figure 7 , the TG, T-CHO, and LDL levels of the mice in the alcohol group showed a significant upward trend compared with those of the mice in the normal control group. The levels of the nattokinase treatment group and the positive drug treatment group were significantly lower than those of the alcohol group. The situation of HDL was opposite. Nattokinase can improve the abnormal blood lipid levels caused by alcohol and restore them to normal levels.

Claims

1. Application of nattokinase in the preparation of drugs for alleviating alcoholic liver damage.

2. The use according to claim 1, characterized in that: The manifestation of alleviating alcoholic liver damage disease is that nattokinase restores the body weight level in alcoholic liver damage.

3. The use according to claim 1, characterized in that: The manifestation of alleviating alcoholic liver damage disease is that nattokinase alleviates the increase of liver-to-weight ratio in alcoholic liver damage.

4. The use according to claim 1, characterized in that: The manifestation of alleviating alcoholic liver damage disease is that nattokinase alleviates liver fat accumulation in alcoholic liver damage.

5. The use according to claim 1, characterized in that: The manifestation of alleviating alcoholic liver damage disease is that nattokinase restores the orderly arrangement of liver cells in alcoholic liver damage and reduces inflammatory cell infiltration.

6. The use according to claim 1, characterized in that: The manifestation of alleviating alcoholic liver damage disease is that nattokinase improves the antioxidant capacity in alcoholic liver damage.

7. The use according to claim 1, characterized in that: The manifestation of alleviating alcoholic liver damage disease is that nattokinase alleviates liver function impairment in alcoholic liver damage.

8. The use according to claim 1, characterized in that: The manifestation of alleviating alcoholic liver damage disease is that nattokinase restores normal liver blood lipid levels in alcoholic liver damage.

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