Application of nattokinase in preparation of medicine for preventing or treating non-alcoholic fatty liver disease

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to application of nattokinase in preparation of medicine for preventing or treating non-alcoholic fatty liver disease. A high fat diet induced ApoE gene knockout mouse (ApoE- / -mouse) model experiment proves that the nattokinase obviously reduces the levels of serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL) and liver nitric oxide (NO) on the premise of not influencing the body weight, and improves abnormal liver lipid metabolism. Histological analysis shows that the pathological injury of the liver of a mouse in a nattokinase treatment group is obviously reduced, and the liver-to-weight ratio is reduced, the liver cell degeneration is reduced, the lipid droplet accumulation is relieved, and the liver blood sinus structure tends to be normal. The invention provides a safe and effective novel medicine candidate for preventing and treating the non-alcoholic fatty liver disease.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of nattokinase in the preparation of drugs for preventing or treating non-alcoholic fatty liver disease. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) is a metabolic disease characterized by excessive lipid deposition in the liver, with a global prevalence as high as 25%, and is closely related to obesity and insulin resistance. In the past decade, it has been shown that the clinical burden of non-alcoholic fatty liver disease is not limited to liver-related morbidity and mortality, and now there is increasing evidence that non-alcoholic fatty liver disease is a multi-system disease that affects multiple extrahepatic organs and regulatory pathways. Non-alcoholic fatty liver disease has become the main cause of chronic liver disease in many parts of the world, and non-alcoholic fatty liver disease may also lead to an important burden of extrahepatic chronic complications. At present, there are no clinically approved drugs for non-alcoholic fatty liver disease, and the treatment mainly involves lifestyle changes through diet and exercise and intervention with lipid-lowering drugs (such as statins), but there is a risk of liver toxicity. However, it is usually difficult for patients with non-alcoholic fatty liver disease to maintain an improved lifestyle. Therefore, it is of great practical significance to find safe and effective drugs for preventing and treating non-alcoholic fatty liver disease.

[0003] Nattokinase (NK) is a serine protease extracted from natto and is known to have thrombolytic, anti-inflammatory and microcirculation-improving effects. Nattokinase can increase insulin secretion and reduce insulin resistance, thereby improving type 2 diabetes with insulin deficiency. Nattokinase can reverse the decline in bacterial community diversity and intestinal microbial imbalance. However, its therapeutic potential in non-alcoholic fatty liver disease has not been clearly revealed.

[0004] ApoE - / - ApoE- / - mice are an animal model with complete deletion of apolipoprotein E constructed by gene knockout technology. Due to their characteristics of spontaneous atherosclerosis and abnormal lipid metabolism, they have become an important tool for studying cardiovascular diseases, lipid metabolism syndrome and neurological diseases. The present invention uses ApoE- / - mice induced by a high-fat diet to confirm the application of nattokinase in the preparation of drugs for preventing or treating non-alcoholic fatty liver disease. Summary of the Invention

[0005] The present invention for the first time confirms that nattokinase can effectively prevent and treat non-alcoholic fatty liver disease by regulating lipid metabolism, inhibiting oxidative stress and repairing liver pathological damage. Therefore, the present invention provides the application of nattokinase in the preparation of drugs for preventing or treating non-alcoholic fatty liver disease.

[0006] The technical solution adopted by the present invention is:

[0007] Use of nattokinase in the preparation of a medicament for preventing or treating non-alcoholic fatty liver disease.

[0008] Furthermore, in the above-mentioned use, the manifestations of preventing or treating non-alcoholic fatty liver disease include at least one of the following:

[0009] Manifestation 1: Reducing serum total cholesterol, triglyceride and low-density lipoprotein levels, as well as reducing liver nitric oxide, triglyceride and total cholesterol levels;

[0010] Manifestation 2: Alleviating liver pathological damage;

[0011] Manifestation 3: Restoring the normal liver morphology and the ratio of liver weight.

[0012] Even further, in the above-mentioned use, the alleviation of liver pathological damage includes reducing hepatocyte vacuolar degeneration, lipid droplet accumulation and inflammatory infiltration.

[0013] Furthermore, in the above-mentioned use, nattokinase is the only active ingredient in the medicament.

[0014] Furthermore, in the above-mentioned use, the dosage form of the medicament is an injection or an oral liquid.

[0015] Furthermore, in the above-mentioned use, the administration method of the medicament is intraperitoneal injection or oral administration.

[0016] Furthermore, in the above-mentioned use, the medicament contains nattokinase and a pharmaceutically acceptable carrier.

[0017] The use of nattokinase provided by the present invention in the preparation of a medicament for preventing or treating non-alcoholic fatty liver disease has the following beneficial effects:

[0018] 1. Without affecting the body weight of mice, NK reduces the levels of TC, TG, and LDL in the serum, and has a dose-dependence.

[0019] 2. NK can restore the appearance lesions of the liver and reduce the liver weight ratio.

[0020] 3. NK can reduce hepatocyte degeneration and decrease the lipid droplet area of non-alcoholic fatty liver.

[0021] 4. NK can reduce liver lipid accumulation, including reducing the contents of liver TG and TC.

[0022] 5. Inhibiting liver inflammation and oxidative stress: NK reduces the production of liver nitric oxide (NO).

[0023] Nattokinase has the effect of treating non-alcoholic fatty liver disease and can be used as a therapeutic drug for non-alcoholic fatty liver disease, providing a new and better choice for such drugs, health products, etc. Description of the Drawings

[0024] Figure 1 This is the result graph of the effect of nattokinase on the body weight of high-fat diet-induced ApoE gene knockout mice (ApoE - / - mice).

[0025] Figure 2 This is the result graph of the effect of nattokinase on the four lipid parameters of high-fat diet-induced ApoE gene knockout mice (ApoE - / - mice). Among them, A is TG, B is TC, C is LDL, and D is HDL.

[0026] Figure 3 This is the result graph of the effect of nattokinase on the liver morphology of high-fat diet-induced ApoE gene knockout mice (ApoE - / - mice).

[0027] Figure 4 This is the result graph of the effect of nattokinase on the liver weight ratio of high-fat diet-induced ApoE gene knockout mice (ApoE - / - mice).

[0028] Figure 5 This is the pathological result graph of HE staining of the liver of high-fat diet-induced ApoE gene knockout mice (ApoE - / - mice) treated with nattokinase.

[0029] Figure 6 This is the result graph of Oil Red O staining of the liver of high-fat diet-induced ApoE gene knockout mice (ApoE - / - mice) treated with nattokinase.

[0030] Figure 7 This is the effect of nattokinase on the contents of TG and TC in the liver of high-fat diet-induced ApoE gene knockout mice (ApoE - / - mice). Among them, A is TG and B is TC.

[0031] Figure 8 This is the result of the effect of nattokinase on the NO content in the liver of high-fat diet-induced ApoE gene knockout mice (ApoE - / - mice). Detailed implementation manners

[0032] The following examples will further illustrate the present invention, but do not limit the present invention thereby.

[0033] Example 1 Construction and grouping of animal models

[0034] 1. Grouping and Intervention: After 1 week of adaptive feeding with standard feed, mice were randomly divided into 3 groups: Model group (High-fat diet group, HF): Fed with high-fat diet for 16 weeks and intraperitoneally injected with normal saline daily; Low-dose nattokinase group (HF+L-NK): Fed with high-fat diet + nattokinase (5000 FU / kg / every two days, intraperitoneally injected) for 16 weeks; High-dose nattokinase group (HF+H-NK): Fed with high-fat diet + nattokinase (10000 FU / kg / every two days, intraperitoneally injected) for 16 weeks. Nattokinase was purchased from Shuangjun Biotechnology Co., Ltd.

[0035] 2. Sample Collection: After the experiment, the mice were fasted for 24 hours. After anesthesia, serum, aorta and liver tissues were collected. Part of the liver tissue was fixed with 4% paraformaldehyde for pathological analysis, and the rest was stored at -80 °C.

[0036] Example 2 Monitoring of Body Weight Changes

[0037] 1. Weighing Method: The body weight of mice was weighed weekly using a precision electronic balance and recorded to one decimal place.

[0038] 2. Experimental Results: As Figure 1 shown, during the intervention period, the body weights of mice in each group increased with the increase of weeks of age, but there was no significant difference among groups.

[0039] Example 3 Serum Biochemical Detection

[0040] 1. Sample Processing: Blood was collected from the orbital cavity, allowed to stand, and then centrifuged at 4 °C and 3000×g for 15 minutes to separate the serum, which was stored at -80 °C to avoid repeated freezing and thawing.

[0041] 2. Index Detection: Serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) were detected using an automatic biochemical analyzer.

[0042] 3. Experimental Results: As Figure 2 shown, compared with the model group, the levels of TC, TG, and LDL in the nattokinase group decreased in a dose-dependent manner. There was no significant difference in the HDL levels among the three groups, indicating that this intervention had no obvious effect on serum HDL.

[0043] Example 4 Analysis of Liver Morphology and Liver Weight Ratio

[0044] 1. Sample Processing: After anesthesia, the mice were dissected, the liver was completely peeled off, rinsed with normal saline, the surface moisture was blotted dry with filter paper and photographed, and then weighed with an electronic balance.

[0045] 2. Calculation of Liver Weight Ratio: Liver weight ratio (%) = (liver weight / body weight) × 100.

[0046] 3. Experimental Results: As Figure 3 、 4As shown, obvious lesions appeared in the livers of the mice in the model group. The appearance was diffusely enlarged, the color turned yellow, the surface was shiny, and there was an oily feeling. While in the nattokinase group, the livers of the mice tended to be normal with the increase of the administration dose, and the liver weight ratio decreased.

[0047] Example 5 Histopathological Analysis of Liver Tissue by HE Staining

[0048] 1. Experimental method: Take liver tissue, fix it with 4% paraformaldehyde for 24 hours, embed it in paraffin, and the section thickness is 4μm; stain with hematoxylin-eosin, observe and take pictures under an optical microscope.

[0049] 2. Experimental results: As Figure 5 shown, in the model group, a large number of hepatocytes were swollen, showing hydropic degeneration, and the cytoplasm was loose and lightly stained; granulocytes were occasionally seen around the central vein; a small amount of chromatin margination of hepatocyte nuclei was visible. In the low-dose nattokinase group, a large number of hepatocytes showed vacuolar degeneration, and tiny round vacuoles were visible in the cytoplasm; the hepatic sinusoids were not significantly dilated or compressed. In the high-dose nattokinase group, a small number of hepatocytes showed vacuolar degeneration, and tiny round vacuoles were visible in the cytoplasm; the hepatic sinusoids were not significantly dilated or compressed, and no obvious necrosis or inflammatory cell infiltration and other abnormalities were seen.

[0050] Example 6 Lipid Detection of Liver by Oil Red O Staining

[0051] 1. Experimental method: Take fresh liver tissue, embed it in OCT, and prepare 8μm frozen sections; stain with Oil Red O staining solution for 15 minutes, counterstain the nucleus with hematoxylin, and seal the sections with neutral balsam for photographing and analysis.

[0052] 2. Experimental results: As Figure 6 shown, in the model group: The red lipid droplets were densely distributed in the liver tissue, and a large number of obvious red plaques were visible, indicating severe liver fat deposition induced by a high-fat diet. In the low-dose nattokinase group: The number of red lipid droplets decreased compared with the model group, and the staining intensity weakened, indicating that low-dose intervention had a certain improvement effect on liver fat deposition. In the high-dose nattokinase group: The red lipid droplets further decreased, and the stained area was sparser, showing that high-dose intervention had a more significant improvement effect on liver fat deposition, and the degree of fat accumulation was the lowest. By comparison, with the intervention treatment (increase in NK dose), the liver fat deposition showed a decreasing trend, reflecting the regulatory effect of nattokinase on liver lipid metabolism.

[0053] Example 7 Determination of TC and TG Contents in Liver

[0054] 1. Experimental method: Take 100mg of liver tissue, homogenize the tissue with absolute ethanol, centrifuge and take the supernatant. Detect the levels of TC and TG in the supernatant with TG kit and TC kit respectively. Both the TG and TC kits are purchased from Nanjing Jiancheng Bioengineering Institute.

[0055] 2. Experimental results: AsFigure 7 As shown, the TG content in the liver of the high-fat diet group was 431.4 ± 12.0 μmol / g, the TG content in the liver of the low-dose nattokinase group was 269.8 ± 23.2 μmol / g, and the TG content in the liver of the high-dose nattokinase group was 236.9 ± 20.0 μmol / g. Compared with the model group, the treatment with high-dose nattokinase significantly reduced the level of TG in the liver of ApoE - / - mice (p < 0.0001). The TC content in the liver of the high-fat diet group was 131.4 ± 7.7 μmol / g, the TC content in the liver of the low-dose nattokinase group was 111.8 ± 12.0 μmol / g, and the TC content in the liver of the high-dose nattokinase group was 103.0 ± 11.0 μmol / g. Compared with the model group, the treatment with high-dose nattokinase significantly reduced the level of TC in the liver of ApoE - / - mice (p < 0.001).

[0056] Example 8 Determination of NO Content in the Liver

[0057] 1. Experimental method: The sample treatment was the same as in Example 7, and the NO kit was used to detect the level of NO in the supernatant. The NO detection kit was purchased from Nanjing Jiancheng Bioengineering Institute.

[0058] 2. Experimental results: As Figure 8 shown, the NO content in the liver of the high-fat diet group was 498.7 ± 51.2 nmol / g, the NO content in the liver of the low-dose nattokinase group was 214.1 ± 92.9 nmol / g, and the NO content in the liver of the high-dose nattokinase group was 169.0 ± 39.8 nmol / g. Compared with the model group, the treatment with nattokinase could significantly reduce the NO content in the liver of ApoE - / - mice (p < 0.0001).

Claims

1. Application of nattokinase in the preparation of drugs for preventing or treating non-alcoholic fatty liver disease.

2. The use according to claim 1, characterized in that: The prevention or treatment of manifestations of non-alcoholic fatty liver disease includes at least one of the following: Manifestation 1: Reduce serum total cholesterol, triglycerides and low-density lipoprotein levels, and reduce liver nitric oxide, triglycerides and total cholesterol levels; Performance 2: Alleviate liver pathological damage; Symptom three: Restoration of the liver's normal morphology and liver weight ratio.

3. The use according to claim 2, characterized in that: The mitigation of liver pathological damage includes reducing vacuolar degeneration of hepatocytes, lipid droplet accumulation and inflammatory infiltration.

4. The use according to claim 1, characterized in that: Nattokinase is the only active ingredient in the medicine.

5. The use according to claim 1, characterized in that: The dosage form of the medicine is injection or oral solution.

6. The use according to claim 1, characterized in that: The drug is administered by intraperitoneal injection or oral administration.

7. The use according to claim 1, characterized in that: The medicine comprises nattokinase and a pharmaceutically acceptable carrier.

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