Use of benzethonium chloride in preparing medicine for improving or treating non-alcoholic fatty liver disease

Abstract

The present invention relates to a use of benzethonium chloride in preparing a drug for improving or treating non-alcoholic fatty liver disease, and relates to the technical field of new uses of products. The present invention discovers for the first time a new use of benzethonium chloride for improving or treating non-alcoholic fatty liver disease. Benzethonium chloride can reduce hepatocyte lipid deposition and regulate hepatocyte lipid metabolism disorder by intervening in a cell lipid deposition model; it can improve liver lipid accumulation in mice caused by lipid metabolism disorder by administering through the digestive tract, and can improve liver coefficient, liver function, hepatic lobular inflammation, and liver fibrosis. The benzethonium chloride of the present invention can be used for the preparation of drugs for improving or treating non-alcoholic fatty liver disease. The benzethonium chloride of the present invention can inhibit the expression of human apolipoprotein C3, thereby reducing the generation of lipids, and is applied to drugs for treating non-alcoholic fatty liver disease, opening up a new use of benzethonium chloride and providing a new drug option for treating non-alcoholic fatty liver disease.

Description

Technical Field

[0001] The invention relates to the technical field of new product applications, and in particular to an application of benzethonium chloride in preparing a medicament for improving or treating non-alcoholic fatty liver disease. Background Art

[0002] Benzethonium chloride, molecular formula C 27 H 42 NO2·Cl is easily soluble in water to form a foamy soapy solution. It is also soluble in ethanol, acetone, and chloroform, and is often used as a bactericidal disinfectant. Benzethonium chloride is also used as a preservative because of its good surface activity. It is widely used in cosmetics, personal care products, antiseptics, water treatment and algae removal. Pharmacological studies have also shown that benzethonium chloride can inhibit the proliferation of both sensitive and resistant medulloblastomas and the Shh signaling pathway (Cheng Xi. A new Shh signaling pathway inhibitor, benzethonium chloride, inhibits the Shh signaling pathway by promoting Gli3 processing to treat medulloblastoma [D]. Suzhou University, 2022.).

[0003] So far, in addition to the above uses, there is no report on the role of benzethonium chloride in the treatment of lipid metabolism diseases, such as non-alcoholic fatty liver disease. The current methods for preventing and treating non-alcoholic fatty liver disease include strengthening early monitoring to prevent the occurrence of the disease, or using other drugs for the treatment of lipid metabolism-related diseases. These drugs are not targeted at non-alcoholic fatty liver disease, and the treatment effect is often poor, and most of them have certain side effects. Therefore, it is urgent to find drugs that can be used for non-alcoholic fatty liver disease. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a use of benzethonium chloride in preparing a medicament for improving or treating non-alcoholic fatty liver disease.

[0005] In order to achieve the above object, the technical solution of the present invention is as follows:

[0006] Disclosed is a use of benzethonium chloride in preparing a medicine for improving or treating non-alcoholic fatty liver disease.

[0007] Further: Use of benzethonium chloride in preparing a medicine for improving or treating hepatic lobular inflammation.

[0008] Further: Use of benzethonium chloride in preparing medicine for improving or treating liver fibrosis.

[0009] The beneficial effects of the present invention are:

[0010] The present invention discovers for the first time a new use of benzethonium chloride, namely, the therapeutic effect of benzethonium chloride on non-alcoholic fatty liver disease. Benzethonium chloride can reduce hepatocyte lipid deposition and regulate hepatocyte lipid metabolism disorder by intervening in a cell lipid deposition model; and can improve liver lipid accumulation in mice caused by lipid metabolism disorder by administration through the digestive tract, and can improve liver coefficient, liver function, hepatic lobular inflammation, and liver fibrosis. Therefore, the benzethonium chloride involved in the present invention can be used for the preparation of medicines for improving or treating non-alcoholic fatty liver disease.

[0011] The benzethonium chloride involved in the present invention can inhibit the expression of human apolipoprotein C3, thereby reducing the generation of lipids, and is applied to drugs for treating non-alcoholic fatty liver disease, thus opening up new uses of benzethonium chloride and providing a new drug option for treating non-alcoholic fatty liver disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0013] Figure 1 This is a diagram for toxicity analysis on L02 cells.

[0014] Figure 2 Graph showing the effect of BZN on APOC3 expression in FFA-induced L02 cells; wherein, a is the graph showing the effect of BZN on APOC3 mRNA gene expression, b is the graph showing the effect of BZN on APOC3 protein expression, and c is the quantification graph of b.

[0015] Figure 3 Figure 3 is the effect of BZN on the content of triglyceride (TG), total cholesterol (TC), and low-density lipoprotein (LDL-c) in FFA-induced L02 cells; among them, a is the effect of BZN on the triglyceride content of hepatocytes, b is the effect of BZN on the total cholesterol content of hepatocytes, and c is the effect of BZN on the low-density lipoprotein content of hepatocytes.

[0016] Figure 4 Figure 2 is the effect of BZN on lipid accumulation in FFA-induced L02 cells; a is the Oil Red O staining of hepatocytes in the control group, b is the Oil Red O staining of hepatocytes in the high-fat group, c is the Oil Red O staining of hepatocytes in the high-fat + BZN group, and d is the quantification of the Oil Red O staining results of hepatocytes in each group.

[0017] Figure 5Figure 3 is the effect of BZN on the AMPK-ACC signaling pathway in L02 cells; wherein, a is the effect of BZN on the expression of proteins related to the AMPK-ACC signaling pathway, b is the quantification result of the protein expression of ACC relative to β-Tubulin, c is the quantification result of the protein expression of P-ACC relative to β-Tubulin, d is the quantification result of the protein expression of P-AMPK relative to AMPK, e is the quantification result of the protein expression of CPT1 relative to β-Tubulin, and f is the quantification result of the protein expression of APOC3 relative to β-Tubulin.

[0018] Figure 6 Figure 3 is the effect of BZN on the AMPK-SREBP1-FASN signaling pathway in L02 cells; wherein, a is the effect of BZN on the expression of proteins related to the AMPK-SREBP1-FASN signaling pathway, b is the quantification result of the protein expression of P-AMPK relative to AMPK, c is the quantification result of the protein expression of SREBP1 relative to β-Tubulin, d is the quantification result of the protein expression of FASN relative to β-Tubulin, and e is the quantification result of the protein expression of APOC3 relative to β-Tubulin.

[0019] Figure 7 Figure 2 shows the appearance of the liver and liver coefficient of each group of mice; a is the front and back views of the liver of the control group mice; b is the front and back views of the liver of the high-fat group mice; c is the front and back views of the liver of the high-fat + BZN low-dose group mice; d is the front and back views of the liver of the high-fat + BZN high-dose group mice; e is the front and back views of the liver of the high-fat + positive drug group mice; f is the statistical graph of the liver coefficients of each group of mice.

[0020] Figure 8 The figures are the levels of serum aspartate aminotransferase (ALT) and alanine aminotransferase (AST) and the levels of TG, TC, and LDL-c in the liver of each group of mice; among them, a is the graph of serum aspartate aminotransferase (ALT) levels of each group of mice, b is the graph of serum alanine aminotransferase (AST) levels of each group of mice, c is the graph of triglyceride (TG) levels in the liver of each group of mice, d is the graph of total cholesterol (TC) levels in the liver of each group of mice, and e is the graph of low-density lipoprotein (LDL-c) levels in the liver of each group of mice.

[0021] Fig. 9Figure 3 is the effect of BZN on lipid accumulation in the mouse liver; a is the Oil Red O staining image of the liver of the control group mice, b is the Oil Red O staining image of the liver of the high-fat group mice, c is the Oil Red O staining image of the liver of the high-fat + BZN low-dose group mice, d is the Oil Red O staining image of the liver of the high-fat + BZN high-dose group mice, e is the Oil Red O staining image of the liver of the high-fat + positive drug group mice, and f is the quantification image of Oil Red O staining of the livers of each group.

[0022] Fig.10 : The figure shows the effect of BZN on mouse liver lobular inflammation; wherein, a is the hematoxylin & eosin staining of the liver of the control group mice, b is the hematoxylin & eosin staining of the liver of the high-fat group mice, c is the hematoxylin & eosin staining of the liver of the high-fat + BZN low-dose group mice, d is the hematoxylin & eosin staining of the liver of the high-fat + BZN high-dose group mice, e is the hematoxylin & eosin staining of the liver of the high-fat + positive drug group mice, f is the immunohistochemical staining result of the liver of the control group mice, g is the immunohistochemical staining result of the liver of the high-fat group mice, h is the immunohistochemical staining result of the liver of the high-fat + BZN low-dose group mice, i is the immunohistochemical staining result of the liver of the high-fat + BZN high-dose group mice, j is the immunohistochemical staining result of the liver of the high-fat + positive drug group mice, and k is the quantification of the immunohistochemical staining results of each group.

[0023] Fig.11 Graph showing the effect of BZN on liver fibrosis in mice; wherein, a is the Sirius red staining graph of the liver of the control group mice, b is the Sirius red staining graph of the liver of the high-fat group mice, c is the Sirius red staining graph of the liver of the high-fat + low-dose BZN group mice, d is the Sirius red staining graph of the liver of the high-fat + high-dose BZN group mice, e is the Sirius red staining graph of the liver of the high-fat + positive drug group mice, and f is a quantification graph of the Sirius red staining results of each group.

[0024] Figure 1-11In the table, BZN represents benzethonium chloride; Control represents the control group; FFA represents the cell high-fat group (cells induced with free fatty acids), HFF represents the mouse high-fat group (mice induced with a high-fat diet); FFA+BZN represents the cell high-fat + BZN group; FFA+BZN-L represents the cell high-fat + BZN low-dose group (5 mg / kg / d); FFA+BZN-H represents the cell high-fat + BZN high-dose group (10 mg / kg / d); FFA+MGL-3196 represents the cell high-fat + positive drug group (positive drug name MGL-3196, 10 mg / kg / d); HFF+BZN-L represents the mouse high-fat + BZN low-dose group (5 mg / kg / d); HFF+BZN-H represents the mouse high-fat + BZN high-dose group (10 mg / kg / d); HFF+MGL-3196 represents the mouse high-fat + positive drug group (positive drug name MGL-3196, 10 mg / kg / d); APOC3 represents human apolipoprotein CⅢ, GAPDH represents glyceraldehyde-3-phosphate dehydrogenase; ACC represents acetyl-CoA carboxylase; P-ACC represents phosphorylated acetyl-CoA carboxylase; AMPK represents adenylate-activated protein kinase; P-AMPK represents phosphorylated adenylate-activated protein kinase; CPT1 represents carnitine palmitoyltransferase 1; SREBP1 represents cholesterol regulatory element binding protein 1; FASN represents fatty acid synthase; β-Tubulin represents β-tubulin. DETAILED DESCRIPTION

[0025] The present invention is described in detail below with reference to the accompanying drawings.

[0026] The present invention is based on a cell model for inducing lipid deposition and a mouse model for non-alcoholic fatty liver disease, and uses benzethonium chloride for intervention, thereby detecting the therapeutic effect of benzethonium chloride on non-alcoholic fatty liver disease.

[0027] The present invention first detects the non-toxic concentration of benzethonium chloride to hepatocyte L02 cells by MTT method, and the concentration is 3 μg / mL. Then, the normal human hepatocyte L02 cells are stimulated with free fatty acids to induce a cell lipid deposition model, and a control group and a treatment group are set. Oil red O staining, triglyceride and total cholesterol content determination are performed, and the results show that benzethonium chloride can effectively reduce the hepatocyte lipid accumulation degree caused by free fatty acids. And by Western Blot, benzethonium chloride is detected to inhibit the expression of human apolipoprotein C3, and activates AMPK-AC and AMPK-SREBP1-FASN pathways to regulate the role of liver lipid metabolism. The detailed mechanism is that AMPK can regulate liver lipid metabolism disorders by inhibiting the synthesis of fat and promoting β-oxidation of fatty acids. This process is mainly through the phosphorylation of ACC by activated AMPK, making it inactive. As the rate-limiting enzyme in the fat synthesis pathway, the inhibition of ACC activity further reduces the generation of malonyl CoA, thereby reducing the de novo synthesis of fat. In addition, malonyl CoA is an inhibitor of CPT1 (promoting β-oxidation of fatty acids), so the activation of AMPK can promote the expression of CPT1 and promote β-oxidation of fatty acids, thereby improving NAFLD. On the other hand, the activation of AMPK can also inhibit the activity of SREBP1, which plays an important role in regulating the synthesis of fatty acids in the liver. For example, SREBP1 can promote the expression of its downstream fatty acid synthase FASN, thereby promoting the synthesis of fatty acids. When the activity of SREBP1 is inhibited, the synthesis of fatty acids will also be inhibited.

[0028] The present invention also induced a non-alcoholic fatty liver disease model by feeding mice with a high-fat diet, and simultaneously administered benzethonium chloride through the digestive tract. The liver coefficient of mice was counted, the levels of liver enzymes and lipid metabolism-related indicators were measured, and the mice were paraffined and frozen for slice preparation. The results of Oil Red O staining, HE staining, Sirius red staining, and immunohistochemistry showed that benzethonium chloride can also improve the related symptoms of non-alcoholic fatty liver disease in mice, and the improvement effect of the high-dose group (10 mg / kg / d) was more significant.

[0029] In the following examples, BZN is also used to represent benzethonium chloride. The L02 cells used in the following examples are derived from L02 cells passaged and frozen in the School of Life Sciences of Changchun Normal University.

[0030] Example 1 Non-toxic concentration of benzethonium chloride to hepatocytes

[0031] L02 cells were seeded at an appropriate density in a 96-well plate. BZN solution (concentration gradient of 1, 2, 3, 4, 6, 8 μg / mL) was diluted with complete culture medium (a mixture of high-glucose DMEM containing 20% ​​FBS by volume and 1% penicillin-streptomycin by volume). At the same time, the control group was still an equal volume of DMSO dilution. To ensure the accuracy of the experiment, 3 replicates were set for each group. MTT test results are shown in Figure 1 The results showed that the non-toxic concentration of benzethonium chloride to the hepatocyte cell line L02 cells was 3 μg / mL.

[0032] Example 2 Effects of benzethonium chloride on lipid metabolism indicators

[0033] L02 cells were inoculated in a six-well cell culture plate. After culturing for 24 hours, 7.5 mM FFA was diluted to a concentration of 600 μM with complete culture medium (a mixture of high-glucose DMEM containing 20% ​​FBS by volume and 1% penicillin-streptomycin by volume), the original culture medium was discarded, and replaced with FFA (free fatty acid) diluent. The BZN group was added with 600 μM FFA diluent and 3 μg / mL BZN diluent, and the control group was added with an equal volume of BSA diluent. After 48 hours, the cells of each group were collected into 1.5 mL EP tubes and marked, then washed twice with PBS and the supernatant was removed. 200 μL of PBS was added again, the cells were resuspended by blowing, and the cell suspension was placed on ice. The cells were disrupted using an ultrasonic cell disruptor, and the homogenate was directly measured. Results are shown in Figure 3 The results showed that benzethonium chloride could significantly reduce the levels of TG, TC and LDL-c.

[0034] Example 3 Oil Red O staining of hepatocytes

[0035] Remove the culture medium from the L02 cells treated accordingly in Reference Example 2, and gently wash the cells three times with PBS to prevent the cells from falling off. Then add 500 μL of cell fixative (4% paraformaldehyde by mass) to each well, fix for 30 minutes in the dark, discard the fixative, gently add Oil Red O working solution (Solebo G1263) along the wall of the plate, stain for 10 minutes at room temperature in the dark, discard the staining solution, add 500 μL of 60% isopropanol to differentiate and remove excess dye, and then quickly absorb the isopropanol. Finally, seal the slides with glycerol, observe under a microscope, and take photos for record. The staining results are shown in Figure 4 In ad, the results showed that benzethonium chloride could significantly reduce the lipid accumulation in L02 cells.

[0036] Example 4 Mechanism of Benzethonium Chloride in Regulating Disorders of Lipid Metabolism in Hepatocytes

[0037] Phosphatase inhibitors (Biyuntian ST640-50g) were added to the protein lysis buffer (NaCl 150mM, glycerol 0.1mM, HEPES-NaOH 50mM, MgCl2·6H2O 1.5mM, 1% Triton X-100 0.4mM, NaF 0.1M, EGTA 1mM, DTT 0.1mM, PMSF 100mM), and the cells were lysed on ice for 30min. Centrifuge at 12000 rpm for 10min in a 4℃ centrifuge. The supernatant was transferred to a new EP tube, and then 4× protein loading buffer (Tris-HCl 3ML, glycerol 4ML, β-mercaptoethanol 3ML, SDS 0.8g, bromophenol blue 0.01g) was added in proportion and mixed thoroughly. Then, the mixture was incubated in a metal bath at 100℃ for 10 min to ensure protein denaturation. Western Blot detection was performed, and the results are shown in Figure 2 Medium AC, Figure 5 Middle AF, Figure 6 ae, the results showed that benzethonium chloride could significantly inhibit the expression of human apolipoprotein C3, and activate AMPK-AC and AMPK-SREBP1-FASN pathways to regulate liver lipid metabolism.

[0038] Example 5 Benzethonium chloride improves liver lipid deposition in mice with non-alcoholic fatty liver disease (NAFLD)

[0039] Male C57BL / 6 mice, aged 5-6 weeks, were provided by Liaoning Changsheng Biotechnology Co., Ltd. The specially prepared HFF high-fat diet was provided by Jiangsu Medison Biomedicine Co., Ltd.

[0040] After 7 days of adaptive feeding, 40 healthy male C57BL / 6 mice aged 5-6 weeks were randomly divided into five groups: control group (normal diet), model group (HFF high-fat diet), BZN low-dose group (HFF high-fat diet + BZN 5 mg / kg / d), BZN high-dose group (HFF high-fat diet + BZN 10 mg / kg / d) and MGL-3196 positive control group (HFF high-fat diet + MGL-3196 10 mg / kg / d), each group containing 8 mice. At the same time as modeling, the control group and model group were gavaged with an equal volume of saline, and the modeling time was 8 weeks. After the last administration, the mice were fasted for 12 hours, and then anesthetized with 4% chloral hydrate, blood was collected through the heart, and the supernatant was collected for separate use; the complete liver was removed and photographed and frozen for later use.

[0041] Statistical mouse liver coefficient: liver coefficient = liver weight / body weight * 100% (see Figure 7 ).

[0042] Determination of serum ALT, AST, liver TG, TC content in mice (see Figure 8 ).

[0043] Figure 7 The data in the af showed that compared with the control group, NAFLD mice had increased liver weight due to lipid accumulation, and the liver coefficient of mice was about 10%. The liver coefficients of the BZN groups and the positive drug groups were significantly reduced, and the downward trend in the BZN high-dose group was more obvious, indicating that BZN and positive drugs had significant improvements on the liver coefficients of NAFLD mice.

[0044] Figure 8 The data in the ae show that ALT and AST are usually used as indicators of liver function testing. ALT reflects the damage of liver cells, and AST reflects the necrosis of liver cells. When the liver is damaged and inflammation occurs, ALT and AST will be released from liver cells into the blood, and the level of liver enzymes in the serum will increase immediately. Compared with the control group, the levels of ALT and AST in the serum of mice in the model group were significantly increased; compared with the model group, the levels of ALT and AST in the low-dose BZN group decreased, but did not reach statistical significance, and the high-dose BZN group decreased significantly. The ALT level in the positive drug group decreased significantly, while the AST level increased abnormally. This shows that high-dose BZN can significantly improve liver damage in mice.

[0045] Oil Red O staining (see Fig. 9 (a-f): Before staining, the frozen sections were taken out of the refrigerator and rewarmed at room temperature for 1 hour; PBS was soaked for 20 minutes to remove OCT; formaldehyde-calcium solution was fixed for 10 minutes; PBS was soaked for 6 minutes; then differentiated with 60% isopropanol for 2 minutes; then stained with Oil Red O working solution for 30 minutes; soaked again with 60% isopropanol for 30 seconds to make the stroma differentiation clear; rinsed once with PBS, and sealed with glycerol gelatin. The results of liver coefficient, ALT, AST, TG, TC, LDL-c content, and Oil Red O showed that lipid deposition in the liver of mice was significantly reduced and liver function was improved after benzethonium chloride treatment.

[0046] Example 6 Benzethonium chloride improves hepatic lobular inflammation in mice with non-alcoholic fatty liver disease

[0047] HE staining: Place the paraffin sections in hematoxylin staining solution for 10 minutes, then rinse the remaining stain gently with tap water, dip in differentiation solution (1% hydrochloric acid by volume), take out immediately, soak in tap water for 15 minutes to reverse blue, transfer the slides to eosin staining solution for 1 minute, and then rinse gently with tap water again to wash away excess dye; then transfer the sections to new 95% ethanol solution for 4 minutes; anhydrous ethanol solution for 4 minutes; xylene I and xylene II for 5 minutes each; finally, open the fume hood and seal the sections with neutral gum sealing agent;

[0048] Immunohistochemistry: antigen repair, inactivation of endogenous peroxidase, incubation with antibodies, color development. Fig.10 (ak) showed that benzethonium chloride significantly reduced liver lobular inflammation in mice.

[0049] Example 7: Benzethonium chloride improves fibrosis in mice with non-alcoholic fatty liver disease

[0050] Use a pipette to draw up the Sirius red dye solution and drop it on the tissue. Drip it at room temperature and avoid light for 1 hour. Rinse the floating color with running water. Then transfer the slices to 95% ethanol solution for 4 minutes, anhydrous ethanol solution for 4 minutes, xylene I and xylene II for 5 minutes each. Seal the slices with neutral gum. Sirius red results (see Fig.11 (a-f) showed that benzethonium chloride significantly improved the degree of liver fibrosis.

[0051] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. Use of benzethonium chloride in preparing a medicament for improving or treating non-alcoholic fatty liver disease.

2. The use according to claim 1, characterized in that Improves hepatic lobular inflammation caused by non-alcoholic fatty liver disease.

3. The use according to claim 1, characterized in that Improve liver fibrosis caused by non-alcoholic fatty liver disease.

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