CN113679731A

CN113679731A - Application of prunasin in preparation of medicine for treating fatty liver disease and hepatic fibrosis

Info

Publication number: CN113679731A
Application number: CN202111046696.8A
Authority: CN
Inventors: 刘伟; 刘平; 张定棋; 王长虹; 张聪聪; 陈佳美; 杨海琳; 王永丽; 戚胜兰
Original assignee: Shuguang Hospital Affiliated to Shanghai University of TCM
Current assignee: Shuguang Hospital Affiliated to Shanghai University of TCM
Priority date: 2021-09-06
Filing date: 2021-09-06
Publication date: 2021-11-23
Anticipated expiration: 2041-09-06
Also published as: CN113679731B

Abstract

The invention relates to application of prunasin in preparing a medicament for treating fatty liver disease and hepatic fibrosis. The invention discovers that for a mouse non-alcoholic steatohepatitis model induced by high-fat high-sugar diet, the intervention of the prunasin can obviously improve the pathological change of liver tissues, inhibit the lipid aggregation of the liver tissues, reduce the TG content of the liver tissues, reduce the activity of AST and ALT in serum and the content of TG, TC and LDL in the serum, and the effect is obviously better than that of a positive control drug; for a mouse chemical injury hepatic fibrosis model induced by carbon tetrachloride and a rat cholestatic hepatic fibrosis model constructed by bile duct ligation, the intervention of the elaeagnus mollis glucoside can obviously improve the pathological change of liver tissues, inhibit collagen deposition, reduce the content of Hyp in the liver tissues, reduce the activity of AST and ALT in serum and the total bilirubin level, and the effect is obviously better than that of a positive control medicament. The combination of the prunasin and clinical drugs shows that the prunasin and sorafenib can obviously improve the hepatic fibrosis induced by carbon tetrachloride and show synergistic effect.

Description

Application of prunasin in preparation of medicine for treating fatty liver disease and hepatic fibrosis

Technical Field

The invention relates to the technical field of medicines, in particular to application of prunasin in preparing a medicine for treating fatty liver disease and hepatic fibrosis.

Background

The Prunasin (Prunasin) exists in kernels of apricot, peach, loquat, green plum, waxberry and other plants, is reported only in the metabolic process of some medicines at present, and related pharmacological and pharmacodynamic studies are rarely reported. The chemical structure of prunasin is shown in figure 1.

Fatty liver disease refers to a disease in which fat is accumulated in liver cells too much due to various causes, and is a second-largest liver disease that is second to viral hepatitis and seriously threatens the health of people in China. Fatty Liver Disease (FLD) including non-alcoholic fatty liver disease (NAFLD) and Alcoholic Liver Disease (ALD), has an increasing incidence year by year, and has become a global important health problem, wherein non-alcoholic steatohepatitis has become the leading cause of biochemical index abnormality of the first chronic liver disease and the health examination liver in our country, and the incidence of non-alcoholic fatty liver disease of patients with diabetes, obesity and dyslipidemia is more significant. NAFLD comprises a clinical pathological syndrome in which a series of diffuse hepatocyte large-vesicular steatosis resulting from accumulation of fat in the liver becomes a major lesion, and is a hereditary-environmental-metabolic-stress related disease, and the disease spectrum comprises non-alcoholic fatty liver disease (NAFL), non-alcoholic steatohepatitis (NASH) and related hepatic fibrosis, cirrhosis and hepatocellular carcinoma. ALD is a liver disease caused by long-term drinking, and its pathological changes are mainly bullous mixed hepatocellular steatosis, and are classified into Alcoholic Fatty Liver (AFL), Alcoholic Hepatitis (AH), and alcoholic cirrhosis.

Hepatic fibrosis refers to the condition that hepatic cells are continuously and repeatedly necrosed or stimulated by inflammation when various chronic liver diseases occur, so that a body has repair reaction, a large amount of fibers are proliferated and are relatively or absolutely deficient in fiber degradation, and extracellular matrix is deposited in the liver in a large amount. Hepatic fibrosis is a common pathological stage of various chronic liver diseases, including viral hepatitis, FLD, autoimmune liver diseases, drug-induced hepatitis and the like. Liver fibrosis can further progress to cirrhosis, liver failure, and even liver cancer.

At present, most of the drugs for treating hepatic fibrosis or non-alcoholic fatty liver disease are in clinical trials and experimental research stages, and no biological and chemical drugs aiming at hepatic fibrosis or liver lipid deposition are recommended to be used clinically. The natural medicine has less toxic and side effects and has outstanding advantages in treating diseases.

At present, the report of the rosa laevigata glycoside for treating the liver diseases is not seen.

Disclosure of Invention

The invention aims to provide the application of the prunasin in preparing the medicine for treating fatty liver disease and hepatic fibrosis aiming at the defects in the prior art.

In a first aspect, the present invention provides an application of prunasin in preparing a medicament for treating Fatty Liver Disease (FLD) or hepatic fibrosis (liver fibrosis).

As a preferred example, the fatty liver disease is non-alcoholic fatty liver disease (NAFLD) or Alcoholic Liver Disease (ALD); the hepatic fibrosis is chemical injury hepatic fibrosis or cholestatic hepatic fibrosis.

More preferably, the non-alcoholic fatty liver disease is non-alcoholic simple fatty liver (NAFL), non-alcoholic steatohepatitis (NASH) or its associated liver fibrosis and cirrhosis; the alcoholic liver disease is Alcoholic Fatty Liver (AFL), Alcoholic Hepatitis (AH) or alcoholic cirrhosis.

More preferably, the non-alcoholic fatty liver disease is caused by a high-fat high-sugar diet.

As another preferred example, the prunasin is the only active ingredient.

In a second aspect, the invention provides a pharmaceutical composition for treating hepatic fibrosis, wherein the active ingredients of the pharmaceutical composition comprise prunasin and sorafenib.

In a third aspect, the invention provides an application of the pharmaceutical composition in preparing a medicament for treating hepatic fibrosis.

In a fourth aspect, the invention provides an application of prunasin in preparing an experimental reagent for researching a pathological mechanism of fatty liver disease or hepatic fibrosis.

As a preferable example, the pathological mechanism of fatty liver disease or hepatic fibrosis is studied, and the study is in vitro or in vivo.

As another preferred example, the in vivo study is based on a high fat diet-induced non-alcoholic steatohepatitis animal model, a carbon tetrachloride-induced liver fibrosis animal model, or a cholestatic liver fibrosis animal model established by bile duct ligation.

As used herein, the term "drug" refers to a substance used for the prevention, treatment and diagnosis of diseases; the experimental reagent is a pure chemical used for realizing chemical reaction, analytical assay, research and test, teaching experiment and chemical formula based on the purpose of experiment.

The invention has the advantages that:

1. the prunasin is found to have a better treatment effect on the non-alcoholic fatty liver disease for the first time. Has obvious improvement effect on abnormal biochemical indexes of the damaged liver and obviously reduces liver lipid accumulation. The completed animal model experiment shows that for a mouse non-alcoholic steatohepatitis model induced by high-fat high-sugar diet, the intervention of the cerasus serrulata glycoside can obviously improve the hepatic cell steatosis, the hepatic cell ballooning and inflammatory cell infiltration, inhibit the hepatic tissue lipid aggregation, reduce the hepatic tissue triglyceride content, reduce the activities of serum aspartate aminotransferase and glutamate pyruvate transaminase, and reduce the contents of serum triglyceride, total cholesterol and low-density lipoprotein.

2. The harpagoside is found to have better treatment effect on hepatic fibrosis for the first time, has obvious improvement effect on abnormal biochemical indexes of liver injury, and obviously reduces the deposition of liver collagen fibers. The completed animal model experiments show that the hepatic fibrosis model induced by carbon tetrachloride and the rat cholestatic hepatic fibrosis model induced by bile duct ligation can obviously improve the pathological change of liver tissues, inhibit the collagen deposition, reduce the content of hydroxyproline in the liver tissues, and reduce the activities of serum aspartate aminotransferase and glutamate pyruvate transaminase and the level of total bilirubin after the intervention of using prunasin.

3. The therapeutic effect of the combination of the rosaniline and clinical drugs on liver diseases is inspected, and the rosavin and sorafenib have synergistic effect, so that the carbon tetrachloride-induced liver fibrosis can be obviously improved, and the treated mouse chemical injury liver fibrosis basically recovers to be normal.

In conclusion, the rosaniline can be used as an active ingredient to prepare a medicament for treating a plurality of liver diseases, including chronic liver diseases such as fatty liver disease, drug-induced hepatitis, viral hepatitis, autoimmune liver disease and the like. The application of the composition of the prunasin and the sorafenib in treating hepatic fibrosis can achieve the aims of reducing toxicity and improving efficiency.

Drawings

FIG. 1 shows the chemical structural formula of prunasin;

FIG. 2H & E staining of liver pathology sections of a high fat and high sugar diet induced non-alcoholic steatohepatitis model;

FIG. 3 liver cryosection oil-red O staining of a high fat and high sugar diet induced non-alcoholic steatohepatitis model;

FIG. 4H & E staining of liver pathology sections of a carbon tetrachloride-induced liver fibrosis model;

FIG. 5. sirius red staining of liver pathological section of carbon tetrachloride-induced liver fibrosis model;

FIG. 6 bile duct ligation to construct H & E staining of liver pathological section of cholestatic liver fibrosis model;

FIG. 7. bile duct ligation constructing liver pathological section sirius red staining of cholestatic liver fibrosis model;

FIG. 8H & E staining of liver pathological section with combined drug evaluation in carbon tetrachloride-induced liver fibrosis model;

FIG. 9. carbon tetrachloride-induced liver fibrosis model evaluation of drug combination liver pathology section sirius red staining;

in the above figures, prunasin-L: the low dose group of rosavin, rosavin-M: the dose group of the rosa laevigata, the rosa laevigata-H: the high dose group of prunasin.

Detailed Description

The following detailed description of the present invention will be made with reference to the accompanying drawings.

Example 1

1. Purpose of the experiment: investigation of effects of prunasin on high-fat high-sugar induced non-alcoholic steatohepatitis of mice

2. The experimental method comprises the following steps:

experimental animals: the experimental animals used in the invention are C57BL/6 mice, the weight is 20-22g, and the male animals are provided by Shanghai Jihui experimental animal feeding Limited company and are fed in the experimental animal center of Shanghai medical university.

The experimental low-fat control feed and high-fat feed were purchased from Research Diets, ltd, fructose and sucrose from Sigma, obeticholic acid from british biotechnology, ltd, and prunasin from yunnan west biotechnology, ltd, with structures determined by NMR and MS and purities of greater than 98% determined by HPLC area normalization.

48C 57BL/6J mice, SPF grade, were divided into normal control group, high-fat high-sugar model group, low-dose group of acteoside (acteoside-L), medium-dose group of acteoside (acteoside-M), high-dose group of acteoside (acteoside-H), and 8 Obeticholic acid group. The model was constructed by feeding the control group with a low-fat control diet and the remaining groups with a high-fat diet and high-sugar (fructose and sucrose) drinking water for 20 weeks. From the 14 th week, while feeding corresponding feed, the control group and the model group are administered with equal volume of 0.3% CMC-Na solution, the lakuranetin group is administered with intragastric administration of 1.25, 2.5 and 5mg/kg of 0.3% CMC-Na suspension lakuranetin solution, and the obeticholic acid group is administered with 10mg/kg of 0.3% CMC-Na suspension obeticholic acid solution, 1 time per day for 6 weeks. All mice were sacrificed at the end of week 20, serum and liver tissue were retained and serum liver function and blood lipid, liver tissue triglyceride content (TG), liver histopathology (H & E) and liver tissue oil red O staining were examined.

The statistical method comprises the following steps: statistical analysis is carried out on the data by adopting SPSS 20.0 software, non-parameter inspection is carried out on the measured data, and the data are obtained

As shown, the One-way ANOVA combined with the LSD multiple comparison analysis was used in the multiple comparison analysis between groups, and the difference was statistically significant with P < 0.05.

3. The experimental results are as follows:

3.1 Effect of Orientin on mouse serology-related indices

Compared with a control group, the serum aspartate Aminotransferase (AST), the serum glutamate pyruvate transaminase (ALT), the Total Cholesterol (TC) and the low-density lipoprotein (LDL) of the model group are remarkably increased (P is less than 0.001); compared with the model group, the levels of AST, ALT, TC and LDL in the prunasin group are obviously reduced (P <0.05, P <0.01 and P <0.001), and the prunasin high-dose group is reduced more obviously than the obeticholic acid group, which is shown in table 1.

TABLE 1 serology-related index Change

Note: indicating, ## P <0.001, compared to the control group; represents P <0.05, P <0.01, P <0.001 compared to the model group.

3.2 Effect of Orientin on Triglycerides (TG)

Compared with the normal group, the content of TG in serum and liver tissues of the mice in the model group is remarkably increased (P <0.001), and the content of TG in each dose group and the obeticholic acid group of the prunasin is remarkably lower than that in the model group (P <0.05, P <0.01 and P <0.001), wherein the reduction of the prunasin group is more obvious, and the table 2 is specifically shown.

TABLE 2 serum and liver TG content

3.3 Effect of Orientin on histological changes in mouse liver

H & E staining of liver tissues shows that the control group of mice shows that the morphological structure of the liver tissues is normal, the hepatocyte cords are radially arranged around the central vein, no inflammatory cells infiltrate around the blood vessels, and a certain amount of small vacuoles exist. Compared with a control group, liver tissues of the model group mice can be seen in hepatocyte steatosis, a large amount of fat vacuoles, hepatocyte ballooning and inflammatory cell infiltration; compared with a model group, the hepatic tissue steatosis and inflammatory cell infiltration of mice of each dose group of the prunasin and the obeticholic acid group are obviously improved. And the prunasin group is improved more obviously than the obeticholic acid group (figure 2). NAS integral judgment is carried out on H & E stained liver tissues, the result is shown in a table 3, the NAS integral of a model group mouse is more than 4 points, the success of model building of the mouse NASH model is prompted, and NAS scores can be obviously reduced by each drug group (P is less than 0.01 and P is less than 0.001).

TABLE 3 mouse liver histology NAS integration

Note: indicating, ## P <0.001, compared to the control group; representation P <0.01, P <0.001 compared to model groups.

3.4 Effect of Orientin on liver lipid deposition in mice

The result of liver tissue oil red O staining is shown in figure 3, and compared with the control group, a large amount of red fat drops are deposited in the liver tissues of the mice in the model group, and the liver tissues are different in shape and size and are arranged closely; and compared with the model group, the liver tissue lipid drop deposition of mice in each dose group of the prunasin and the obeticholic acid group is obviously reduced. The results of positive area ratio of oil red O staining show: compared with a control group, the oil-red O staining area ratio of the model group is obviously increased (P is less than 0.001), the oil-red O staining area ratio of each dose group of the prunasin is obviously reduced (P is less than 0.05, P is less than 0.01) compared with the liver tissue of mice in the obeticholic acid group, and the prunasin group is improved more obviously, which is shown in a table 4.

TABLE 4 liver tissue oil-Red-O staining area ratio

Note: indicating, ## P <0.001, compared to the control group; represents P <0.05, P <0.01 compared to model groups.

4, experimental conclusion:

the prunasin has good therapeutic effect on non-alcoholic steatohepatitis, and can be used for preparing medicine.

Example 2

1. Purpose of the experiment: investigating the effect of laevigata on carbon tetrachloride-induced hepatic fibrosis

2. Experimental methods

Experimental carbon tetrachloride (CCl)₄) Olive oil was purchased from chemical agents ltd; sorafenib was purchased from Sigma.

C57BL/6J mice, SPF grade, divided into normal control group, CCl₄The model group comprises a low-dose group of the rosaniline (laevigatin-L), a medium-dose group of the rosaniline (laevigatin-M), a high-dose group of the rosaniline (laevigatin-H) and a sorafenib group, wherein each group comprises 8 of the rosaniline, the sakurin, the sakuranex and the sakura. The molding mode is as follows: the control group was injected with olive oil into the abdominal cavity, and the other groups were administered with 15% CCl₄Olive oil was injected intraperitoneally at 2mL/kg 3 times a week for 6 weeks. From the 4 th week, the control group and the model group are respectively administered with equal volume of 0.3% CMC-Na solution, the lakuranetin group is administered with 1.25, 2.5 and 5mg/kg of 0.3% CMC-Na prepared suspension lakuranetin solution by intragastric administration, the sorafenib group is administered with 10mg/kg of 0.3% CMC-Na prepared suspension sorafenib solution by intragastric administration, 1 time per day, and 3 weeks are intervened. All mice were sacrificed under anesthesia at the end of week 6, serum and liver tissue were retained, and serum liver function, liver hydroxyproline content (Hyp) and liver histopathology (H) were examined&E)。

3. The experimental results are as follows:

3.1 Effect of Orientin on serological indices

Compared with a control group, the activity of AST and ALT in the serum of the model group is obviously increased, and the TBiL content is obviously increased (P is less than 0.001); compared with the model group, the levels of AST, ALT activity and TBiL in each dose group of the prunasin are obviously reduced (P is less than 0.05, P is less than 0.01, and P is less than 0.001), and the specific table is shown in table 5.

TABLE 5 mouse serological index Change

3.2 Effect of Orientin on Hyp content in liver tissue

Compared with the control group, the liver tissue Hyp content of the model group mice is obviously increased (P <0.001), while the Hyp content of the individual dose groups of the rosavin is obviously lower than that of the model group (P <0.05, P <0.01 and P <0.001), which is shown in Table 6.

TABLE 6 Hyp content of liver tissue

3.3 Effect of Orientin on liver histological Change

H & E staining shows that the hepatic lobules of the normal mice have clear structures, and the hepatic cell cables are arranged from the central vein to the periphery in a radial mode; the liver cells of the model group mice are necrotic widely, normal lobular structures of the liver disappear, and a large amount of inflammatory cells infiltrate into the fibers at intervals; hepatic cell necrosis and inflammatory cell infiltration were significantly reduced in each dose group of prunasin compared to the model group (fig. 4). Sirius red staining showed that the liver of normal mice only seen a small amount of collagen deposition in the area of the junction and around the central vein; the liver of the mouse in the model group has a large number of fiber intervals to form a pseudo-lobular structure; compared with the model group, the concentration of collagen in each dose group of the prunasin is obviously reduced, the fiber interval is loose, and the complete false lobular structure is less visible (figure 5). Results of whole-slice scanning semi-quantitative analysis of sirius red staining slice show that the collagen area of the model group is significantly increased (P <0.001) compared with the normal group, and the collagen area of each dosage group of prunasin is significantly decreased (P <0.05, P <0.01, P <0.001) compared with the model group, which is shown in Table 7.

TABLE 7 ratio of areas of positive for sirius red staining

4. And (4) experimental conclusion:

the prunasin has good therapeutic effect on hepatic fibrosis, and can be used for preparing medicine.

Example 3

1. Purpose of the experiment: investigating the effect of the prunasin on cholestatic liver fibrosis established by bile duct ligation

2. Experimental methods

Experimental animals: the experimental animal used in the invention is a Wistar rat with the weight of 160-.

Wistar rats are 48, SPF (specific pathogen free) grades, and are divided into a normal control group, a bile duct ligation model group, a rosavin low-dose group (rosavin-L), a rosavin medium-dose group (rosavin-M), a rosavin high-dose group (rosavin-H) and a DAPT group, wherein the total number of the rats is 8. The molding mode is as follows: rats in the model group, the laevigat-L group, the laevigat-M group, the laevigat-H group and the DAPT group underwent bile duct ligation under anesthesia, while rats in the normal group underwent bile duct pseudosurgery as a control. From 3 weeks, the normal group and the model group are administered with equal volume of 0.3% CMC-Na solution, the elaeagnus conferta roxburghii glycoside group is administered with 1.25, 2.5 and 5mg/kg of 0.3% CMC-Na suspension elaeagnus persicarini glycoside solution respectively by intragastric administration, the intragastric administration volume is 20mL/kg, the DAPT group is administered with 30mg/kg of DAPT solution by intraperitoneal injection, the injection volume is 1mL/kg, 1 time a day, and 3 weeks are intervened totally. All rats were sacrificed under anesthesia at the end of week 6, serum and liver tissue were retained and examined for serum liver function, liver hydroxyproline content (Hyp) and liver histopathology (H & E).

3. The experimental results are as follows:

3.1 Effect of Orientin on serological indices

Compared with a normal group, the activity of AST and ALT and the content of TBil in the serum of the model group are obviously increased (P < 0.001); compared with the model group, the serum AST and ALT activities and TBil content of each dose group of the prunasin are remarkably reduced (P <0.05 and P <0.01), and the specific table is shown in Table 8.

TABLE 8 changes in serological indices in rats

3.2 Effect of Orientin on Hyp content in liver tissue

The liver tissue Hyp content of the rats in the model group is obviously increased (P <0.001) compared with that in the normal group, while the Hyp content of the rats in each dose of the prunasin is obviously lower than that in the model group (P <0.05, P <0.01 and P <0.001), which is shown in Table 9.

TABLE 9 rat liver Hyp content

3.3 Effect of Orientin on liver histological Change

H & E staining shows that the hepatic lobules of the rats in the normal group have clear structures, and the hepatic cell cables are arranged from the central vein to the periphery in a radial mode; the bile duct cells of the rat in the model group are hyperplastic, the liver cells are widely necrotic, the fiber interval is formed, and a large amount of inflammatory cell infiltration can be seen; in each dose group of prunasin, bile duct cell proliferation, hepatocyte necrosis, fibroseptal formation and its inflammatory cell infiltration were significantly improved compared to the model group (fig. 6). Sirius red staining showed that normal rat liver only seen a small amount of collagen deposition in the area of the assembled tract and around the central vein; the liver of the rat in the model group shows a large amount of collagen deposition, and the collagen deposition is widely deposited near the hyperplastic bile canaliculus; the collagen deposition was significantly improved in each dose group of prunasin compared to the model group (fig. 7). Results of whole-slice scanning semi-quantitative analysis of sirius red staining slice show that the collagen area of the model group is significantly increased (P <0.001) compared with the normal group, and the collagen area of each dose group of prunasin is significantly decreased (P <0.05, P <0.01) compared with the model group, which is shown in Table 10.

TABLE 10 ratio of areas of positive for sirius red staining

4. And (4) experimental conclusion:

the prunasin has good therapeutic effect on cholestatic hepatic fibrosis, and can be used for preparing medicine.

Example 4

1. Purpose of the experiment: investigating the effect of the combination of laevigat and sorafenib on carbon tetrachloride-induced hepatic fibrosis

2. The experimental method comprises the following steps:

Experimental carbon tetrachloride (CCl)₄) Olive oil was purchased from chemical agents ltd; the rosavin is purchased from Yunnan Xili biotechnology limited, the structure is determined by NMR and MS, and the purity is more than 98 percent determined by HPLC area normalization; sorafenib was purchased from Sigma.

40C 57BL/6J mice, SPF grade, divided into normal control group, CCl₄Model group, prunasin group, sorafenib group, prunasin + sorafenib group, 8 per group. The molding mode is as follows: the control group was injected with olive oil into the abdominal cavity, and the other groups were administered with 15% CCl₄Olive oil was injected intraperitoneally at 2mL/kg 3 times a week for 6 weeks. From the 4 th week, the control group and the model group are filled with 0.3% CMC-Na solution with the same volume, the lakuranetin group is filled with 5mg/kg of suspended lakuranetin solution prepared by 0.3% CMC-Na through intragastric administration, the sorafenib group is filled with 10mg/kg of suspended sorafenib solution prepared by 0.3% CMC-Na through intragastric administration, and the lakuranetin + sorafenib group is filled with 2.5mg/kg of suspended lakuranetin solution prepared by 0.3% CMC-Na and 5mg/kg of suspended sorafenib solution prepared by 0.3% CMC-Na through intragastric administration. 1 time daily, intervene for 3 weeks. All mice were sacrificed under anesthesia at the end of week 6, serum and liver tissue were retained, and serum liver function, liver hydroxyproline content (Hyp) and liver histopathology (H) were examined&E)。

The statistical method comprises the following steps: using SPSS 20.0 software statistically analyzing the data, non-parameter testing the measured data, and analyzing the data

3. The experimental results are as follows:

3.1 serological index of mice in each group

Compared with a control group, the activity of AST and ALT in the serum of the model group is obviously increased, and the TBiL content is obviously increased (P is less than 0.001); compared with the model group, the serum AST, ALT activity and TBiL content of the prunasin group are obviously reduced (P <0.01, P <0.001), the serum AST, ALT activity and TBiL content of the prunasin + sorafenib group are also obviously reduced (P <0.001), and the combination of the prunasin and sorafenib shows a synergistic effect, which is specifically shown in the table 11.

TABLE 11 mouse serological index Change

3.2 liver tissue Hyp content of mice in each group

Compared with a control group, the content of Hyp in liver tissues of mice in a model group is remarkably increased (P <0.001), the content of Hyp in a laevigat group is remarkably lower than that in the model group (P <0.001), the content of Hyp in the laevigat + sorafenib group is also remarkably lower than that in the model group (P <0.001), and the combination of the laevigat and sorafenib shows a synergistic effect, which is specifically shown in Table 12.

TABLE 12 Hyp content of liver tissue

3.3 histological Change of liver of mice in groups

H & E staining shows that the hepatic lobules of the normal mice have clear structures, and the hepatic cell cables are arranged from the central vein to the periphery in a radial mode; the liver cells of the model group mice are necrotic widely, normal lobular structures of the liver disappear, and a large amount of inflammatory cells infiltrate into the fibers at intervals; compared with the model group, hepatic cell necrosis and inflammatory cell infiltration of the prunasin group are obviously reduced, hepatic cell necrosis and inflammatory cell infiltration of the prunasin + sorafenib group are most obviously reduced, hepatic lobule structures are clear, and hepatic cell cords are arranged radially from central veins to the periphery (figure 8). Sirius red staining showed that the liver of normal mice only seen a small amount of collagen deposition in the area of the junction and around the central vein; the liver of the mouse in the model group has a large number of fiber intervals to form a pseudo-lobular structure; compared with the model group, the deposition of the collagen in the laevigat group is obviously reduced, the fiber interval is loose, the complete false lobular structure is rare, and the deposition of the collagen in the liver of the laevigat + sorafenib group is obviously reduced and is close to that of the liver of the normal group of mice (figure 9). Results of whole-slice scanning semi-quantitative analysis of sirius red staining slice show that the collagen area of the model group is significantly increased (P <0.001) compared with the normal group, the collagen area of the laevin group is significantly reduced (P <0.001) compared with the model group, and the collagen area of the laevin + sorafenib group is also significantly reduced (P <0.001), specifically shown in Table 13.

TABLE 13 ratio of areas of positive for sirius red staining

4. And (4) experimental conclusion:

the combination of the prunasin and the sorafenib has obvious treatment effect on hepatic fibrosis and shows synergistic effect.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.

Claims

1. Application of prunasin in preparing medicine for treating fatty liver disease or hepatic fibrosis is provided.

2. The use of claim 1, wherein the fatty liver disease is a non-alcoholic fatty liver disease or an alcoholic liver disease; the hepatic fibrosis is chemical injury hepatic fibrosis or cholestatic hepatic fibrosis.

3. The use of claim 2, wherein the non-alcoholic fatty liver disease is non-alcoholic simple fatty liver, non-alcoholic steatohepatitis or liver fibrosis and cirrhosis associated therewith; the alcoholic liver disease is alcoholic fatty liver, alcoholic hepatitis or alcoholic cirrhosis.

4. The use of claim 2, wherein the non-alcoholic fatty liver disease is caused by a high-fat, high-sugar diet.

5. The use according to claim 1, wherein the prunasin is the only active ingredient.

6. The pharmaceutical composition for treating hepatic fibrosis is characterized in that the active ingredients of the pharmaceutical composition comprise prunasin and sorafenib.

7. Use of the pharmaceutical composition of claim 6 for the preparation of a medicament for the treatment of liver fibrosis.

8. Application of prunasin in preparing experimental reagent for researching pathological mechanism of fatty liver disease or hepatic fibrosis is provided.

9. Use according to claim 8, for studying the pathological mechanism of fatty liver disease or liver fibrosis, in vitro or in vivo.

10. The use according to claim 9, wherein the in vivo study is based on a high fat diet-induced non-alcoholic steatohepatitis animal model, a carbon tetrachloride-induced liver fibrosis animal model or a cholestatic liver fibrosis animal model established by bile duct ligation.