| 【Purpose】This experiment aims to study the effect of ginkgolide B on NAFLD by replicating the rat model of non-alcoholic fatty liver disease.This experiment further explores whether the effect of GB on NAFLD rats is related to the regulation of PERK/eIF2α/ATF4/CHOP signaling pathway,inhibition of endoplasmic reticulum stress,and thus reduction of inflammatory response and hepatic cell apoptosis.【Methods】1.Prevention and treatment effect of GB on NAFLD rats84 SPF male SD rats were randomly divided into two groups after purchase.There were 14 rats in the normal group and 70 rats in the model group.Among them,the normal group was fed with common diet for 8 weeks.The model group was fed with high fat and high sugar diet for 8 weeks.Replication of NAFLD rat model.After 8 weeks,2 rats were taken from the normal group,and10 rats from the model group were selected for model verification(The criteria for successful modeling are as follows: liver pathology shows a large number of fat vacuoles.).After successful modeling,the rats in the model group were randomly divided into 5 groups.These five groups are model group,ginkgolide B low-dose group(GB-L),ginkgolide B medium-dose group(GB-M),ginkgolide B high-dose group(GB-H)and positive drug stimulant Vastatin group.There were 12 rats in each group.The normal group and the model group were intragastrically administered with2 m L/d of normal saline.The simvastatin group was intragastrically administered with2.0 mg/kg/d simvastatin suspension.GB treatment groups were given GB suspension(0.5,1.0,2.0 mg/kg/d)by intragastric administration respectively.1 time a day for 8weeks.During the experiment period,the general situation of the rats was observed every day.Regularly measure the body weight and food intake of rats.After 8 weeks of treatment,the serum and liver tissue of rats were collected to detect the following indicators: liver weight,liver index.HE and Oil Red O staining were used to observe the fatty degeneration and inflammatory infiltration of rat liver tissue.Detection of lipid metabolism indicators such as high-density lipoprotein cholesterol,total cholesterol,low-density lipoprotein cholesterol and triglyceride in rat serum and liver by using a biochemical kit microplate reader.Liver function levels such as serum alanine aminotransferase and aspartate aminotransferase were detected by biochemical kit microplate reader.Observation of the preventive and therapeutic effect of GB on NAFLD rats.2.GB regulates PERK/eIF2α/ATF4/CHOP pathway inhibits ERS in NAFLD rats,reduces inflammatory response and liver cell apoptosisThe content of inflammatory factors interleukin-6(IL-6),interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)in serum and liver of rats were detected by ELISA method.Detection of GRP78,PERK,p-eIF2α,ATF4,CHOP,Bcl-2 and Bax protein expression in rat liver by Western Blot.Immunohistochemical method was used to detect the integrated optical density of GRP78,PERK,p-eIF2α,ATF4,CHOP,Bcl-2 and Bax in rat liver tissue.Exploring the therapeutic mechanism of GB on NAFLD rats.【Results】1.The therapeutic effect of GB on NAFLD ratsA rat model of NAFLD was successfully replicated by feeding a high-fat and high-sugar diet for 12 weeks.Observation of the general conditions of the rats during the feeding period showed that: The rats in the normal group have a good mental state.Compared with the normal group,the mental state of rats in the model group was poor.The mental state of the rats in each treatment group was significantly improved compared with the model group.The results of liver index showed: Compared with normal rats,the body weight and liver index of model rats were obviously increased(P<0.05).Compared with the rats in the model group,the body weight and liver index of the rats in the positive drug simvastatin group and GB treatment groups were significantly lower(P<0.05).Pathological HE staining results showed: Compared with the normal group,there were a large number of fat vacuoles and infiltration of inflammatory factors in the liver cells of the rats in the model group.Compared with the rats in the model group,the GB treatment groups and the simvastatin group significantly reduced a large number of fat vacuoles and infiltration of inflammatory factors in the liver of the rats.Pathological oil red O staining results showed:Compared with the normal group,a great deal of orange-red lipid droplets can be seen in the liver cells of the rats in the model group.Compared with the rats in the model group,the orange-red lipid droplets in the liver cells of the rats in the GB treatment groups decreased in a dose-dependent manner.Liver function results show: Compared with the normal group,the serum ALT and AST liver function indexes in the model group were significantly increased(P<0.05).Compared with the model group,the ALT and AST indexes of rats in the simvastatin group and GB treatment groups were significantly decreased(P<0.05).Lipid metabolism results show: Compared with the normal group,the levels of TG,TC and LDL-C in the serum and liver of the rats in the model group were significantly increased(P<0.05),and the levels of HDL-C were significantly decreased(P<0.05).Compared with the rats in the model group,the levels of TG,TC and LDL-C in the serum and liver of the rats in the GB low,middle and high dose group and the simvastatin group showed a significant decrease trend(P<0.05).Compared with model group,the serum and liver levels of HDL-C in GB high-dose group and simvastatin group were increased(P < 0.05).The HDL-C levels in serum and liver of rats could be increased in low and medium dose GB groups,but there was no statistical significance compared with model group.2.GB regulates PERK/eIF2α/ATF4/CHOP pathway inhibits ERS in NAFLD rats,reduces inflammatory response and liver cell apoptosisWB detection result shows that: Compared with the rats in the normal group,the expression levels of GRP78,PERK,p-eIF2α and ATF4 proteins in the liver tissue of the rats in the model group were increased(P<0.05).Compared with the model group,the protein expression levels of GRP78,PERK,p-eIF2 α and ATF4 in the liver tissue of rats in GB low,middle,high dose groups and positive drug simvastatin group were decreased(P<0.05).Immunohistochemical examination result showed that: Compared with normal group,the cumulative optical density of GRP78,PERK,p-eIF2α and ATF4 in the liver tissue of rats in model group was increased(P<0.05).Compared with model group,the cumulative optical densities of GRP78,PERK,p-eIF2α and ATF4 in the liver tissue of GB treatment groups and positive drug simvastatin group decreased(P<0.05).The results of ELISA test show that: compared with the rats in the normal group,the levels of inflammatory factors IL-1β,TNF-α and IL-6 in the serum and liver of the rats in the model group showed a significant upward trend(P<0.05).Compared with the rats in the model group,the levels of IL-1β,TNF-α and IL-6 in the liver and serum of rats in the simvastatin group and GB low,medium and high treatment groups showed a significant downward trend(P<0.05).WB test showed: compared with normal group,the expression level of CHOP and pro-apoptotic protein Bax in liver tissue of rats in model group was increased(P<0.05),and the expression level of anti-apoptotic protein Bcl-2 was decreased(P<0.05).Compared with the rats in the model group,the expression levels of CHOP and Bax in the liver tissue of rats in each dose group of GB and the positive drug simvastatin group was decreased(P<0.05),while the expression level of Bcl-2increased(P<0.05).Immunohistochemical detection shows: compared with normal group,the cumulative optical density of CHOP and pro-apoptotic protein Bax in liver tissue of rats in model group was increased(P<0.05),and that of anti-apoptotic protein Bcl-2 was decreased(P<0.05).Compared with model group,the cumulative optical density of CHOP and Bax in liver tissue of rats in GB low,medium and high dose groups and positive drug simvastatin group decreased(P<0.05),and the cumulative optical density of anti-apoptotic protein Bcl-2 increased(P<0.05).【Conclusion】1.GB can improve the lipid metabolism of NAFLD rats fed a high-fat and high-sugar diet,reduce lipid deposition in rats,and protect liver function,thereby playing a role in the treatment of NAFLD.2.The mechanism of GB treatment on NAFLD rats may be to regulate PERK/eIF2 α /ATF4/CHOP signal pathway,inhibit ERS,and then reduce inflammatory reaction and hepatocyte apoptosis. |
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