Expression And Significance Of MTOR In Nonalcoholic Steatohepatitis

Objective:Non-alcoholic steatohepatitis(Nash)is one of the most common chronic liver diseases in clinic,and its pathogenesis has not been fully elucidated.Mammalian target of rapamycin(mTOR)is an important regulator of cell proliferation,differentiation,apoptosis and protein synthesis.In this subject,through the establishment of NASH animal model,the therapeutic effect of mTOR inhibitor Rapamycin(RAPA)on nonalcoholic steatohepatitis is observed,and the relationship between mTOR and nonalcoholic steatohepatitis is discussed,aiming to provide new targets and intervention measures for clinical treatment of nonalcoholic fatty liver disease.Methods:(1)Establishment of animal model: 20 female Wistar rats were randomly divided into two groups: experimental group(RAPA intervention)and control group(NASH model group).In addition,a blank control group was set up,10 rats in normal group(normal diet).Normal group was fed with common feed,while control group and experimental group were fed with high-fat feed.After feeding for 6 weeks,the rats in the experimental group were intraperitoneally injected with rapamycin at a dose of 1 mg kg-1d-1.The rats in the normal group and the model group were intraperitoneally injected with the same volume of normal saline for 4 weeks.Blood and liver tissue samples of three groups of rats were collected.(2)The contents of Total Cholesterol(TC),Aspartate Aminotransferase(AST),Alamine Aminotransferase(ALT)and Triglyceride(TG)in serum of rats were determined by a full-automatic biochemical analyzer.HE and Masson staining methods were used to observe the pathological changes of liver tissues of rats in each group under optical microscope.Immunofluorescence staining and fluorescence microscopy were used to compare the expression of mTOR in liver tissues of rats in each group.The level of mTOR protein in liver tissues of rats in each group was detected by Western blot.(3)SPSS 23.0 statistical software was used to process the data.the data was expressed by mean soil standard deviation(x ± s),one-way ANOVA was used for comparison among groups,and LSD was used for comparison between the two groups,p<0.05 was statistically significant.Results:(1)The contents of TC,TG,AST and ALT in the serum of the rats in the experimental group were significantly lower than those in the control group(p<0.05).the contents of TC,TG,AST and ALT in the serum of the rats in the experimental group were significantly higher than those in the normal group(p<0.05).compared with the normal group,the contents of TC,TG,AST and ALT in the serum of the rats in the control group were significantly increased(p<0.05).(2)HE staining of liver tissue.The experimental group showed structural changes of hepatic lobules,fatty degeneration of hepatocytes and a few necrosis of hepatocytes,but the pathological changes were less severe than those inthe model group.In the control group,fatty liver tissue,liver lobule structure disorder,extensive fatty degeneration of liver cells,ballooning degeneration and liver cell necrosis were observed.NAS score >4,NASH model successfully established.Normal group showed complete and clear structure of liver lobule,neat liver plate and no cell necrosis.(3)Masson staining comparison of rats in each group.In the experimental group,the structure of hepatic lobules was changed,and some hepatocytes had less fat vacuoles in cytoplasm,fatty degeneration of hepatocytes and a few necrosis of hepatocytes,but the lesion was less severe than that in the control group.In the control group,hepatic lobule structure was disordered,lobule structure partially disappeared,fat vacuoles of different sizes appeared in most hepatocytes,the boundaries between cells were blurred,and there were extensive hepatocyte steatosis,ballooning degeneration and hepatocyte necrosis.At the same time Masson staining showed perisinusoidal fibrosis or periportal fibrosis.Masson staining of liver tissue in normal group rats showed no obvious abnormality and no fibrous connective tissue deposition.(4)Immunofluorescence staining of liver tissue.In the experimental group,vacuoles formed after fat dissolution were observed,and mTOR protein expression was not found.A large number of fat vacuoles,mTOR expression and strong fluorescence intensity were observed in the liver tissue of the control group.No mTOR protein was expressed in normal group.(5)Western blot results showed that the mTOR content in the liver tissue of rats in the control group was the highest,followed by that in the experimental group and the lowest in the normal group.The level of mTOR protein in the experimental group was significantly lower than that in the control group(p<0.05),and the level of mTOR protein in the control group was significantly higher than that in the normal group(p<0.05).Conclusion:High fat diet leads to nonalcoholic steatohepatitis in rats,and mammalian target protein of rapamycin(mTOR)inhibitor rapamycin can reduce mTOR expression.The expression of mTOR protein is reduced,and lipid deposition and inflammatory damage in rat NASH liver tissue are relatively reduced.The expression of mTOR may be an important signal pathway for NASH.