Differential Expression Of TGF-β-Smad Signaling Pathway Related Cytokines In The Intervention Of Non-alcoholic Fatty Liver Disease In Mice

Objective: Discuss the intervention of aerobic exercise training and Chinese medicine on the formation of non-alcoholic fatty liver in mice that was induced by high fat diet.And preliminary reveal the mechanism of aerobic exercise training and Chinese medicine on improving the cellular factors of non-alcoholic liver injury in mice.Methods: Experimental object :80 male mice of KM species(Weight 18~22g),feed at room temperature(23 C —25 C)and under the natural light.Established the model of non-alcoholic fatty liver which was induced by high glucose and high fat diet.Eliminated injury mice and kept the mice which were successful modeled. Randomly divided those mice into 5groups(12 mice per group).The groups were named as Negative control group(OJ),Positive control group(Y),Traditional Chinese medicine group(M),Aerobic exercise group(E),Aerobic exercise+ Traditional Chinese medicine group(E+M).Establish a mouse model of non-alcoholic fatty liver disease.The high fat diet has been modified slightly from the non-alcoholic fatty liver animal model of high fat emulsion lavage which had been reported.lavaged once at 1 pm everyday.Established the model of non-alcoholic fatty liver after5 weeks of continuous lavage. Randomly selected 5 mice after the end of modeling and Selected fresh liver tissue for HE test.Group E and group E+M aerobically exercise in a horizontal animal run stage,In the first three days,Both groups were trained for 30 min on the stage and the speed was 8m/min. Time increased to 60 min and the speed increased to 12m/min from 4-6day. Keep it until the seventh weeks. The best medicine effective dose according to body surface area and mice by gavage dose equivalent conversion. Before gavage with 0.5 ml orally.(the concentration of 0.2^g/g in weight). The dose of group E+M was(200ml/kg body weight), before the experiment is equipped with the required concentration of irrigation(total volume0.5ml). Formulated the drug of required concentration before experiment(0.5mL). Group OJ and group E took same amount of normal saline. The positive control group were gavaged with silymarin suspension by human and mouse 0.5ml/(reduced surface area equivalent to 20 times the human use).Each group was administered for 6 weeks. Mice were kept moving during training.6 weeks later, The liver mitochondria were isolated from mice after a fast of one night and recorded the change of behavior.Observed the microstructure morphology of livertissue injury in mice. growth factor TGF-β1 and protein expression level were detected with immunohistochemistry.Expression level of smad2、smad3 mRNA in hepatic tissue were detected with RT-PCR. Serum aspartate aminotran sferase(AST),Alanine Amiotransferase(ALT), Triglyceride(TG) and Serum total cholesterol(TC), Superoxide Dismutase(SOD),Malondialdehyde(MDA) were detected with Biochemical analysis.Results:(1)The HE stain displayed that the degrees of fatty liver lesion of group OJ was Significantly higher than group Y. mouse hepatocytes became fibrosis and degeneration of liver cells appeared. the degrees of fatty lesion was reduced in group M and group Y compared with group OJ. the degrees of fatty liver lesion was reduced in group E and group E+M compared with group OJ.There was no statistically significant difference between normal group and group(E+M)/E. hepatic cells of group OJ arranged in disorder. Degeneration and fibrosis of liver cells was obvious in group Y and group E. Observed point or slice necrosis, Structural changes in hepatic lobules and hepatic cells arranged in disorder in group M.there was no obviously regression in the tissue of group E+M,and the Hepatic leaflets was in normal structure, the hepatic cells arranged in order.(2)Serological results showed: Throught the single factor analysis, ALT,AST,TG and TC level were markedly reduced in the group Y,group M,group E and group E+M. The obvious synergy between Traditional Chinese medicine and Aerobic exercise reduced the level of ALT,AST,TG and TC.(3)Immunocytochemical results showed that the growth factor TGF-β1 and the expression level of Protein were both Significantly lower than group.(4)RT-PCR results showed :Compared with group OJ,Expression level of smad2、smad4 mRNA were Significantly decreased in group E+M. The combinative intervention model of aerobic exercise and traditional chinese medicine has an apparent protection against NAFLD liver injury of mice.Conclusions: Aerobic exercise combined with traditional Chinese medicine in the intervention group, on NAFLD liver injury are obviously resistant to damage and protective effect of aerobic exercise combined with traditional Chinese medicine intervention can down mouse TGF- 1 protein expression and expression in liver tissue of Smad3 and Smad4 growth factor protein was effectively suppressed, then the impact of TGF beta/ Smad signaling pathway occurs in NAFLD, a regulatory role in the development of prevent or reverse the process of hepatic fibrosis.