Study On The Mechanism Of Sanren Decoction In Prevention Hepatic Fibrosis Based On TGF-β1/Smads Signaling Pathaway

ObjectiveIn this study,dimethylnitrosamine（DMN）-induced hepatic fibrosis（HF）rat model was used to observe the effect of Sanren Decoction intervention on the expression of TGF-β1/Smad3 signaling pathway in HF rats.The effects of Sanren Decoction on the prevention and treatment of HF in rats were investigated from liver function,liver morphology,liver fibrosis degree and TGF-β1/Smad3 signaling pathway,for clinical use of xinfanhua method prevention and treatment of liver fibrosis beneficial exploration.MethodsForty-eight male Wistar rats at the age of 21 days were divided into blank group,model group,sanren Decoction groups(4.1g·kg^-1,8.2g·kg^-1,16.4g·kg^-1)and colchicine group(1.0×10^-4g·kg^-1)by random number method,with 8 rats in each group.The blank group was intraperitoneally injected with normal saline at 1.6 m L·kg-1 two days a week,and the other groups were intraperitoneally injected with 5%dimethylnitrosamine at 1.6 m L·kg^-1for four weeks.Since the modeling,sanren Decoction dose groups and colchicine group were given drug intervention according to the corresponding dose.Blank group and model group were given the same volume of sterilized drinking water for 4 consecutive weeks.Liver pathology of rats in each group was observed by HE,Sirius red and MASSON staining.Liver function（ALT,AST,PDGF）and fibrosis degree（HA,LN,TIMP-1,MMP-1）were detected by enzyme-linked immunosorbent assay（ELISA）to evaluate the liver function of rats in each group.The gene expression levels of TGF-β1,Smad3 and Smad7 were determined by PCR,and the protein expression levels of Smad3 andα-SMA were determined by WB method.Results1.General conditions:Naked eye observation showed that with the extension of modeling time,rats in the model group showed withered and yellow hair,decreased diet and activity,sticky and smelly stool,yellow urine,progressive weight decline,and group warming,etc.Compared with the model group,the above conditions of rats in sanren Decoction concentration groups and colchicine group were significantly improved.2.Comparison of liver morphology:By observing the results of HE staining and Masson staining under light microscope,it was found that the liver tissue lesions of the model group were significant,and the liver damage of the sanren Decoction groups and colchicine groups was lighter than that of the model group.3.Comparison of serum liver function and fibrosis degree:Compared with blank control group,the contents of ALT,AST,PDGF,HA,LN and TIMP-1 in serum of model group were increased（P<0.01）,while the content of MMP-1 was decreased（P<0.01）.Compared with model group,serum ALT,AST,HA and LN of colchicine and Sanren Decoction groups were significantly decreased（P<0.01）;PDGF decreased in the low and medium concentrations of Sanren Decoction groups（P<0.01）,while there was no statistical difference in PDGF between the colchicine group and the high concentration group compared with the model group.There was no statistical difference in serum MMP-1 between low and medium sanren Decoction groups and model group,but it was significantly increased in high and colchicine groups（P<0.01）.Compared with model group,TIMP in sanren Decoction low concentration group was decreased（P<0.05）,and significantly decreased in medium and high concentration groups and colchicine groups（P<0.01）.4.Comparison of m RNA expression levels of TGF-β1,Smad3 and Smad7:TGF-β1,Smad3 and Smad7 m RNA in model group were up-regulated compared with blank control group（P<0.01）,TGF-β1 and Smad3 m RNA in sanren Decoction and colchicine groups were significantly down-regulated compared with model group（P<0.01）,and Smad7 m RNA was significantly up-regulated compared with model group（P<0.01）.5.Comparison ofα-SMA and Smad3 protein expression levels:The expression ofα-SMA and Smad3 protein in the model group was significantly increased compared with that in the blank control group（P<0.01）,the expression ofα-SMA protein in the low concentration group of Sanren Decoction was lower than that in the model group（P<0.05）,and the expression ofα-SMA protein in the medium and high concentration groups of Sanren Decoction and colchicine groups was significantly decreased compared with that in the model group（P<0.01）.Smad3 protein expression in sanren Decoction groups and colchicine group was significantly lower than that in model group（P<0.01）.Conclusions1.Sanren decoction can inhibit TGF-β1/Smads signal pathway can significantly improve the liver morphological structure,liver inflammation and reduce the degree of liver fibrosis,so as to protect liver function.2.Low and medium concentration Sanren decoction has better effect on improving liver inflammation in HF rats,medium and high concentration Sanren decoction has better effect on improving liver fibrosis in HF rats,and medium concentration Sanren decoction has the best effect on preventing and treating DMN induced HF rats.