Effects Of Removing Dampness And Clearing Vessels And Networks Method On Mice With Nonalcoholic Fatty Liver Fibrosis Based On The Hedgehog Signal Transduction Pathway

Objective: According to the etiology and pathogenesis of liver fibrosis in traditional Chinese medicine,the influences of dispelling dampness,regulating collagements and removing dampness and regulating collagements on the expression of Hedgehog pathway in liver tissue of mice with liver fibrosis in non-alcoholic fatty liver disease were observed and evaluated respectively,and their mechanism of action was further studied and discussed,so as to clinically apply dispelling dampness,regulating collagements and removing dampness and regulating collagements to treat liver fibrosis.Provide some theoretical support.Methods: Sixty adult male C57BL/6J mice were randomly divided into normal group,model group,western medicine group,Qushi group,Tongluo group and Qushi Tongluo group.Normal group and model group were administrated with distillation,western medicine group was administrated with colchicine,Qushi group was administrated with Yinchenhao Decoction,Tongluo group was administrated with Xue’s Sanjia San,and Qushi Tongluo group was administrated with self-designed Fang Yinchen Sanjia San.Except the normal group,all the patients were fed adequate methionine choline deficiency diet(MCD diet)for8wk.The administration group was also given corresponding traditional Chinese medicine and colchicine daily gavage intervention,and killed at the end of 8wk.Serum aspartate aminotransferase(AST),alanine aminotransferase(ALT),total bilirubin(TBil),liver fibrosis indexes hyaluronic acid(HA),laminin(LN)and type III procollagen(PCⅢ)were determined by enzyme-related immunosorbent assay.The content of hydroxyproline(HYP)in mouse liver was determined by alkaline hydrolysation.HE and Masson staining were used to observe the pathological changes of liver tissue of mice in each group.The expression levels of ligand Shh,nuclear transcription factors Gli1,Gli2,and α-smooth muscle actin(α-SMA)were detected by q PCR and Western blot.Results:1.Serum liver function test results: AST,ALT and TBil of mice in the model group were significantly higher than those in the normal group(P < 0.05).Compared with the model group,AST,ALT and TBil of all drug groups were decreased to varying degrees(P<0.05),among which the decrease was most significant in the Qushi Tongluo group,but there was no significant difference between the Qushi and Tongluo groups.2.Results of liver fibrosis indexes: compared with normal group,HA,LN and PCⅢ in model group were significantly increased(P<0.05);Compared with model group,the liver fibrosis indexes HA,LN and PCⅢ in all medication groups were significantly decreased(P<0.05),especially in Qushi Tongluo group.Compared with western medicine group,the levels of HA,LN and PCⅢ in all TCM treatment groups were significantly decreased(P<0.05),and the levels of LN and PCⅢ in Qushi Tongluo group were significantly decreased compared with Western medicine group(P<0.05).3.Compared with normal group,HYP content in model group was significantly increased(P < 0.05).Compared with model group,HYP in all medication groups was significantly decreased(P < 0.05),especially in Qushi Tongluo group.Compared with western medicine group,the levels of HYP indexes in all TCM treatment groups were significantly decreased(P < 0.05),but there was no difference between Qushi group and Tongluo group.4.Histopathological results: HE and Masson staining results showed that the liver fibrosis in the drug treatment group was improved to varying degrees,among which the steatosis,watery degeneration and liver cell necrosis in the Qushi Tongluo group were most significantly reduced compared with the model group,and the improvement effects in the western medicine group,the Qushi and Tongluo groups were similar.5.The m RNA expression levels of Gli1,Gli2,Shh,α-SMA in the model group were significantly higher than those in the normal group(P < 0.05).Compared with the model group,the expressions of Gli1,Gli2,Shh,α-SMA in all drug treatment groups were decreased.Compared with western medicine group,the expressions of Shh,Gli1,Gli2 and α-SMA in Qushi Tongluo group,Tongluo group and Qushi Tongluo group were decreased(P<0.05),and the expression levels of Shh,Gli1,Gli2 and α-SMA in Qushi Tongluo group were most significantly decreased(P < 0.05),but there was no significant difference between Qushi group and Tongluo group.6.Gli1,Gli2,Shh,α-SMA protein expression Compared with the normal group,Gli1,Gli2,Shh,α-SMA protein expression in the model group was significantly increased(P<0.05).Compared with the model group,Gli1,Gli2,Shh,α-SMA protein expression in all drug treatment groups was decreased,Gli1,Gli2,Shh,α-SMA in Qushi Tongluo group were most significantly decreased(P<0.05).Conclusion:1.Yinchenhao Decoction,Xue’s Sanjia San and Yinchen Sanjia San can all improve the morphological structure of liver,improve liver function and reduce the degree of liver fibrosis by inhibiting Hedgehog signaling pathway,indicating that the proper application of dispelling dampness method and Tongluo method can effectively prevent and treat liver fibrosis and protect liver function.2.Dispelling dampness and Tongluo methods can significantly reduce liver function indexes,liver fibrosis and HYP indexes in mice with liver fibrosis of non-alcoholic fatty liver disease,and reduce the expression levels of Gli1,Gli2,Shh,α-SMA.The best curative effect is obtained from the combination of dispelling dampness and Tongluo drugs,suggesting that the combination of dispelling dampness and Tongluo drugs has the best curative effect,and dispelling dampness and Tongluo method has the most significant curative effect on improving liver fiber indexes.3.Dispelling dampness method and Tongluo method have synergistic effect,and the combined use is the best.Dispelling dampness and Tongluo method has good curative effect on liver function indexes,liver fibrosis and HYP indexes,and the comprehensive curative effect is better than colchicine in western medicine group.4.Dispelling dampness and Tongcollateral can significantly down-regulate the expression levels of Gli1,Gli2,Shh,α-SMA in Hedgehog signaling pathway,and play an important role in inhibiting HSC activation,which may be one of the mechanisms of dispelling dampness and Tongcollateral therapy against liver fibrosis.