Effect Of Zhuanggan Zhuyu Decoction On Expression Of Shh,Smo,Gli1 And Bcl-2 In Rats With Hepatic Fibrosis

Objective: In this study,CCl4 modeling method was used to induce a rat model of liver fibrosis,to explore the effects of Zhuanggan Zhuyu Decoction on Shh,Smo,Gli1 and Bcl-2 in rats with liver fibrosis,and then to explore the mechanism of Zhuanggan Zhuyu decoction in the treatment of liver fibrosis,in order to provide objective basis for Zhuanggan Zhuyu decoction to prevent and treat liver fibrosis.Methods: Fifty SD rats were randomly selected from 60 SD rats to replicate liver fibrosis model by CCl4 induction method,and the remaining 8were blank group.The modeling rats were injected with 40% CCl4 peanut oil solution subcutaneously,with the first injection ratio of 0.5m L/100 g and the subsequent injection ratio of 0.3m L/100 g,three times a week.At the 6th and8 th week of modeling,3 modeling rats were selected for liver tissue HE and Masson detection to observe whether the modeling was completed.After successful modeling,the rats were randomly divided into model group,Zhuanggan Zhuyu decoction low-dose group,Zhuanggan Zhuyu decoction medium-dose group,Zhuanggan Zhuyu decoction high-dose group and colchicine group.The doses of Zhuanggan Zhuyu decoction in high-dose,medium-dose and low-dose groups were 26.46g/kg,13.23g/kg and 6.625g/kg,respectively,while the doses of colchicine group were 0.126g/kg.The blank group and model group were given the same amount of normal saline once a day for 6 weeks.After the last administration of the drug,the rats were deprived of water for 12 h,and their weights were weighed and recorded.The rats were anesthetized and blood from abdominal aorta was taken,and the upper serum was centrifuged for 2h at room temperature.The liver function in dexes such as ALB(alanineaminotransferase),AST(aspartateaminotransferase)and TBIL(total bilirubin)were detected by automatic biochemical instrument.The rat livers were divided into two parts,one was fixed with 10%formaldehyde,and the other was stored in a-80℃ refrigerator.The fixed liver tissues were histopathologically sliced and stained by HE and Masson,and the pathological status of the liver tissues was observed under the microscope.The protein and m RNA expression levels of Shh,Gli1,Smo and Bcl-2 in the liver tissues of rats placed in the refrigerator at-80℃ were detected by WB and RT-PCR.Results: 1.The health condition of rats in all treatment groups was significantly improved compared with that before treatment,the hair color changed from yellow to white,the diet increased,the mental condition became better,the activity ability and stool quality were significantly improved.The trend of body weight increase in treatment groups was higher than that in model group,but lower than that in blank group,without statistical significance.2.Compared with blank group,the level of serum ALT and AST in model group was significantly increased(P<0.01),and the level of TBIL was increased(P<0.01),suggesting serious liver tissue injury in model group.Compared with model group,the serum ALT and AST levels in Zhuanggan Zhuyu decoction medium and high dose groups were significantly decreased(P<0.01),and the serum TBIL levels were decreased;Compared with model group,the serum ALT level and AST level of Zhuanggan Zhuyu decoction low-dose group were significantly decreased(P<0.01)and decreased(P<0.05),but there was no statistical significance in the comparison of TBIL.The AST and ALT levels of colchicine group were decreased compared with model group(P<0.05),but there was no statistical significance in TBIL compared with model group.3.HE and Masson results showed that obvious fibrous tissue hyperplasia,swelling and necrosis of liver cells occurred in liver tissues of the hepatic fibrosis model group,forming mesh fibrous septa,infiltration of inflammatory cells,expansion of the portal area,and formation of false lobules.After drug intervention,compared with the model group,the number of liver cell necrosis and deformable cells decreased,the degree of inflammatory cell infiltration decreased,the proliferation of fibrous tissue and the area of fibrous septum area decreased,and no false lobules were observed in the Zhuanzhuyu decoction treatment groups.Compared with the model group,colchicine group showed a certain improvement in the area of fibrous septum.4.WB detection results showed that compared with blank group,Shh protein expression,Smo protein expression,Gli1 protein expression and Bcl-2 protein expression were increased in model group(P<0.01).Compared with model group,Shh,Smo,Gli1 and Bcl-2 protein expression in colchicine group were decreased(P<0.01).Compared with model group,Shh protein expression,Smo protein expression,Gli1 protein expression and Bcl-2 protein expression were decreased in Zhuangganzhuyu decoction low-dose,medium-dose and highdose groups(P<0.01).5.PCR results showed that compared with blank group,Shh m RNA expression,Smo expression,Gli1 expression and Bcl-2 m RNA expression were significantly decreased in model group(P<0.01).The expression of Shh m RNA,Smo m RNA,Gli1 m RNA and Bcl-2 m RNA were decreased(P<0.01)in Zhuangganzhuyu decoction groups(P<0.01).The m RNA expression of Shh,Smo,Gli1 and Bcl-2 in colchicine group was decreased(P<0.01).Conclusion(s): 1.Zhuanggan Zhuyu Decoction has as significant therapeutic effect on rats with liver fibrosis,which can improve the liver function of rats with liver fibrosis,reduce the degree of liver tissue fibrosis,and has a good effect on liver protection.The therapeutic effect of Zhuanggan Zhuyu decoction group was better than colchicine group.2.Zhuanggan Zhuyu Decoction may regulate hepatic fibrosis by down-regulating the expression of Hedgehog signaling pathway molecules Shh,Smo,Gli1 and Gli1 target gene Bcl-2.