Research On The Effect Of "Chai Jie Yi Gan Xian" On Hepatic Fibrosis In Rat Model

Objective:Through observing the therapeutic effect of "Chai Jie Yi Gan Xian" on indexes of hepatic fibrosis model in rats,to investigate the effect of "Chai Jie Yi Gan Xian" on PDGF and TGF-beta in rat with hepatic fibrosis and its mechanism of inhibiting rat HSC-T6 cell activation and proliferation,which showed the feasibility of "Chai Jie Yi Gan Xian" in the treatment of liver fibrosis,in order to provide a kind of compound Chinese medicine preparation feasible for clinical treatment of hepatic fibrosis,and also to make a scientific evaluation,and basis for provide experimental in it widely used in clinic.Method:Animal experiments: 60 SD rats,male,were randomly divided into 6groups,10 rats in each group.Were blank control group,model group,positive group(bjjw group),"Chai Jie Yi Gan Xian" low,medium and high dose groups.Except for the blank group,all the other groups were given 3% thioacetamide intraperitoneal injection molding,160mg/Kg,once every other day for 6 weeks.Fourth weeks,positive group of Biejiajian pills to 1g/Kg·d(medium dose)by gavage,"Chai Jie Yi Gan Xian" low(1.99g/Kg·d),medium(3.98g/Kg·d)and high(7.96g/Kg·d)dose groups were given "Chai Jie Yi Gan Xian",by gavage,for 5 weeks.In the last 24 hours after gavage,with chloral hydrate(normal saline into the concentration of 4% rats per 100 g rats were injected with 1ml)lightly anesthetized rats,the serum and liver tissue,said electronic weighing wet liver weight and calculate the liver index;automatic analysis method for detection of rat liver function index(ALT,AST,ALP);enzyme linked immunosorbent assay of hepatic fibrosis serum markers(LN,HA,CIV,PCIII);liver slices were stained with Masson,to observe the degree of hepatic fibrosis;immunohistochemistry was used to detect the expression of PDGF and TGF-beta 1 in liver after drug treatment.Cell test: HSC-T6 cell lines were purchased from the Shanghai cell bank,10% fetal bovine serum DMEM culture,37 degrees,5%CO2,saturated humidity.Collect the logarithmic growth phase were seeded in 96 well plates,each hole of 5x104 cells.Add the corresponding concentration of the experimental drug(dissolved in normal saline after filtration by 0.2um membrane).Drug effects after 24 h through the proliferation of HSC cells was measured by CCK-8.Western blot to detect the expression of TGFbeta 1 and alpha-SMA in HSC cells.Apoptosis flow cytometric detection of HSC cells.Result:(1)The rat liver fibrosis model: compared with the control group,each group of rat liver index increased significantly(P<0.01),compared with the model group,"Chai Jie Yi Gan Xian" high dose group decreased most significantly;rat liverslices stained with Masson showed: model group liver color dark red,visible liver cell swelling,degeneration and wide the optical fiber hyperplasia,the formation of fibrous septa,different degree of each treatment group of rat liver tissue inflammatory pathological changes were hyperplasia of fibrous tissue decreased,especially in "Chai Jie Yi Gan Xian" middle and high dose group was most obvious.It was suggested that both the model group and the treatment group were successful,and the positive drug group and the "Chai Jie Yi Gan Xian" dose group could play a different therapeutic effect on the development and development of liver fibrosis.(2)Rat serological test: automatic analysis was used to detect the liver function of rats.Compared with the control group,the serum concentration of rats in the model group in ALP,AST and ALT were increased significantly.Compared with model group,each group in ALP,AST,ALT were concentration decreased to different extent(P<0.05),of which "Chai Jie Yi Gan Xian" in the middle and high dose group were significantly reduced(P<0.01).Enzyme-linked immunosorbent assay was used to detect the serological markers of liver fibrosis in rats.Compared with the blank group,the LN,HA,CIV and PCIII in the model group increased significantly.Compared with the model group,the LN,HA,CIV and PCIII indexes in each drug delivery group decreased significantly(P < 0.05),and the most significant decrease was in the "Chai Jie Yi Gan Xian" high dose group(P < 0.01).(3)The expression of PDGF and TGF-beta 1 in liver tissues of rats was detected by immunohistochemistry.Compared with the blank group,the PDGF and TGF-beta 1were significantly increased in the model group.Compared with the model group,the expression of PDGF could be decreased by both the positive drug group and the "Chai Jie Yi Gan Xian" dose group(P<0.01).The expression of TGF-beta 1 could be decreased in the positive drug group and the "Chai Jie Yi Gan Xian" medium and high dose group(P < 0.01).(4)Cell experiments showed that: compared with the control group,200,100,50mg/L concentration of "Chai Jie Yi Gan Xian" can inhibit the proliferation of HSC-T6 cells(P<0.05);"Chai Jie Yi Gan Xian" in the medium and high dose group can downregulate the expression of TGF-beta 1 in HSC-T6 cells,decrease the protein expression of alpha-SMA(P < 0.05).Flow cytometry showed "Chai Jie Yi Gan Xian" had no significant effect on the apoptosis of HSC-T6 cells.Conclusion:(1)"Chai Jie Yi Gan Xian" can significantly inhibit and alleviate TAA induced liver fibrosis in rats,reduce liver index,liver function index and liver fibrosis index,protect and improve liver function.(2)"Chai Jie Yi Gan Xian" play a role in anti hepatic fibrosis by inhibiting the expression of PDGF and TGF-beta 1 and inhibiting the proliferation of hepatic stellate cells.