The Study Of Mechanism Of Xiaochaihutang On The Hepatic Fibrosis Involved Nrf2/ARE Signal Pathway

Objective:To study the effect and mechanism of Xiaochaihutang on hepatic fibrosis in rats.Methods:This experiment is divided into two parts:1.In vivo experiments:Healthy male Sprague Dawley rats were randomly divided into 6 groups:Vehicle control group,CCl₄control group（model group）,XCHT（2.5 g/kg）treatment group,XCHT（5 g/kg）treatment group,XCHT（10 g/kg）treatment group and silybin treatment group.In addition to the Vehicle control group,the liver fibrosis model was established by repeated injection of 50%CCl₄4ml/kg,olive oil dissolution,intraperitoneal injection twice a week within 9 weeks.All rats were sacrificed on the ninth weekend and the blood and live samples were obtained.Liver function was detected by HE staining and Masson trichrome staining.The expression of alpha-smooth muscle actin（α-SMA）was detected by immunohistochemical method.The method of Real-time RT-PCR and Western-Blot were used to detect the changes of Nrf2 pathway,such as the Nrf2,Nqo1,GCLM,GCLM,Keap-1,Nqo-1,HO-1 and so on.2.In vitro experiments:Serum containing drug was prepared by serum pharmacological method,and rat hepatic stellate cells were cultured with drug containing serum.The survival rate of hepatic stellate cells was detected by CCK-8 method and the expression ofα-SMA protein was detected by immunofluorescence.The method of Real-time RT-PCR and Western-Blot were used to detect the changes of Nrf2 pathway,such as Nrf2,Nqo1,GCLM-1,Keap1,Nqo-1,HO-1,and so on.HSCs were treated with gene silencing method to observe the effect of Xiaochaihutang after Nrf2 gene knockdown on the proliferation and activation of HSC cells.Results:1.In vivo experiments.The results of liver function and liver fibrosis test showed that compared with the vehicle group,the ALT in the model group was significantly higher than that in the vehiclel group,indicating that repeated intraperitoneal injection of CCl₄resulted in liver injury and hepatic fibrosis.Compared with the model group,the ALT in the Xiaochaihutang treatment group decreased significantly,which indicated that Xiaochaihutang could improve the ALT elevation induced by CCl₄,and the Xiaochaihutang（10 g/kg）treatment group could significantly reduce the serum HA,PCIII,LN concentration,which indicated that Xiaochaihutang could reduce the degree of liver fibrosis.The results of HE staining and Masson staining showed that:compared with the vehicle group,the model group produced fatty degeneration,inflammatory cell infiltration and hepatic fibrosis,compared with the model group,Xiaochaihutang and silybin treatmentgroupcouldimprovethepathologicalchanges.Theresultsof immunohistochemistry showed that the expression ofα-SMA in the model group was much higher than that in the vehicle group.After 4 weeks of treatment,the expression ofα-SMA in the Xiaochaihutang（10 g/kg）treatment group was significantly decreased.The results of real-time RT-PCR showed that:After liver fibrosis induced by CCl4,the expression of Nrf2,GCLC,GCLM gene was significantly decreased in the liver compared with the vehicle group.Compared with the model group,the Xiaochaihutang（10g/kg）treatment group could increase the expression of the GCLM,Nrf2,Nqo-1 and GCLC.The results showed that Xiaochaihutang could activate Nrf2 pathway.Western blotting showed that:Compared with the model group,the Xiaochaihutang（10 g/kg）treatment group could significantly increase the protein expression of Nqo-1,GCLC and GCLM in the liver.At the same time,Xiaochaihutang（10 g/kg）treatment group also increased the expression of HO-1 protein in liver.This is consistent with the results of real-time RT-PCR,indicating that Xiaochaihutang activated the Nrf2 pathway.2.In vitro experiments.CCK-8results show that:The inhibitory rates of HSCT6 cells cultured with 10%volume fraction of Xiaochaihutang containing drug serum for 48 h were 10.30,14.60,16.50,respectively,and the highest inhibition rate was 20.30%at 60 h.The results showed that XCHT could inhibit the proliferation of HSCT6 cells.ELISA results showed that:compared with the normal group,XCHT and silybin containing serum significantly reduced the production of Col-I in HSCT6 cells cultured for 60 h.The results of immunofluorescence showed that compared with the normal group,the expression ofα-SMA was significantly reduced in the XCHT group and the silybin group.The results of real-time RT-PCR showed that the level of Nrf2 gene in the nucleus of HSCT6 cells and the level of Nqo-1 gene in the cytoplasm of HSCT6 cells were significantly increased in XCHT group compared with the normal group.The results showed that the serum of XCHT activated the Nrf2 pathway of HSCT6 cells.The results of Western blotting showed that the protein expression ofα-SMA was significantly decreased in XCHT group and silybin group compared with the normal group.XCHT group significantly increased the protein expression of Nrf2,Nqo-1,GCLC,GCLM,which is consistent with the results of PCR.The results of gene silencing experiment showed that:Compared with negative control group,the protein expression of Nrf2,Nqo-1,GCLC,GCLM in XCHT groupwas significantly decreased in the cells.The protein expression ofα-SMA was significantly increased,the cell survival rate was increased and the Col-I concentration increased.Conclusion:1.Xiaochaihutang improved the liver function and the degree of hepatic fibrosis of chronic hepatic fibrosis model induced by CCl4;2.Xiaochaihutang can inhibit the activation of hepatic stellate cells;3.Xiaochaihutang is related to the mechanism of anti-hepatic fibrosis and the up-regulation of Nrf2 pathway to resist oxidative stress.