Effects Of Haobie Yangyin Ruanjian Fang On Inhibition Of Hepatic Fibrogensis Induced By Carbon Tetrachloride Through Influenced The Nrf2/NQO1 Signal Pathway

Hepatics fibrosis?HB?,as a common pathological change in all chronic liver disease,is caused by a variety of chronic pathogenic factors such as?cholestasis,chemical toxicities or certain metabolic diseases?,leading to the Extracellular matrix?ECM?synthesis increased,.the imbalance between the synthesis and degredation of ECM,caused a unusual deposition in the liverl.Researches have shown that the pathological changes in liver fibrosis are reversible,.But if there is no treatment,it will develop into liver cirrhosis or even liver cancer which is serious and irreversible.Hepatic stellate cell?HSC?,as the main source of ECM,its activation and proliferation has become the center event of the development of liver fibrosis.In this study,CCl₄?Carbon tetrachloride?was used as a stimulant to activate HSC,and Haobie Yangyin Ruanjian Fang was treated as a therapeutic drug.PurposeThe inhibition effect of Nrf2/NQO1 pathway on hepatic fibrosis was preliminarily revealed by investigating the relieved degree of oxidative damage induced by CCl₄-induced liver fibrosis via the treatment of Hao Bie Yang Yin Ruan Jian Fang.Methods1.the human LX-2 cells were selected as the experimental subjects in vitro.Divided the cells into normal control group,model control group,Haobie Yangyin Ruanjian Fang group,Haobie Yangyin Ruanjian Fang.+ CCl₄ group.Then the proliferation rate of LX-2 were measured by MTT assay;the activity of LDH and content of Hyp were measured by colorimetry assay.2.The human LX-2 cells were selected as the experimental subjects in vitro.Divided the cells into normal control group,model control group,CCl₄+Haobie Yangyin Ruanjian Fang group,CCl₄+dicoumarol+Haobie Yangyin Ruanjian Fang group,Sulforaphane + CCl₄ +Haobie Yangyin Ruanjian Fang group,CCl₄ +ATRA+Haobie Yangyin Ruanjian Fang group and CCl₄+dicoumarol+ATRA+Haobie Yangyin Ruanjian Fang group.Then the proliferation rate of LX-2 was determined by MTT assay.The content of Hyp was measured by colorimetry assay.The proliferation of LX-2 was determined by MTT assay.Western Blot was used to evaluate theexpression level of ?-SMA,Nrf2 and NQO1.3.In vivo,the SPF grade healthy Wistar rats were selected as the research object.The rat model of hepatic fibrosis induced by CCl₄ was established.The rats were divided into normal control group,model control group,colchicine group,The rats in the middle and low dose groups were followed for 6 weeks.All the rats were normal feeding for another week and then the liver tissue of each rats were collected.The content of Hydroxyproline?Hyp?was determained by colorimetric method,The expression of Nrf2 and NQO1 in liver tissue was detected by Western blot.What's more,the distribution of Nrf2 and NQO1 in liver tissue was detected by immunohistochemistry.Results1.When compared with the normal control group,the proliferation rate of LX-2 in the other three groups was much higher,the difference was statistically significant?P<0.05?,The content of Hyp was also significantly increased?P <0.05?,While the difference about the activity of LDH among the four groups was not significant?P >0.05?,When compared with the model control group,the content of Hyp was much higher than that of CCl₄ + Sulforaphane group and CCl₄ + Hao Bie Yang Yin Ruan Jian Fang group?P <0.05?.While the expression level of ?-SMA was the highest in the model control group?P <0.05?,and the expression level of Nrf2 and NQO1 was the lowest in that group?P <0.05?.2.Compared with the control group,the LX-2 proliferation rate of the other five groups was significantly higher than that of the control group?P <0.05?.The content of Hyp was also significantly increased?P <0.05?.The second one is the CCl₄ +Sulforaphane group,the survival rate of LX-2 cells of which was significantly lower than that of CCl₄ + Haobie Yangyin Ruanjian Fang group?P <0.05?,.And the inhibitory rate of LX-2 and the content of Hyp were significantly higher than that of CCl₄ + decoumarin + ATRA + Haobie Yangyin Ruanjian Fang group,the difference was statistically significant?P <0.05?.The expression levels of Nrf2 and NQO1 were much up-regulated?P <0.05?.3..Compared with the normal group,the content of Hyp of the model control group was significantly increased,the expression levels of Nrf2 and NQO1 weremuch decreased,?P <0.05?.and the ascites and hepatic fibrosis classification were serious in the model group.Compared with the model control group,the content of Hyp of the three therapeutic dose groups of Haobie Yangyin Ruanjian Fang was significantly decreased,?P <0.05?.the expression levels of Nrf2 and NQO1 were much increased,?P <0.05?.and the degree of liver fibrosis was also alleviated.Those show that Haobie Yangyin Ruanjian Fangsignificantly relieved the degree of liver fibrosis caused by the CCl₄ compound factors.Conclusions:1.Combained the results of vivo and viro,Haobie Yangyin Ruanjian Fang could influence the expression of Nrf2/NQO1 pathway,Then the proliferation of LX-2 was inhibited.At the same time,down-regulate the expression of ?-SMA,thus inhibiting the proliferation of LX-2 and production of Hyp induced by CCl₄?2.Haobie Yangyin Ruanjian Fang specifically activited the Nrf2/NQO1 pathway and up-regulated the expression level of Nrf2,which leads to the downstream target protein NQO1 much increased.Then effectively inhibited hepatic fibrosis.