Effects Of Hbyy Prescription On Inhibition Of Hepatic Fibrogensis Through Nrf2/HO-1 Signaling Pathway

Due to the process of liver fibrosis accompanied with fibrosis,the central mechanism of liver fibrosis is the activation of hepatic stellate cells?HSCs?.The ativation of HSCs is regulated by various factors,which oxidative stress plays a vital role.Oxidative stress is mediated by reactive oxygen species.Under normal condition,the level of ROS production and clearance is a dynamic banlance in the body.When the liver is exposed to xenobiotics,the banlance of ROS production and its removal is broken,which ultimately leads to oxidative damage of large Mecular substances in cells and liver dysfunction.Nuclear factor erythroid 2 related factor 2?Nrf2?is an essential regulatory protein of endogenous antioxidant defense system.In the condition of oxidative stress,nuclear transcription factor Nrf2 translocates to nucleus and combines with antioxidant response element?ARE?,and then regulates the transcription of downstream antioxidant enzymes HO-1 gene.There are various drugs that induce Nrf2 activation.In this article we chose tradition Chinese medicinal herbs Haobie Yangyin Ruanjian Prescription?HBYY?as a tool drug.ObjectiveTo investigate the effect of Haobie Yangyin Ruanjian Prescription on lipopolysaccharide-induced liver injury.This study preliminarily reveals that the mechanism of Nrf2-HO-1signaling pathway on inhibition of inflammation and oxidative stress under the impact of the tranfer of Nrf2 from cytoplasm to nucleus,which provides the suggestion for the prevention and treatment of Haobie Yangyin Ruanjian Prescription in autoimmune hepatitis.Methods1.Choose LX-2 as the research object.LX-2 were incubated with various concentration of H₂O₂?10?M/L?50?M/L?100?M/L?200?M/L?500?M/L?.After24h,MTT colorimetric assay was employed to detect the proliferation of LX-2 so that we can confirm the optimal concentration of H₂O₂ used to activate LX-2 and proper reaction time.2.Choose LX-2 as the research object.LX-2 were divided into control group and experimental groups.The experimental groups.The experimental groups including H₂O₂-treated groups and Co-administration of H₂O₂ and HBYY.The rate of proliferation of LX-2 was measured by MTT assay.Colorimetric method was employed to determine the activity of LDH;the experssions of Nrf2,HO-1 and TNF-?were evaluated by western blotting.Digestive method was adopted to determine the content of hydroxyproline.3.Using concanavalin A intravenous injection to induce liver fibrosis model in mice.The experimental groups including normal group,model group,HBYY-treated group,Fu fang bie jia ruan gan pian-treated group and colchicine group.Immunohistochemical staining was used to detect Nrf2,HO-1 protein experssion.HE staining was used to detect the effect of HBYY on liver tissue morphology induced by ConA in liver fibrosis mice.The effect of HBYY on the hepatic collagen fibers induced by ConA in liver fibrosis mice was detected by Sirius red staining.The levels of HA and PC?in serum were measured by radioimmunoassay.Diggestive method was adopted to determine the content of liver tissues hydroxyproline.The experssions of Nrf2 and HO-1 were evaluated by western blotting.Results1.HBYY inhibited the proliferation and function of LX-2 cells:compared with the control group,HBYY can inhibit the proliferation of LX-2 cells significantly with serine palmitoyl transferase,and the inhibition value in a concentration and time dependent manner.HBYY increased the experssion of Nrf2 and HO-1 and decreased the experssion of TNF-?;compared with the control group,the activity of LDH has no significant change;After HBYY treated LX-2 cells 24 hours,the content of Hyp in the supernatant was significantly decreased with dose-dependent manner.2.Using concanavalin A intravenous injection to induce liver fibrosis model in mice.Immunohistochemistry results showed that compared with model group,the expression of Nrf2 and HO-1 were increased in HBYY-treated group and the low dose was the most significant.HE staining and Sirius red staining showed that HBYY could relieve the damage caused by ConA to the liver structure and reduce the degree of liver fibrosis.Determination of the content of hydroxyproline in liver tissue by enzyme digestion method.The results showed that the content of hydroxyproline in HBYY-treated group was lower than model group.The results of radioimmunoassay for detection of HA and PC?showed that the content of HA and PC?in serum was significantly lower in HBYY-treated group than that in model group.Conclusions1.HBYY can activated Nrf2/HO-1 signaling pathway and promote the transcription of Nrf2.2.On the one hand,HBYY can decrease the proliferation of LX-2 cells in a dose and time-dependent manner.One the other hand,the expression of Nrf2 and HO-1protein was up-regulated and the expression of TNF-?protein was down regulated.3.Through inhibiting the proliferation of LX-2 cells and inducing cell apoptosis,HBYY can inhibit the formation of extracellular matrix.4.The content of serum HA and PC?in ConA-induced liver fibrosis model was significantly decreased by HBYY.