Discovery Of Active Components And Confirmation Of Action Target Of Lignans In Schisandra Chinensis Attenuating Liver Fibrosis

Objective:To explore the main pharmacodynamic components of Lignans in Schisandra chinensis attenuating Liver Fibrosis.The experiment was carried out in vitro and in vivo experiments by gene knockout or knockdown technique to verify its target of anti-liver fibrosis of Lignans in Schisandra chinensis.Methods:1.Firstly,CCK-8 experiment and real-time PCR were used to screen the active components of Lignans in Schisandra chinensis and to determine the main pharmacodynamic components that play an important role in attenuating Liver Fibrosis.2.Secondly,CRISPR/Cas9 technology was used to construct CB2-/-mouse model,and the liver fibrosis model of normal mice and gene knockout mice was established intraperitoneal injection of carbon tetrachloride carbon（CCL₄）.We set up seven groups（WT+CD、CB2-/-+CD、WT+CCl₄、CB2-/-+CCl₄、WT+CCl₄+JWH133、WT+CCl₄+Sch B、CB2-/-+CCl₄+Sch B）.The animal experiment was carried out for 49days.At the end of the experiment,liver tissues were taken for HE and Masson staining to observe the pathological changes of liver tissues in each group.ELISA was used to determine ALT and AST in serum of mice.Western blot was used to detect the protein expression level ofα-SMA and collagen I.In vivo to verify the anti-fibrosis effect of pharmacodynamic components combined with CB2.3.Finally,RNAi,ELISA and q PCR were used to conduct cell experiments to verify the anti-fibrosis effect of pharmacological ingredients based on target CB2 in vitro.Results:1.In eight kinds of Lignans in Schisandra chinensis（Schisandrin A、Schisandrin B、Schisandrin C、Schizandrol A、Schizandrol B、Schisanrherin A、Schisantherin B、Schisanhenol）screening of its main efficacy components Schisandrin B which is play the important role in attenuating Liver Fibrosis.2.CB2-/-mouse model was successfully constructed.ALT and AST levels in liver fibrosis model group were significantly higher than those in normal group（P<0.05）,suggest the animal pathological model was successfully established.Animal experiments showed that Schisandrin B binding targets CB2,can effectively reduce the expression level of ALT and AST in liver fibrosis model mice,and significantly improve the pathological changes of liver fibrosis in mice organization,reduce liver fibrosis in miceα-SMA and collagen I protein expression.3.Subsequent cell experiments showed that Schisandrin B targeting CB2 receptor could significantly inhibit the release of LPS-induced inflammatory cytokines（IL-6、IL-1β、TNF-α、i NOS）and its inhibitory effect was dose-dependent;After transfection with CB2 si RNA,the expression of proinflammatory factor was further increased,and the anti-inflammatory effect of Schisandrin B was significantly reduced.In conclusion,these findings suggest that Schisandrin B can significantly reduce liver fibrosis via inhibiting macrophagocyte polarization by targeting CB2 receptors.Conclusions:Schisandrin B is the main active ingredient of Lignans in Schisandra chinensis which is play the important role of attenuating liver fibrosis.In vitro and in vivo experiments showed that Schisandrin B can effectively attenuating liver fibrosis by targeting CB2 receptors.