Hepatoprotective Action And Mechanism Of Salidroside Against Hepatic Fibrosis

Background and objective:Liver fibrosis is a kind of pathological condition,which is caused by the invasion of various kinds of stimulation factors,which is characterized by over production and deposition of extracellular matrix and proliferation and activation of hepatic stellate cells.Without intervention,liver fibrosis often develops to liver cirrhosis,portal hypertension,liver cancer and so on.However,the process of liver fibrosis can be reversed,so it is necessary to focus on the treatment of hepatic fibrosis and its mechanism.Rhodiola rosea is a kind of medicinal herbs with lots of biological effects,including immune regulation,scavenging free radical,anti-tumor,anti-inflammation,improving sleep,enhancing memory,hepatoprotective action,anti-fatigue effect,adjusting the balance of energy metabolism and other pharmacological effects.Salidroside is the most effective component of Rhodiola rosea.Salidroside has protective effect on liver fibrosis,but the research on its protective mechanism is little.The study is to investigate the effect of salidroside on hepatic fibrosis and the mechanism by studying the cytokines,inflammasome,signaling pathway and epithelial mesenchymal transition and other aspects through constructing mice model of hepatic fibrosis and human liver cell line L02 model of hepatic fibrosis in vitro induced by TGF-?,in order to deepen our understanding on pharmacological action of salidroside and mechanism of liver fibrosis,.Method:Liver fibrosis model was established by subcutaneous injection of CCl₄ in mice and inducing human liver cell line L02 in vitro by TGF-?.Detect alanine aminotransferase?ALT?and aspartate aminotransferase?AST?,hyaluronic acid?HA?,laminin?LN?and pathological change by HE staining and Masson staining to evaluate the effect of salidroside on liver fibrosis.To study the possible mechanism of the protective effect of salidroside on liver fibrosis,the profibrogenic cytokines?TNF-?,IL-6 and IL-1??were detected by ELISA assay,and the expression of NLRP3,ASC,caspase-1,IL-1?;NF-?B,I?B and p-NF-?B,p-I?B;TGF?,smad3 and p-smad3;MMP2 and MMP9;?-SMA and type I collagen were detected by Western blot.Results:1.In mice models of hepatic fibrosis,AST and ALT,HA and LN in CCl₄ group were significantly higher than those in the control group,there was significant difference between the two groups?P<=0.01?.AST,ALT,HA and LN were significantly reduced in salidroside?20 mg/kg?group and?40mg/kg?group compared with CCl₄ group,which has statistical significance?P<0.01?.In HE staining,CCl₄ group showed hepatic lobules were unclear,liver cell cord was disordered,hepatic sinusoid was disappeared,and liver cells showed moderate or severe edema,degeneration and fatty change and multiple ballooning degeneration,accompanied with inflammatory cell infiltration.Salidroside?20 mg/kg?group and?40 mg/kg?group significantly reduced the degree of liver fibrosis,with mild ballooning degeneration and a small amount of inflammatory cell infiltration.In Masson staining,a large number of collagen fibers proliferated in the CCl₄ group,while in the salidroside?20 mg/kg?group and?40 mg/kg?group,collagen fibers proliferated less.2.In mice models of liver fibrosis,TNF-?,IL-6 and IL-1?in serum and liver of CCl₄ group were significantly higher than the control group,there was statistical significance?P<0.01?;TNF-?,IL-6 and IL-1?were decreased statistically in salidroside?20 mg/kg?group and?40 mg/kg?group compared with CCl₄group?P<0.01?.In liver tissue of group CCl₄,the expression of NLRP3,ASC,caspase-1 and IL-1 beta;p-NF-?B,p-I?B;TGF-?and p-Smad3;MMP2 and MMP9;?-SMA type and I collagen were significantly higher than that in the control group,there was statistical significance?P<0.01?,while decreased in salidroside?20mg/kg?group and?40mg/kg?compared with CCl₄ group,with statistical significance?P<0.01?.3.In human liver cell line L02 model of hepatic fibrosis in vitro induced by TGF-?,the content of ALT and AST in the supernatant of model group was significantly higher than that in the control group,with statistical significance?p<0.001?.Compared with the model group,salidroside?20?M and 40?M?could significantly reduce the levels of ALT and AST,with statistical significance.Under the stimulation of TGF-?,the expression of IL-6,IL-1 beta and TNF-?in the supernatant of L02 cells was significantly higher?p<0.001?,while the concentrations of IL-1,IL-6 and TNF-?were significantly lowered in salidroside group?p<0.001?.4.In human liver cell line L02 model of hepatic fibrosis in vitro induced by TGF-?,the expression of NLRP3,ASC,caspase-1 and IL-1 beta;p-NF-?B,p-I?B;TGF-?and p-Smad3;MMP2 and MMP9;?-SMA type and I collagen were significantly higher than that in the control group,there was statistical significance?P<0.001?,while decreased in salidroside?10?M?20?M?40?M?group compared with the model group,with statistical significance?p<0.01 in the expression of caspase-1 in salidroside 10?M group,and P<0.001 in the rest?.Conclusion:1.Salidroside has a protective effect on hepatic fibrosis and effectively reduces the liver injury.2.Salidroside may play a role in anti-liver fibrosis by inhibiting the production of proinflammatory cytokines TNF-?,IL-6 and IL-1,and inhibiting the activation of NLRP3 inflammasome.3.In mice models of liver fibrosis,Salidroside may reduce the expression of MMP2 and MMP9,maintain the balance between MMPs and TIMPs,inhibit the epithelial mesenchymal transition?EMT?process,and then play a role in anti-hepatic fibrosis.4.Salidroside may play a role in anti-liver fibrosis by inhibiting NF-?B and TGF-?/Smad3 signaling pathways and the inflammatory response mediated by them.5.The protective effects of salidroside against hepatic fibrosis is complex with a multi-level,multi-target and multi-pathway.The interaction and mutual influence among the mechanism,interwoven into a huge protective network of salidroside against hepatic fibrosis.