Rotundine Inhibits Hepatic Fibrogenesis Through CB1/TGF-β1/Smad3 Signaling Pathway

Liver fibrosis is a common result of chronic liver injury caused by a number of factors such as hepatitis B or C virus infection,nonalcoholic fatty liver disease,alcoholic liver disease,schistosomiasis and drug damage,and chronic fibrosis can cause liver cirrhosis or even hepatocellular carcinoma.Quiescent HSCs（hepatic stellate cells）activation is the defining event in the development of liver fibrosis.Once activated,HSCs will lead to the excessive accumulation of extracellular matrix（ECM）and eventually lead to liver fibrosis.Therefore,the preferred therapeutic target for the treatment of hepatic fibrosis is inhibition of the activation of HSCs.Two types of cannabinoid receptors（CB1 and CB2）are expressed in HSCs and may play an important role in chronic liver disease,which have opposing activities.CB1promotes liver fibrosis while CB2 inhibits liver fibrosis.Genetic or pharmacological inactivation of CB1 receptors can inhibit the activation of HSCs by lowering hepatic transforming growth factor（TGF-β1）,the key activator of HSCs,and then decrease hepatic fibrogenesis.Therefore,CB1 receptor antagonism is a new strategy for the treatment of liver fibrosis.Rotundine,also known as levo-tetrahydropalmatine,is the effective part of tetrahydropalmatine,which is extracted from Rhizoma Corydalis.It has analgesic,sedative and sedative effects.Furthermore,it has protective effects on CCl₄-induced liver injury and Con A（concanavalin A）-induced acute autoimmune liver injury in mice.However,the effect of Rotundine on hepatic fibrogenesis is unclear.In this thesis,HSC-T6 cells were cultured in vitro,and HSC-T6 cells were treated with Rotundine to explore the effect of Rotundine on liver fibrosis and further clarify its mechanism.Objective:The purpose of this study was to investigate the effect of Rotundine on hepatic fibrogenesis,and to elucidate the mechanism of Rotundine inhibiting hepatic fibrogenesis by downregulating CB1/TGF-β1/Smad3 signaling pathway.This study provides a new theoretical basis for the treatment of liver fibrosis with Rotundine.Method:1.HSC-T6 cells were cultured in vitro.The effects of Rotundine on the proliferation of HSC-T6 cells were measured by MTT assay at different concentrations and times.The best concentration and time were selected according to the experimental results,and then the optimal concentration and time were used to act on HSC-T6 cells to obtain the IC₅₀.LDH activity in HSC-T6 cell culture medium was detected by colorimetry to evaluate the cytotoxicity of the drug.The content of Hyp in HSC-T6 cell culture medium was determined by enzyme digestion to evaluate the inhibitory effect of drugs on collagen secretion.The expression of CB1 in HSC-T6was detected by immunofluorescence.Western blot was used to detect the protein expression of collagen I andα-SMA,it is proved that the drug can inhibit the synthesis of collagen and activation in HSC-T6 cells.2.HSC-T6 cells were cultured in vitro.HSC-T6 cells was divided into control group,Rotundine middle dose group（62.5μm）,Rotundine high dose group（125μm）,Rimonabant（Rimo）group and Rotundine middle dose group+Rimonabant（Rimo）group.Rimonabant is a CB1 receptor specific inhibitor.The protein expression determination ofα-SMA,collagenⅠ,CB1,TGF-β1 and p-Smad3 by western blot.3.HSC-T6 cells were cultured in vitro.The experiment was divided into control group,Rotundine middle dose group（62.5μm）,Rotundine high dose group（125μm）,ACPA group and Rotundine high dose group+ACPA group.ACPA is a CB1 receptor specific agonist.The protein expression ofα-SMA,collagenⅠ,CB1,TGF-β1 and p-Smad3 by western blot.Results:1.Compared with the control group,Rotundine can significantly inhibit the proliferation of HSC-T6 cells.When the concentration of Rotundine is 31.25μm,62.5μm and 125μm and the time is 48 h,the effect is obvious（P<0.05）.And,the effect has a certain degree of dose-dependent.2.Compared with the control group,after treatment with with low,medium and high dose of Rotundine for 48 hours,the content of Hyp decreased significantly in HSC-T6 cells（P<0.05）.And with the increase of Rotundine concentration,Hyp content is lower.There was no significant difference in LDH activity between the Rotundine groups and the control group（P>0.05）,which showed that the inhibition of Rotundine on HSC-T6 cell proliferation and its influence on the indexes were not caused by drug cytotoxicity.3.Compared with the control group,the protein expression level ofα-SMA and collagen I in HSC-T6 cells treated with low,middle and high dose of Rotundine for48 hours was significantly decreased（P<0.05）,which proved that Rotundine could inhibit the fibrogenesis by inhibiting the activation of HSCs and the synthesis of collagen.4.Compared with the control group,the protein expression of CB1,TGF-β1 and p-Smad3 in HSC-T6 cells could be significantly reduced by treating with low,middle and high dose Rotundine for 48 hours（P<0.05）.The results of immunofluorescence showed that CB1 was expressed in the cytoplasm of HSC-T6 cells.5.Compared with the control group,the Rotundine middle and high dose group and Rimonabant（Rimo）group can significantly inhibit the proliferation of HSC-T6cells,reduce the content of Hyp and downregulate the protein expression levels ofα-SMA,collagen I,CB1,TGF-β1 and p-Smad3（P<0.05）.Rimonabant can significantly downregulate the protein expression of CB1 in HSC-T6 cells.Compared with Rotundine middle dose group and Rimonabant（Rimo）group,middle dose Rotundine+Rimonabant（Rimo）group significantly inhibits proliferation of HSC-T6cells,decreases the content of Hyp and downregulates the protein expression ofα-SMA,collagen I,CB1,TGF-β1 and p-Smad3（P<0.05）.6.Compared with the control group,the Rotundine middle and high dose group can significantly inhibit the proliferation of HSC-T6 cells,reduce the content of Hyp and downregulate the protein expression levels ofα-SMA,collagen I,CB1,TGF-β1and p-Smad3（P<0.05）.ACPA can upregulate the protein expression of CB1 in HSC-T6 cells.Compared with Rotundine high dose group,high dose Rotundine+ACPA group can significantly reverse the inhibition of Rotundine on the proliferation of HSC-T6 cells and the decrease of Rotundine onα-SMA,collagen I,CB1,TGF-β1and p-Smad3 protein expression level（P<0.05）.Conclusion:1.Rotundine can inhibit the proliferation and activation of HSC-T6 cells,at the same time,it can effectively reduce the production and secretion of collagen in HSC-T6 cells,thus,it can exert its inhibitory effect on hepatic fibrogenesis.And this effect is not caused by the drug cytotoxicity of Rotundine.2.Downregulation of CB1 expression can significantly enhance the inhibition of Rotundine on HSC-T6 cell proliferation and collagen synthesis,and the decrease ofα-SMA,collagen I,CB1,TGF-β1 and p-smad3 protein expression level.And upregulation of CB1 expression can significantly reverse the inhibition of Rotundine on HSC-T6 cells proliferation and collagen synthesis,and the decrease of Rotundine onα-SMA,collagen I,CB1,TGF-β1 and p-Smad3 protein expression level.Therefore,Rotundine may inhibit the proliferation and activation of HSC-T6 cells,reduce the production and secretion of collagen and then inhibit the hepatic fibrogenesis by downregulating CB1/TGF-β1/Smad3 signaling pathway.