Study On The Mechanism Underlying That Tetrahydropalmatine Attenuated Acuate And Chronic Liver Injury And Fibrosis

Aims:Liver fibrosis was caused by many factors,mainly characterized by activation of hepatic stellate cells（HSCs）and deposition of extracellular matrix（ECM）.Endoplasmic reticulum（ER）stress played an important role in the activation of hepatic stellate cells and the formation and development of hepatic fibrosis.Tetrahydropalmatine（THP）,as an active component of traditional Chinese medicine,has been used in clinical safety for many years in China.However,the role of THP in liver injury and fibrosis has not been reported.The aim of this study was to investigate the pharmacological effects and mechanism of THP on acute and chronic liver injury and liver fibrosis induced by carbon tetrachloride.Methods:（1）C57BL/6 mice were divided into 3 groups:the Oil group,CCl₄ group and THP group（40 mg/kg）.The mice were given tetrahydropalmatine by gavage every day for 7 days.At the same time,the acute liver injury model was induced by intraperitoneal injection of 20%CCl₄（5μL/g）on the 7th day.（2）40 C57BL/6 mice were randomly divided into 5 groups:Oil group,CCl₄model group,low dose treatment group（THP-L,20 mg/kg）,middle dose treatment group（THP-M,40 mg/kg）,high dose treatment group（THP-H,80 mg/kg）.To induce chronic liver injury and fibrosis,mice in the model group and treatment groups were intraperitoneally injected with 20%CCl₄（5μL/g）,twice a week for six weeks.Meanwhile,the mice in the treatment groups were given THP by gavage every day for 6 weeks.（3）In vitro,human hepatic stellate cell line LX-2 cells were treated with different concentrations of THP（0,2.5,5,10,20μM）,and lasted for 12 or 24 hours.The m RNA and protein expressions of related genes in liver tissues and LX-2 cells were examined by real-time fluorescent quantitative PCR and western blot,respectively.（4）A portion of liver tissue was taken for transcriptome sequencing to predict potential therapeutic targets and explore the molecular mechanisms of THP in the treatment of liver fibrosis.Results:（1）THP attenuated hepatocyte apoptosis and inflammatory cell infiltration in mice with acute liver injury induced by CCl₄ and significantly decreased the serum levels of alanine aminotransferase（ALT）.（2）THP reduced the serum ALT level,down regulate the m RNA levels of inflammation related factors（Ccl2 and Cxcr2）and fibrosis related genes（α-Sma,Collagen I,Pdgf,Timp-1 and Ctgf）in CCl₄ induced chronic liver injury and liver fibrosis mice.（3）Go pathway analysis showed that THP treatment significantly inhibited the signaling pathways related to liver fibrosis,ER stress and inflammation.Protein-protein interaction（PPI）analysis predicted that these genes,such as Egr1,Fos,Ddit3,Hspa5 and Eif2ak3 in endoplasmic reticulum stress pathway,may be the main targets of THP in the treatment of liver fibrosis.（4）THP treatment attenuated hepatic stellate cell activation and fibrosis by inhibiting the genes and proteins expression related to ER stress in vivo and in vitro.Conclusion:THP protected mice liver against CCl₄-induced acute and chronic injury.Moreover,we have for the first time demonstrated that THP has promising therapeutic effects on CCl₄-induced liver fibrosis,and the underlying mechanisms were involved in blockage of ER stress in HSCs.