The Protective Effect And Mechanism Of Sancao Granule Against Concanavalin A-induced Liver Injure In Mice

Background: San-cao Granule(SCG),a traditional Chinese formula,originated the classical prescription “Yugan Fang”,has long been used in clinical for treating autoimmune hepatitis(AIH).It consists of Rubia cordifolia L.,Sieges beckia orientalis L.,Gentiana macrophylla Pall.,Salvia miltiorrhiza Bunge,Forsythia suspensa(Thunb.)Vahl.,and Glycyrrhiza uralensis Fisch with a weight ratio of 6:6:3:3:3:6.There was clinical observations showed the hepatoprotective effect of SCG on AIH by improving liver function and alleviating the clinical symptoms.Objective: The present study was aimed to explore the hepatoprotective effect and mechanisms of Sancao granule(SCG)against Con A-induced liver injury(CILI),which provide a scientific evidence for SCG against autoimmune hepatitis(AIH).Methods: Firstly,Con A was administrated in mice to construct acute liver injury by tail vein injection.Then,based on the model of CILI,we studied the therapeutic effect of SCG on CILI.Combined with molecular biology,the mechanisms of SCG against CILI was investigated.Subsequently,an ultraperformance liquid chromatography with quadrupole time-of-flight mass spectrometry(UHPLC-QTOF/MS)-based metabolomics approach combined with principle component analysis(PCA)and orthogonal projection to latent structures discriminate analysis(OPLS-DA)were integrated applied to obtain metabolites for clarifying mechanisms of disease.Results: In the present study,we demonstrated the protect effect of SCG on CILI,which was through improving the liver function,relieving the pathological changes of liver tissues,decreasing the level of MPO and pro-inflammatory cytokines in liver tissues or serum.The results of molecular biology study showed that the TRAIL-and FASLmediated apoptosis pathway was significantly inhibited by SCG,which indicated the anti-apoptosis effect of SCG on CILI.And the further study showed that the effect of SCG on TRAIL-and FASL-mediated apoptosis pathway was NKT cell dependence.Finally,the metabolomics approach combined with network pharmacology were applied to obtain metabolites and pathways for clarifying mechanisms of disease.The results showed the different expression of 9 metabolites related to 8 pathways among the normal,CILI and SCG groups,which co-related to the anti-inflammatory and antiapoptosis effect of SCG.The network pharmacology showed that the targets of ALOX5、CYP4501A2 、 CYP4503A4 、 FAS 、 CYP4501B1 were demonstrated should be responsible for the therapeutic effect of SCG in treating CILI.Simultaneously,the study also showed that SCG could obviously decrease the high expression of ALOX5,CYP4503A4,CYP4501A2,CYP4501B1,and increase the expression of FAS in mice,which is consistent with the study above that there was a specific hepatoprotective effect of SCG on CILI.Conclusion: the present study demonstrated the protect effect of SCG on CILI,and systematically interpreted the mechanisms.And the study showed the combination of molecular biology,metabolomics and network pharmacology could be a promising approach,which provide insights into mechanisms of SCG in treating CILI.