Combined Transcriptome And Metabolome Analyses Of Mechanisms Of Rhizoma Panacis Majoris Saponins Against Hepatic Fibrosis

Hepatic fibrosis resulting from multiple etiologies,such as viral infections,alcohol,metabolic disorders and so on,may gradually progress to cirrhosis,and if not prevented,it may evolve into hepatocellular carcinoma.Progressive hepatic fibrosis leads to more than one million deaths annually via development of cirrhosis,although no anti-fibrosis therapy is approved by FDA so far^[1].Therefore,how to effectively prevent and treat hepatic fibrosis has become a key issue in hepatic fibrosis treatment.Modern pharmacological studies demonstrates Rhizoma Panacis majoris has hepatoprotective effect^[3].Previous studies have indicated that Rhizoma Panacis majoris saponins(PMS)has a protective effect on both a chronic liver injury and hepatic fibrosis by CCl₄-induced rat model.However,the bioactive constituents and mechanism of PMS anti-hepatic fibrosis are still unclear.Metabolome approach and transcriptome approach focuse on the holistic investigation of gene expression profiling and metabolic profiling of living systems,which are used to reveal the complex regulatory networks of genes and metabolic pathways and to explore potential biomolecules and biological processes.Combination of metabolome and transcriptome were utilized to analyze metabolites and gene expression and to search for the pathways of metabolism.The critical biological processes and signaling pathways were researched to verify and supplement the anti-hepatic fibrosis mechanism of PMS.1.Protective effect of PMS on CCl₄-induced hepatic fibrosisAn hepatic fibrosis by CCl₄-induced rat model was established.Biochemical indicators in serum and liver tissue were detected,paraffin-embeded liver tissues were stained with HE and Masson to observe the level of hepatic fibrosis,and immunohistochemical method was used to detect the expression of?-SMA.Biochemical assays showed that ALT,AST,MDA,HYP,HN,LA,IL-6 and TNF-?were increased,GSH-Px and SOD were decreased.Fibrosis changes in liver morphology were observed in the model group after administration of CCl₄ comparing to sham group.Expression of?-SMA was increased in the model group in immunohistochemical analysis,PMS treatment at three doses ameliorated fibrosis induced by CCl₄.2.Metabolome study of the hepatoprotective effect of PMS on CCl₄-induced hepatic fibrosisMetabolome study results indicated that the endogenous metabolites in the CCl₄-induced group were changed by PMS.Seventeen metabolites in serum and twenty-five metabolites in liver tissue were identified as differential metabolites relating to the anti-fibrotic effect of PMS,which were related with pathways of PUFA metabolism,glycerophospholipid metabolism,tryptophan metabolism,cysteine and methionine metabolism,arginine and proline metabolism,nicotinate and nicotinamide metabolism,nucleic acid metabolism and vitamin B₆ metabolism.PMS play an anti-hepatic fibrosis role by regulating the disordered metabolic state.3.Transcriptome study of the hepatoprotective effect of PMS on CCl₄-induced hepatic fibrosisTranscriptome study in liver showed that PMS mainly regulated 59 key genes in hepatic fibrosis rats.Among them,22 genes were significantly up-regulated including Acaca,Cyp2c13,Cyp3a9,Chka,Ass1,Tdo2 and Ahcy,et al,37 genes were significantly down-regulated including Blnk,Ccl3,Cd3d,Cd74,Clec7a,Cdk1,Birc5and Gabarapl1,et al,which suggested that the mechanisms of PMS anti-fibrosis on CCl₄-induced liver fibrosis may involve regulating dysfunction in cell metabolism,immune response and cell proliferation.4.Verification of the anti-hepatic fibrosis mechanism of PMSCombination of metabolome and transcriptome were utilized to analyze metabolites and gene expression and to search for the pathways of metabolism.The critical potential biomolecules were verifed by Western Blot and RT-PCR analysis.Combination of metabolome and transcriptome find out five key metabolites incluing4-hydroxyretinoic acid,kynurenine,spermidine,D-homocysteine and cis-11,14-eicosadienoic acid,and eight key genes incluing Gpx7,Ifitm1,Avpr1a,Rdh16,Tdo2 Cyp26a1,Pla2g16 and Cyp2c13.The most significantly genes regulated by PMS and associated with hepatic stellate cell activation and liver fibrosis were verified.In this study,five key metabolites and nine key genes were potential biomolecules which indicated that PMS had potential anti-fibrosis effects through regulating retinol metabolism,unsaturated fatty acid metabolism,tryptophan metabolism,arginine and proline metabolism,and cysteine and methionine metabolism.