Research On The Anti-hepatic Fibrosis Effect Of Scutellarin Based On Multiomics

Purpose:To observe the preventive and therapeutic effect of scutellarin（SCU）on liver fibrosis in rats constructed with carbon tetrachloride,we explored the ameliorative mechanism of SCU on liver fibrosis from three aspects of gut microbiomics,metabolomics and transcriptomics,in order to provide a scientific basis for the study of scutellarin on anti-liver fibrosis.Method:1.The rat liver fibrosis model was established by intraperitoneal injection of 40%CCl₄olive oil solution twice a week.Each administration group was given SCU drug and positive drug colchicine by gavage every day.After 10 weeks,feces,serum,liver tissue and colon were collected.The liver indices of the rats in all groups were examined,the hepatic pathological changes were evaluated according to HE and Masson staining,the contents of ALT and AST in rat serum were determined by microplate method;The levels of HA,LN,PC-III,and COL-IV were measured in rats serum by ELISA.2.16S r RNA sequencing was used to analyze the variations of gut microbiota diversity,and fecal microbiota transplantation experiments were applied to verify whether the beneficial effects of SCU on liver fibrosis were dependent on the regulation of gut microbiota.To construct a BALB/c mouse model of liver fibrosis,after that the normal mice（N）and mice with liver fibrosis（M）were pretreated with antibiotics for 7 days to become the NA,MA groups,respectively.Then the gut microbiota of rats in model group was transplanted into NA mice,the gut microbiota of normal rats and gut microbiota of rats intervened by SCU were transplanted into MA mice,respectively.After the last gavage and fasting for 24 h,fresh feces,serum and liver tissues were collected to observe the pharmacodynamic parameters.Spearman correlation analysis was used to analyze the correlation between gut microbiota and liver fibrosis indexes.3.The feces,liver tissues and serum of rats in the normal group,model group and scutellarin group were screened for the differential metabolites by untargeted metabolomics analysis using UHPLC-Q-Exactive MS/MS technology,and the association of differential metabolites with pharmacodynamic indicators was initially analyzed by Spearman correlation analysis.4.The colon and liver samples from the normal,scutellarin group rats were transcriptomically analyzed at the gene level using m RNA-Seq based transcriptomics to screen differentially expressed genes to find the key genes in which scutellarin exerts anti fibrotic effects.Results:1.Scutellarin significantly reduced hepatic indices,liver function indexes and hepatic fibrosis indexes,and improved fibrotic tissue proliferation and reduced collagen deposition in rats with hepatic fibrosis,suggesting that scutellarin can improve hepatic fibrosis induced by carbon tetrachloride in rats.2.The diversity of gut microbiota and the composition of gut microbiota were significantly altered in the liver fibrosis rats,and SCU treatment significantly repaired the gut microbial dysbiosis of the liver fibrosis rats,shifting it toward the normal group.Fecal microbial transplantation assay results showed that after antibiotic intervention,the liver index,liver function and liver fibrosis index in the model group were significantly decreased,and a large amount of collagen deposition appeared;transplantation of the bacterial fluid from model rats into NA significantly increased the liver index,liver function index,and liver fibrosis index in mice,and the histopathology showed inflammatory cell infiltration;compared with the MA group,transplantation of the bacterial fluid from the SCU intervened rats into the MA group resulted in a significant decrease in the liver index,liver function index,and liver fibrosis index,as well as an improvement in fibroplasia.Spearman’s analysis showed that Bifidobacterium and Allobaculum were markedly negatively correlated with AST,ALT,PCIII,Co L-IV,HA and LN.3.The results of untargeted metabolomics analysis showed that 10 biomarkers were identified by the screening in feces,mainly involved in the Metabolic pathways such as Linoleic acid metabolism,α-Linolenic acid metabolism;a total of 6 biomarkers were selected in the liver tissue samples,mainly involved in Glycerophospholipid metabolism,Purine metabolism,and other pathways;7 biomarkers were selected from serum samples,mainly related to pathways involved in Biosynthesis of unsaturated fatty acids andα-Linolenic acid metabolism.4.By transcriptomic analysis,compared with the normal group,451 differential genes were screened in the colon samples of SCU group,of which 375 genes were significantly increased and 76 genes were significantly decreased;The KEGG pathway analysis results suggested that the differential genes mainly involved related pathways such as c GMP-PKG signaling pathway,Vascular smooth muscle contaction,and Neuroactive ligand-receptor interaction.In liver samples,a total of 187 differential genes were selected,of which 63 genes showed significantly increased expression and 124 genes showed significantly decreased expression;The KEGG pathway analysis results showed that the differential genes mainly involved related pathways such as Retinol metabolism,PPAR signaling pathway,and PI3K-Akt signaling pathway.Conclusions:Scutellarin ameliorated liver fibrosis by reshaping gut microbiota,modifying signaling pathways such as PPAR signaling pathway,PI3K-Akt signaling pathway,and regulating metabolic disorders such as Linoleic acid metabolism,Glycerophospholipid metabolism and Biosynthesis of unsaturated fatty acids,suggesting that scutellarin anti-liver fibrosis is achieved through multi-target multi-pathway.