Study On The Intervention Effect And Molecular Target Of Scoparone-the Main Active Ingredient Of Artemisia Annua Soup On Acute Liver Injury

Background: Acute liver injury（ALI）is a functional liver abnormality that may arise from a variety of causes,including viral infection,alcohol or drug abuse.Prolonged and widespread ALI may lead to liver failure or hepatic cirrhosis,which are acute clinical presentations of ALI that is associated with high mortality.Scoparone（6,7-dimethoxycoumarin,Sco）is the main active ingredient in traditional Chinese herbal formulas such as Yin Chen,a component of Artemisia annua soup,and is often used clinically to treat liver dysfunction,cholestasis,and jaundice.Although Sco has good clinical efficacy in the treatment of liver disease,its underlying mechanism remains unclear.Objective: To investigate the interventional effects and molecular targets of Sco,the main active ingredient of Artemisia Annua Soup,on acute liver injury based on Omics and molecular mechanism studies.Methods: This study was divided into two parts,the first part was based on acetaminophen（N-acetyl-p-aminophenol [APAP]）induced acute liver injury in rats,based on gut microbial amplicon sequencing and liver tissue transcriptome sequencing to evaluate the anti-liver injury efficacy of Sco and its effects on gut microflora and liver tissue transcriptome in order to elucidate its effects in rats with acute liver injury;in the second part we（1）validated the expression levels of the potential target gene of Sco identified in the first part,CHI3L1,and its target gene NF-kappa B-inducible kinase（MAP3K14）,by establishing an APAP-induced acute human Hep G2 hepatocyte injury model followed by Sco intervention;（2）Intervention of CHI3L1 expression level in Hep G2 cells to investigate the effect of altered CHI3L1 expression on the proliferation ability of hepatocytes.Results:Part I.（1）Administration of 800 mg/kg APAP successfully established a rat model of acute liver injury,and several liver injury parameters have been alleviated after 14 days of Sco treatment.（2）Sco modulates the dysregulation of gut microbiota in rats with acute liver injury.A total of 16 microbial taxa were significantly altered in the model group relative to the control group and significantly recovered after Sco intervention,including Actinobacteria,Firmicutes,Bacteroidetes,Actinobacteria,Actinomycetales,Coriobacteriales,Coriobacteriaceae,Micrococcaceae,Bacillaceae,Bacteroidetes,Rikenellaceae,Bacteroides,Alistipes,Corynebacterium_maris_DSM₄5190（species）,uncultured_actinobacterium（species）,Bacillus_sp.N41（species）.（3）Sco modulates transcriptome abnormalities in liver tissues of rats with acute liver injury.Sequencing results showed that mRNA and lnc RNA changed more drastically during liver injury and Sco intervention,which could well differentiate samples between groups.Compared with the control group,there were 264 significantly up-regulated coding genes and 116 significantly down-regulated coding genes in the model group;compared with the model group,there were 55 up-regulated coding genes and 169 down-regulated coding genes in the Sco treatment group.Among them,50 genes were aberrantly expressed in the model group but significantly recovered after Sco intervention;based on the functional enrichment analysis of these 50 genes,the most significantly enriched GO pathway was identified as NF-kappa B-inducible kinase activation pathway（GO:0007250,P = 0.0006）,including differentially expressed genes mas1（mas1 proto-oncogene,the G protein-coupled receptor）and Chi3l1（Chitinase 3 Like 1）.Both genes were significantly upregulated in the model group but significantly downregulated after Sco intervention;compared with the control group,475 significantly upregulated lnc RNAs and185 significantly downregulated lnc RNAs were found in the model group;compared with the model group,123 upregulated lnc RNAs and 249 downregulated lnc RNAs were found in the Sco treatment group Among them,54 lnc RNAs were significantly up-regulated in the model group but significantly down-regulated after Sco intervention;39 lnc RNAs were significantly down-regulated in the model group but significantly up-regulated after Sco intervention;7 pairs of up-regulated mRNA-lnc RNA pairs were co-expressed in the model and control groups with cis-regulation,and 5 pairs of down-regulated mRNA-lnc RNA pairs were cis-regulated co-expressed;in the Sco and model groups,one pair of up-regulated mRNA-lnc RNA pairs were cis-regulated co-expressed and three pairs of down-regulated mRNA-lnc RNA pairs were cis-regulated co-expressed;in the model and control groups,a total of 56 ce RNA interaction networks were identified;in the Sco treatment and model groups,a total of 9 ce RNA interaction networks were identified.（4）This study also found a strong correlation between differential microbial and differential transcriptional RNAs among the three groups,suggesting a synergistic regulation of the gut microbial-liver tissue transcriptome in APAP-induced liver injury and Sco intervention in liver injury.Part II.（1）Hep G2 cells were given 20 m M APAP and an acute liver injury cell model was successfully established.After Sco intervention,the levels of liver injury indexes ALT and AST were reduced,and the proliferation capacity of hepatocytes was enhanced.（2）Transcripts and protein expression levels of CHI3L1,MAP3K14（target of CHI3L1）and IKKa（substrate of MAP3K14）were elevated in the model group relative to the control group and Sco decreased their expression level in a dose-dependent manner.（3）Using si RNA to reduce CHI3L1 mRNA expression levels in cells of the APAP-treated group,a decrease in CHI3L1 mRNA and protein expression levels was observed,along with a decrease in MAP3K14 mRNA and protein expression levels and an alleviation of the damage phenotype and an increase in the proliferation capacity of hepatocytes.Conclusions.（1）Sco intervention can alleviate APAP-induced liver injury and liver function impairment,dysregulation of gut microbial homeostasis,and expression anomalies of genes related to NF-kappa B-induced kinase activation pathway and glycolysis/gluconeogenesis pathway in liver tissues of rats.（2）APAP-induced acute liver injury upregulated the expression of CHI3L1 gene,which inhibited the proliferation capacity of hepatocytes by further upregulating MAP3K14 gene expression,thus manifesting the liver injury phenotype;treatment of Sco,probably by downregulating CHI3L1 and decreasing MAP3K14 expression,enhanced the proliferation and replication ability of hepatocytes.