Mechanisms Study Of Hepatotoxicity Induced By Dioscorea Bublifera L. Based On Digital Gene Expression Library

Objective:The present study was designed to detect the gene expression profiles associtaed with Dioscorea bulbifera L.(DB)-induced liver injury in mice by digital gene expression(DGE),a next-generation sequencing technology,and analyzed its molecular mechanisms.Then explored a potential compound of traditional Chinese medicines(TCMs)for DB by literature anlysis.Methods:(1)Forty SPF Kunming female mice(6 weeks old,20±2 g)were purchased from Shanghai Slack Laboratory Animal Co.Ltd.(animal certification number:2015000533474).Animals were randomly divided into the control group,high,moderate and low dose groups(n=10 in each group).Mice in the latter three groups were administered intragastrically(i.g.)with DB decoction(24,12 and 6 g/kg/d,respectively)for 21 consecutive days,while the control group were given equal volume of pure water.After experiment the mice were killed,and their livers were collected.The optimum dose to induce hepatotoxicity was chosed by compared the treated groups and the control group in terms of mice weight gain,liver index,serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)levels and liver tissue pathology after H&E staining.(2)Two DGE libraries of mice liver in the control and the DB group were created by RNA-seq,following with data analysis and differential gene expression analysis.Then,Gene ontology(GO)enrichment and kyoto encyclopedia of genes and genomes(KEGG)pathway analyses were implemented on all differentially expressed genes to eluciate their biological functions and processes.Finally,these representative differentially expressedgenes in the main metabolic pathways were validated by quantitative real-time polymerase chain reaction(qRT-PCR).(3)Literature analysis:Obtained TCMs with the efficacy of against DB induced hepatotoxicity by literature search.Then searched their chemical ingredients and the mechanisms of hepatoprotective,and discussed the regulation of those main active constituents on the signal pathways that had been validated in previous study.Results:(1)Obvious liver injury was observed only in the high-dose group,which mainly manifested as decreased weight gain,elevated liver index and serum ALT and AST levels(P<0.05).Histopathological changes were apparent in liver tissue,with destruction of structure,liver cells swelling and cord derangement and infiltration of neutrophilic granulocytes,as well as hepatic cells were degenerated obviously(P<0.05).(2)A total of 13,214,693 and 11,124,617 raw reads were generated from the control and DB exposure DGE libraries respectively,with 12,819,933 and 10,786,300 clean reads left for assembly after removing adaptor related,containing N and low quality reads.According to the statistical result of the two DGE libraries,302 genes were detected differentially expressed with 148 up-regulated and 164 down-regulated.And 9537 high-quality sequences were observed co-expression among two DGE libraries,with 702 in the DB group only and 539 in the normal group only,after clustering analysis.GO annotation there mainly had molecular function and biological process.All differentially expressed genes were mapped to 140 pathways of KEGG database,and the top 8 significantly enriched KEGG pathways were retinol metabolism,arachidonic acid metabolism,PPAR signaling pathway,drug metabolism-cytochrome P450,metabolism of xenobiotics by cytochrome P450,steroid hormone biosynthesis,glutathione metabolism and biosynthesis of unsaturated fatty acids.After qRT-PCR varification,five representative genes were significantly up-regulated campared to control group,including CD36,SCD2,CYP2E1,CYP3A44,CYP4A,while GPX was down-regulated(P<0.05).(3)The TCMs commonly used as hepatotoxic antidote to DB including Glycyrrhiza,Angelica sinensis,Scutellaria baicalensis,cortex Phellodendri,fructus Schizandrae and Grateloupia filicina,etc.,especially the first two.The active ingredients contained could alleviate liver damage through regulating the different signal pathways of hepatic injury caused by DB,so as to exert the protective effects for the liver.Conclusions:The results showed that DB may induce hepatotoxicity by the following four pathways:firstly,calcium homeostasis unbalance emerged in hepatocytes and mitochondria after mice were exposured to DB for some time by influencing the arachidonic acid metabolic pathway,which resulting in mitochondria dysfunction and hepatocyte apoptosis.Secondly,DB may promote the produce of free radicals by inducing or inhibiting the gene expression of multiple CYP4A isoenzymes,CD36 and SCD2 in the PPAR signaling pathway.Thirdly,DB may affect the metabolism of CYP450 enzymes,which causing the accumulation of poisonous metabolites.Finally,the activity of glutathione-dependent antioxidant enzymes were regulated directly by DB.Seven TCMs(including glycyrrhiza,angelica sinensis,scutellaria baicalensis,cortex phellodendri,fructus schizandrae and Grateloupia filicina)could reverse liver damage based on their antioxidant properties and the funtions of regulating the metabolism of CYP450 enzymes.So we can well take advantage of these TCM with similar efficacy but different roperties and flavors to develop a new compound as a potential remedy for injury in liver.