Study On The Mechanism Of Bear Bile Powder Regulating Bile Acid Metabolic Network Based On Bi-omic

Bear bile powder(BBP),one of the most valuable medicine,has been widely used in the clinical treatment of various hepatobiliary system diseases.Our group had previously confirmed the efficacy of BBP in alleviating ANIT-induced intrahepatic cholestasis,but its therapeutic mechanisms were still unclear.The core of cholestasis is the imbalance of the bile acid pool homeostasis in vivo.Bile acid pool homeostasis is precisely regulated by a variety of metabolic enzymes,transporters and nuclear receptors.Therefore,the establishment of a quantitative bile acid metabolic network consisting of "bile acid pool-transporters,metabolic enzymes-nuclear receptors" is conducive to the accurate evaluation of the disease status of cholestasis and the effective mechanism of drug.This thesis was based on nontargeted metabolomics and shotgun proteomics to investigate the potential pathological markers of cholestasis and the effective mechanism of BBP in alleviating intrahepatic cholestasis.The present research further constructed widely targeted bi-omics combining metabolomeproteome to validate the significantly varied metabolites and differential expressed proteins(DEPs),and investigated the inter-relationship between differential metabolites and DEPs,in order to achieve quantitative evaluation of the pathological state of cholestasis and elucidation of the therapeutic mechanisms of BBP.The detailed research contents and results are as follows:1.Study on amelioration of cholestasis by BBP based on untargeted metabolomics and shotgun proteomicsThe intrahepatic cholestasis model was established by ANIT.The untargeted metabolomics and shotgun proteomics strategies based on LC-MS technology were used to collect plasma metabolite information and liver tissue protein information of rats,respectively,to study the effects of BBP on the metabolome and proteome group of cholestatic rats.Non-targeted metabolomics results showed that the model group significantly altered bile acid,lysophosphatidylethanolamine and lysophosphatidylcholine,and the bile acid pool was significantly enlarged in vivo,with the most significant changes in taurine-conjugated and sulfated bile acids.The main metabolic pathways involved were primary bile acid biosynthesis,taurine and hypotaurine metabolism,fatty acid degradation,glycerophospholipid metabolism and biosynthesis of unsaturated fatty acid.UDCA and BBP administration regulated 19 and 39 metabolites,respectively,restoring the disordered bile acids in the model group and regulated TCA,dehydrocholanic acid,β-MCA,24-oxocholesterol,UDCA,G-β-MCA.and GUDCA jointly.UDCA and BBP were also able to act on the primary bile acid biosynthetic metabolic pathway,taurine and hypotaurine metabolism,biosynthesis of unsaturated fatty acid,along with pentose and gluconate interconversion pathways.It was hypothesized that BBP ameliorate cholestasis by reducing the direct toxic effects of bile acid components and the bile acid-induced inflammatory damage.Shotgun proteomics results showed the cholestatic rats up-regulated of 77 protein groups and down-regulated of 50 protein groups,including adaptive upregulation of metabolic enzymes such as BAAT,SULT2A2,and UGT2,which are related to bile acid metabolism,as well as downregulation of the expression levels of the hepatocyte reabsorption transporter OATP1A4/5,partially alleviating liver injury.The model rats significantly decreased insulin secretion,bile secretion and steroid hormone biosynthesis metabolic pathways and significantly increased peroxisomal pathways.UDCA down-regulated metabolic pathways such as bile secretion,and proximal tubular bicarbonate reclamation,and upregulated metabolic pathways such as insulin signaling pathway,glutathione metabolism and AMPK signaling pathway.BBP administration significantly reduced the elevated peroxisome pathway in the model group and upregulated the complement pathway.The results of metabolomic-proteomic joint analysis showed that both UDCA and BBP ameliorate cholestasis by modulating primary bile acid biosynthetic pathway,which was altered the model group,changing the composition and size of the bile acid pool in vivo and exerting an ameliorative effect on cholestasis.2.Establishment of widely targeted metabolomics and widely targeted proteomics analysis methodsThe results of non-targeted metabolomics and shotgun proteomics showed that the differential metabolites in model vs.control,UDCA vs.model,and BBP vs.model were bile acids,and the DEPs were dominated by a variety of phase Ⅰ and Ⅱ metabolizing enzymes and transporters involved in bile acid metabolism.Therefore,the pMRM ion lists of bile acid components were organized,combing the MS1 and MS2 provided by Qtof-MS of the differential metabolites with the chemical composition and in vivo metabolite analysis method of BBP constructed by our group previously.QTRAP-MS was used to characterized plasma QC samples,and finally a widely targeted metabolomics analysis method of 178 bile acid compounds was established.Skyline software was used to obtain the corresponding unique peptide Q1→Q3 ion pairs and CE values of the differential proteins to obtain the list of pMRM ions.The liver tissue QC samples were tested using QTRAP-MS to remove false positive ions,and finally 139 peptides were confirmed for widely targeted proteomics analysis method.3.Study on amelioration of cholestasis by BBP based on widely targeted bi-omicsThe constructed widely targeted metabolomics analysis and widely targeted proteomics analysis were applied to rat plasma metabolome and liver tissue proteome analysis,respectively,to validate the non-targeted results,and to elucidate the mechanism of BBP in alleviating cholestasis.Widely targeted metabolomics results showed the bile acid pool in cholestatic rats was enlarged,and taurine-conjugated and sulfated bile acids were generally elevated,suggesting that they could be potential biomarkers of cholestasis.Some bile acid levels fell back after UDCA and BBP administration,exerting an ameliorating effect on cholestasis.In addition,the levels of TUDCA,and other conjugated bile acid were increased after administration of BBP,which may exert hepatocyte-protective effects by promoting extracellular secretion of bile,promoting enterohepatic circulation,and alleviating inflammatory responses.Widely targeted proteomic results showed significant changes in 10 proteins in the model group rats,including upregulation of CYP4A2 and CYP2C11 involved in arachidonic acid metabolism,retinol metabolism,and inflammatory mediator regulation of TRP channels,and other proteins involved in metabolic pathways.The expression levels of BAAT,CYP4A2 and Calponin-3 decreased towards the normal group after UDCA administration,meanwhile,the expression of one protein was upregulated and five proteins were downregulated in liver tissues.The differential proteins were involved in primary bile acid biosynthesis,bile secretion,PPAR signaling pathway,arachidonic acid metabolism and peroxisome pathways.The administration of BBP significantly upregulated the expression of 7 proteins in the liver tissue of cholestatic rats,and enrichment analysis revealed molecular functions involving oxygen binding,βamyloid binding,and serine-type endopeptidase inhibitor activity.Widely targeted bi-omics results showed that the cholestasis involved primary bile acid biosynthesis,taurine and hypotaurine metabolism.UDCA and BBP could regulate the above two pathways,suggesting that BBP has similar regulatory effects with its main component UDCA.In summary,this thesis screened the plasma differential metabolites and DEPs in vivo after cholestasis and BBP administration based on non-targeted metabolomics and shotgun proteomics.Establishment of widely targeted metabolomics for bile acids and widely targeted proteomics for metabolic enzymes and transporters in bile acid metabolic network by pathway analysis.The results showed that the bile acid pool was significantly enlarged in cholestasis,and free bile acids and various conjugated bile acid elevated,tentatively confirming that sulfated bile acids and taurine-conjugated bile acid can be used as potential biomarkers of cholestasis.BBP can ameliorate the composition of the bile acid pool in vivo,possibly by regulating bile acid synthesis and enterohepatic circulation.In addition,the conjugated bile acids in BBP may exert hepatoprotective effects by alleviating the inflammatory response.