Study On The Mechanism Of Action Of Sinisan In The Intervention Of ANIT-induced Cholestatic Liver Disease In Rat

Background:Cholestasis is a pathological condition in which the formation,secretion,and excretion of bile in the liver were obstructed,resulting in the inability of bile flow from liver to duodenum while accumulates in blood circulation.Nowadays,the pathogenesis of this disease is unclear,generally considered to be related to autoimmune imbalance and chronic inflammation,etc.,and safe and effective therapeutic drugs were still deficient in clinical application.Sini San(SNS)is derived from the‘Treatise on febrile Diseases’,which has the effect of soothing the liver,regulating the spleen,relieving the depression and heat dissipation,and was widely used in hepatobiliary diseases.At present,there were few literatures on the chemical ingredients of SNS,and the pharmacodynamic study and potential mechanism of SNS in improving cholestatic hepatitis were still not illuminated.Objective:In this study,we used liquid chromatography-mass spectrometry(LC-MS)to qualitatively analyze the chemical ingredients of SNS in water-extraction and their constituents absorbed into blood,predicted its mechanisms against cholestatic hepatitis by network pharmacology and further evaluated and validated the mechanisms of SNS in vivo and vitro by metabonomics and molecular biological technique to demonstrate the material basis and mechanism of SNS against cholestatic hepatitis.Methods:(1)The chemical components and blood components of SNS were qualitatively analyzed by LC-MS.Combined with the fragmentation patterns of relevant ingredients,they were further identified by comparing their retention time and fragmentation information with the related references and available reference substance.The disease targets were obtained from Online Mendelian Inheritance in Man(OMIM)and GeneCards database.These targets were performed protein-protein interaction(PPI)analysis via searching a tool for recurring instances of neighbouring genes(String)database and core targets were screened through Cytoscape 3.7.2.Then the core targets were carried out Gene ontology(GO)and Kyoto encyclopedia of genes and genomes(KEGG)pathway enrichment analysis by DAVID.And the compound-targetpathway network was constructed by using Cytoscape 3.7.2,and the potential active components of SNS were screened by molecular docking.(2)The α-naphthalene isothiocyanate(ANIT)induced cholestatic liver injury rat model was used to evaluate the protective effect of SNS on cholestatic hepatitis.The hepatoprotective and choleretic effects of SNS were evaluated by examining the liver index,animal behavior,bile flow,serum biochemical indexes,pathological changes of liver tissue,inflammatory and antioxidant factors.(3)Based on UPLC-Q-E-Orbitrap MS,non-targeted metabonomics was used to analysis the serum metabolic profile of SNS in cholestasis rats.Combined with principal component analysis and partial least squares analysis to screen and identify potential differential metabolites and observed the potential metabolic pathways.According to the results of network pharmacology and non-targeted metabonomics,bile acid targeted metabonomics was used by LC-MS to screen the bile acid metabolites of SNS against cholestasis.The effect of SNS on bile acid homeostasis in cholestatic hepatitis rats were explored.(4)Real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)and western blot were used to detect the effects of SNS on the expression of bile acid receptors,transporters,metabolic enzyme,nuclear transcription factors and antioxidant enzymes in rats with cholestatic hepatitis.The protective mechanism of SNS on cholestatic hepatitis was also explored.HepG2 cells were stimulated by chenodeoxycholic acid(CDCA)to induce cholestasis in vitro,and the activities of SNS blood components were evaluated by cell counting kit-8(CCK8)assay and enzyme-linked immune sorbent assay(ELISA).RT-PCR and western blot were detected to verify the effect of SNS on the expression of related genes and proteins in FXR signal pathway.Results:(1)Fifty chemical constituents in SNS were preliminarily identified by LC-MS,including 32 flavonoids,9 triterpenoids,6 monoterpenes,1 coumarin,1 alkaloid and 1 organic acid.Among them,23 chemical constituents were firstly discovered in SNS,and 14 blood components were identified.The above 14 blood components were predicted to act on 62 potential targets.By searching the disease databases,a total of 1912 disease targets were obtained.There were 34 core genes(including FXR,BSEP,MRP2 were highly correlated with cholestatic hepatitis)of SNS potential targets against cholestatic hepatitis were obtained by intersecting disease targets with active component targets.Enrichment analysis results showed that 34 core genes significantly regulated 165 GO entries,including regulation of bile acid and bile acid transport,bile acid signal pathway,inflammatory response,oxidative stress,regulation of bile acid biosynthesis,cholesterol metabolism,etc.Furthermore,they could also mediate 40 KEGG pathways,including primary bile acid biosynthesis,bile secretion,sphingolipid signal pathway,cholesterol metabolism,etc.By constructing and analyzing the compound-targetpathway network and molecular docking,hesperidin,paeoniflorin,glycyrrhizin,saikosaponin A and naringin were discovered for acting on multiple targets or exerting lower affinities.Therefore.they were considered as the potentially bioactive components of SNS on cholestatic hepatitis.(2)SNS could significantly restore the abnormal increase of biochemical indexes(including AST,ALT,ALP,y-GT,TBA,TBIL and DBIL)on cholestasis,increased bile flow,reversed the degree of liver pathological injury,reduced the release of inflammatory factors TNF-α,IL-1β and IL-6,decreased the levels of ROS and MDA,increased the levels of SOD and GSH-Px in liver and exerted antioxidant activity.These results showed that SNS has a certain protective effect on ANIT-induced cholestatic hepatitis and reduced cholestatic liver injury caused by inflammation and oxidative stress.(3)The serum non-targeted metabolites results showed that 24 differential metabolites were screened out among normal group,model group and SNS group.KEGG analysis showed that these differential metabolites were mainly related to primary bile acid biosynthesis,arachidonic acid metabolism and sphingomyelin metabolism.Bile acid-targeted metabonomics showed that compared with control group,the levels of 11 bile acids such as TCA and TCDCA in the liver of cholestasis rats were dramatically increased,while 7 bile acids such as GCA and TDCA were significantly decreased.Compared with model group,SNS could decrease the levels of TCA,TCDCA,T-α-MCA,T-β-MCA,THCA,CA,CDCA and HCA,and increase the levels of GCA and GUDCA in the liver of cholestasis rats.(4)RT-PCR and western blot results demonstrated SNS could significantly up-regulated the expression of Fxr,Pxr,Shp,Bsep and Mrp2 and down-regulated the expression of Cyp7a1,Cyp8b1 and Ntcp in liver.inhibiting bile acid synthesis.increasing bile mobilities and alleviating cholestatic liver injury induced by ANIT.SNS could also uptake the expression of Nqo1,Nrf2 and Ho-1 so that to alleviate oxidative stress induced by ANIT.The mechanism of SNS on cholestatic hepatitis in rats might be related to the activation of Fxr and Nrf2 signal pathways.SNS containing serum group could reduce the levels of AST,ALT,ALP,TNF-α,IL1β and IL-6 in the supernatant of HepG2 cells stimulated by CDCA,and reduce the secretion of TBA in model cells,which were helped to alleviate inflammatory reaction and hepatocyte injury.RT-PCR and western blot results showed that compared with model group,SNS containing serum group significantly up-regulated the expression of FXR,SHP,BSEP and MRP2,while significantly down-regulated the expression of CYP7A1 and NTCP,inhibit the biosynthesis of bile acid,increase the efflux of bile acid,reduce the accumulation of bile acid in hepatocytes and reduce the toxic effect of bile acid on hepatocytes.Conclusion:In this study,the chemical and blood components of SNS were qualitatively analyzed by LC-MS.The protective effect of SNS on cholestatic hepatitis were evaluated by ANIT-induced rat model.Combined with network pharmacology and molecular biology techniques,we found SNS could alleviate cholestasis and oxidative stress injury to protect cholestatic hepatitis by activating FXR and Nrf2 signal pathways.These findings preliminarily illustrated the potential active constituents and mechanism of SNS against cholestatic hepatitis,laid a foundation for the quality control and further scientific research,and provided a theoretical basis for the clinical application of SNS.