Study On The Mechanism In Rats Of Anti-Cholestatic Liver Injury Effect And Pharmacokinetics Of Swertiamarin

Objective: In this paper,swertiamrin was used as the object of study to explore its protective effect on 1-naphthalene isothiocyanate(ANIT)-induced cholestatic liver injury,as it is the highest content of iridoids in Swertia cincta Burk.And its mechanism was explored through the combined application of metabolomics and proteomics.At the same time,through liquid-mass spectrometry(LC-MS)analysis technology to study the pharmacokinetics and metabolites of swertiamarin in vivo.Method:(1)Healthy SD male rats were randomly divided into a blank control group,a model group(ANIT,45 mg/kg),a positive drug group(UDCA,80 mg/kg),a low-dose group(swertiamarin,80 mg/kg),a medium-dose group(swertiamarin,100 mg/kg)and a high-dose group(swertiamarin,150 mg/kg).The rats were given oral administration for consecutive 7 days,and ANIT was given to model on the fifth day.Collect serum and determine biochemical indicators.At the same time,the collected liver tissue was stained with hematoxylin and eosin to preliminarily determine the protective effect of swertiamarin.(2)Rat liver,serum and urine samples of each group were processed by precipitation method and then analyzed by LC-MS analysis.The obtained data were preprocessed for principal component analysis and orthogonal partial least-squares discriminant analysis,with VIP > 1.5,P < 0.05 was used as the condition to screen different metabolites.Differential metabolites were identified by searching comparison of HMDB and Metlin database or comparing with standard compounds.(3)The liver samples of the rats in the blank group,model group,positive drug group and the medium-dose group of swertiamarin were extracted,enzymatically digested and labeled,and then separated by high-p H reversed-phase liquid chromatography.The obtained fraction passed Nano-liquid chromatography tandem Q Exative mass spectrometry analysis,and the mass spectrometry data obtained were analyzed qualitatively and quantitatively with Mascot software.P < 0.05 and FOLD CHANGE > 1.2(or < 0.83)were used as screening criteria.Some differential proteins were verified by Western blotting and real-time quantitative reverse transcription polymerase chain reaction.At the same time,the relevant proteins were verified again by constructing L-02 cell model experiments in vitro.(4)An ultra-high-performance liquid chromatography coupled with tandem triple quadrupole mass spectrometry method was established for the determination of swertiamarin in rat plasma,and it was successfully used for pharmacokinetic study after oral administration(50 mg/kg,100 mg/kg,150 mg/kg)and tail vein administration(2 mg/kg).At the same time,the metabolites in serum,bile,urine and feces of rats were detected by UPLC-Q/TOF method after oral administration of swertiamarin(100 mg/kg).Results: Swertiamarin can reduce the contents of various serum biochemical indicators raised in rats to a certain extent,and can reduce the degeneration and necrosis of some liver cells,which shows a certain protective effect.After administration of swertiamarin,primary bile acid synthesis,glycerophospholipid metabolism,sphingolipid metabolism,and retinol metabolism pathways were regulated in rats with cholestatic liver injury.The expression of upstream FXR,CYP7A1 and CYP8B1 increased,the expression of NTCP,CYP27A1 decreased,and the content of downstream taurocholic acid and tauroursodeoxycholic acid decreased,and the homeostasis of bile acid metabolism was improved in vivo.At the same time,BSEP and MRP2 expression increased,and glutathione level decreased.After oral administration,the peak concentration of swertiamarin could be reached around 1h,and the half-life was also around 1h.Moreover,the bioavailability is only about 8%.Moreover,six different metabolites were found after oral administration of swertiamarin,which involved metabolic pathways including deglycosylation,glycosylation and nitrogen hybridization.Conclusion: Swertiamarin has a certain anti-cholesteric liver injury effect,can improve the dysfunction of bile acid production and excretion caused by cholestatic liver injury,regulate primary bile acid synthesis,glycerophospholipid metabolism,sphingolipid metabolism and retinol metabolic pathways.Reverse the expression of FXR and its related proteins,and improve the accumulation of bile acids in vivo.The drug is quickly absorbed and eliminated in vivo and with a low bioavailability.