Study On The Anti-hepatic Fibrosis Effect And Pharmacokinetics Of Picroside ?

Objective: Liver fibrosis is a pathological process of abnormal liver structure or function resulted by excessive accumulation of extracellular matrix.Picroside I,isolated from Picrorhiza scrophulariiflora Pennell,has cholagogic and hepatoprotective effects,which could be served as a candidate drug of hepatic fibrosis.The study would explore whether picroside I protected against thioacetamide(TAA)-induced liver fibrosis and investigate the mechanism of drug action by metabolomic and proteomic analyses.On the other hand,the pharmacokinetics and metabolic profiles of picroside I in vivo were conducted by mass spectrometry.Method: 1.Mice were randomly divided into six groups: control group;model group(TAA);positive group(TAA + S-adenosyl-L-methionine 10 mg/kg);low-dose group(TAA + picroside I 25 mg/kg);middle-dose group(TAA + picroside I 50 mg/kg);and high-dose group(TAA + picroside I 100 mg/kg).TAA was given intraperitoneally at a dose of 200 mg/kg three times a week,and SAMe or picroside I was given orally once a day.After 8 weeks,serum transaminase and liver fibrosis markers were detected,and hematoxylin–eosin and sirius red staining were performed to observe the morphological change of liver tissue.2.Mice urine,serum,and liver samples in each group were pretreated by precipitation method and analyzed by liquid-chromatography coupled with tandem mass spectrometry.The preprocessed data were processed by principal component analysis and orthogonal partial least-squares discriminant analysis.Putative differential metabolites were selected according to VIP > 1 and p < 0.05.Differential metabolites were identified by searching HMDB and Metlin database or comparing with standard compounds.3.Mice liver samples in control,model,positive,and high-dose group were extracted,digested,and labeled,then separated by high p H reverse phase liquid chromatography.The collected fractions were analyzed by nano liquid-chromatography coupled with Q-Exactive mass spectrometer.Mass spectra data were analyzed using Mascot software for protein identification and quantification.Differentially expressed proteins were selected according to p < 0.05 and fold change < 0.83(or > 1.2).The important differentially expressed proteins would be validated by Western blot and real-time quantitative reverse transcription polymerase chain reaction analyses.4.An ultra-high-performance liquid chromatography coupled with tandem triple quadrupole mass spectrometry method was built to detect picroside I in rat plasma,and the method was applied to pharmacokinetics study of picroside I orally administered(75 mg/kg)to rats.Besides,ultra-high-performance liquid chromatography coupled with tandem quadrupole time-of-flight mass spectrometry was used to detect the metabolites of picroside I in rat bile,urine,serum,and feces after oral administration.Result: Picroside I could reverse increasing mice serum transaminase and liver fibrosis markers(collagen type IV,N-terminal peptide of type III procollagen,laminin,or hyaluronic acid)induced by liver fibrosis,reduce the soakage of inflammation cell and accumulation of collagen fiber.After medication of picroside I,citrate cycle,branched amino acid metabolism,and glycerophosphatide metabolism of liver fibrosis mice were regulated;upstream CYP8B1 was glutathione level was up-regulated,and downstream cholic acid and taurocholic acid were down-regulated;glutathione level was up-regulated,and transglutaminase was down-regulated;upstream sphingosine kinase 2 was up-regulated,and downstream sphingomyelin level was down-regulated.After oral administration,picroside I was quickly absorbed and reached the maximum plasma concentration in 30 min,then eliminated quickly.Fifteen metabolites of picroside I in vivo were detected,and the metabolic pathways included hydroxylation,deoxygenation,glucuronidation,sulfation,and methylation.Conclusion: Picroside I may have an anti-hepatic fibrosis effect,which can reverse the perturbation of energy,lipid,and glutathione metabolism induced by hepatic fibrosis.Picroside can also regulate sphingolipid signaling pathway,primary bile acid biosynthesis,and PPAR signaling pathway.The drug is quickly absorbed and eliminated,and possibly metabolized rapidly in vivo.