| [BACKGROUND AND OBJECTIVE]Cyclosporine A?CsA?is an immunosuppressive drug commonly applied after solid organ transplantation to prevent rejection,but its usage is limited due to hyperbilirubinemia,hypercholesterolemia and cholestatic liver injury.Clinical studies have shown that Yinzhihuang?YZH?,a prescription of traditional Chinese medicine,enhances bilirubin and bile acids clearance and is widely used to treat neonatal jaundice or cholestasis.The purpose of the current study was to determine the protective effect of YZH on CsA-induced cholestatic liver injury and explore its underlying molecular mechanisms in vivo and vitro.[METHODS]1.Thirty two male Sprague-Dawley rats?250±20g?were divided into 4 groups:Control group,CsA group,YZH group and UDCA group.Control group were given0.5%CMC-Na for 10 days;CsA group were administrated by oral gavage of CsA(30mg·kg-1,twice daily,dissolved in 0.5%CMC-Na)for 10 days;YZH group were administrated by oral gavage of YZH(5 mL·kg-1,twice daily),and administrated with CsA(30 mg·kg-1,twice daily)for 10 days;UDCA group were administrated by oral gavage of UDCA(25 mg·kg-1,twice daily),and administrated with CsA(30 mg·kg-1,twice daily)for 10 days.Rat liver function and pathological changes in hepatic tissue were assessed using biochemical tests and HE staining.2.Pharmacodynamics indicators:?1?Rat liver function were assessed using biochemical tests for the levels of TBIL,DBIL,IBIL,TBA,ALT and AST in rat serum;?2?The pathological changes of hepatic tissue were assessed using HE staining;?3?The levels of oxidative damage indexes such as superoxide dismutase?SOD?,reduced glutathione?GSH?and malondialdehyde?MDA?in liver were detected by ELISA.3.LC-MS/MS was developed for the simultaneous determination of the component of bile acids in rat serum,and the content of serum bile acids of control group,CsA group,YZH group and UDCA group was measured and analyzed.4.RT-PCR and western blotting were employed to detect the expression of Fxr,Mrp2,Bsep,Ntcp,Oatp2,Cyp7a1 and Ugt1a1 in rat liver.In addition,immunohistochemistry?IHC?was used to examine the expression of Bsep and Mrp2in liver tissues.5.For the in vitro experiment,LO2 cells were used to investigate the active pharmaceutical ingredients and elucidate their mechanisms.Real-time PCR and western blotting were employed to detect the expression of FXR,MRP2,BSEP,NTCP,OATP2,CYP7A1 and UGT1A1 in LO2 cells.[RESULTS]YZH apparently mitigated serum biochemical parameters such as ALT,AST,TBIL,DBIL and TBA levels in CsA-induced intrahepatic cholestasis rats.In addition,CsA significantly increased SOD activity and GSH level in rat liver.Furthermore,H&E experiment revealed that YZH treatment remarkably alleviated necrotic and degenerative changes of hepatobiliary cells,the bile duct proliferation,and the inflammatory cell infiltration,indicating the hepatoprotective effects of YZH involved in CsA-induced cholestasis.In this experiment,LC-MS/MS was successfully established to determine concentrations of 15 bile acids in rat serum.It was found that CsA could significantly increase the content of CA,GCDCA,TCDCA and TCA in rat serum,and significantly reduce the content of UDCA.Compared with the CsA group,YZH significantly increased the content of UDCA in rat serum and decreased the contents of CA,GCDCA,TCA and GCA.Targeted protein and gene analysis revealed that CsA markedly decreased the protein expression of upstream nuclear receptor Fxr,and reduced the expression of Mrp2,Bsep,Ntcp and Oatp2 transport system,meanwhile it increased bile acids synthetic enzyme Cyp7a1 protein expression and decreased bilirubin metabolic enzyme Ugt1a1 protein expression.In comparison,YZH treatment enhanced CsA-induced decreased Fxr protein expression,and increased effux transpoters Mrp2 and Bsep protein accumulation,as well as repressed Cyp7a1 protein expression and increased Ugt1a1 protein expression.In addition,the alterations of protein expression levels of FXR,MRP2,BSEP,NTCP,OATP2,UGT1A1 and CYP7A1 in LO2 cells treated by CsA were consistent with these changes in rat liver.YZH pretreated groups showed significantly elevated expression of FXR,MRP2,BSEP and OATP2,together with reduced CYP7A1 enzyme expression and increased UGT1A1 enzyme expression.In addition,the results indicated that geniposidic acid,baicalin and chlorogenic acid were active pharmaceutical ingredients of YZH.Specifically,the expression levels of FXR,MRP2,BSEP,NTCP,OATP2,UGT1A1 and CYP7A1 were promoted or inhibited to varying degrees in LO2 cells treated with these ingredients.[CONCLUSION]YZH may be an effective approach for the prevention and treatment for CsA-induced cholestatic liver injury by reversing the disordered homeostasis of bile acids and bilirubin via regulation of MRP2,BSEP,NTCP and OATP2 transport system and metabolic enzymes CYP7A1 and UGT1A1,as well as upstream nuclear receptors FXR.In addition,geniposidic acid,baicalin and chlorogenic acid were found to be effective ingredients and exert hepatoprotective effect via regulation of metabolic enzymes and transporters of bile acids and bilirubin.In conclusion,Yinzhihuang,as a compound traditional Chinese medicine,may be a potential drug for the treatment of CsA-induced cholestatic liver injury. |
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