敲低PRDX6对利福平诱导HepG2细胞胆汁酸转运体适应性表达的影响

Effect of knockdown of PRDX6 on adaptive expression of bile acid transporter in HepG2 cells induced by rifampicin

目的探讨敲低过氧化物还原酶-6(PRDX6)在利福平(RFP)诱导人肝癌细胞(HepG2)损伤及胆汁酸转运体适应性表达中的作用。方法将处于对数生长期的细胞均匀接种于6孔板中,使用特异PRDX6-siRNA、control-siRNA分别转染HepG2细胞构建敲低组及对照组。给予细胞100μmol/L RFP诱导24 h后,Western blot和qRT-PCR检测各组细胞PRDX6、多药耐药蛋白1(MDR1)、多药耐药相关蛋白2、3、4(MRP2、MRP3、MRP4)、Na+/牛磺胆酸协同转运蛋白(NTCP)的蛋白及基因表达水平;Annexin V-FITC/PI双染法检测各组细胞凋亡率;CCK-8法检测各组细胞增殖变化;试剂盒检测各组细胞培养上清液中细胞损伤标志物丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)、总胆红素(TBIL)、直接胆红素(DBIL)、总胆汁酸(TBA)相对含量变化。结果RFP可诱导HepG2细胞MRP2、MRP3、MRP4、MDR1、NTCP及PRDX6的蛋白和基因表达水平升高(P<0.05),而敲低PRDX6后,MRP2、MRP3、MRP4、MDR1、NTCP的蛋白和基因表达水平均有不同程度的降低(P<0.05)。此外,PRDX6敲低后HepG2细胞凋亡率升高(P<0.05),细胞增殖能力下降(P<0.05),细胞培养上清液中细胞损伤标志物(ALT、AST、TBIL、DBIL、TBA)水平升高(P<0.05)。结论RFP可增加HepG2细胞胆汁酸转运体及PRDX6的蛋白和基因表达量,敲低PRDX6并用RFP诱导后胆汁酸转运体的蛋白及基因表达量降低,同时细胞损伤加重,表明PRDX6在RFP诱导的HepG2细胞适应性反应中发挥保护作用。

Objective To investigate the role of knockdown of peroxiredoxin-6(PRDX6)in injury and adaptive expression of bile acid transporter in human hepatoellular carcinomas(HepG2)cells induced by rifampicin(RFP).Methods Cells in logarithmic growth phase were uniformly inoculated in six-well plates,and HepG2 cells were transiently transfected with specific PRDX6-siRNA and control-siRNA to construct the knockdown group and control group.After 24 h of induction with 100μmol/L RFP,Western blot and qRT-PCR were performed to detect the protein and gene expression levels of PRDX6,multidrug resistance protein 1(MDR1),multidrug resistance-associated proteins 2,3 and 4(MRP2,MRP3 and MRP4),and Na+/taurine taurocholate cotransporter protein(NTCP).Annexin V-FITC/PI double staining assay was used to detect the apoptosis rate of cells in each group;CCK-8 assay was used to detect the changes of cell proliferation in each group;The relative contents of alanine aminotransferase(ALT),aspartate aminotransferase(AST),total bilirubin(TBIL),indirect bilirubin(IBIL)and total bile acid(TBA)in the supernatant of cell culture medium of each group were detected by kits.Results RFP increased the protein and gene expression levels of MRP2,MRP3,MRP4,MDR1,NTCP and PRDX6 in HepG2 cells(P<0.05),while the protein and gene expression levels of MRP2,MRP3,MRP4,MDR1 and NTCP decreased to different degrees after PRDX6 knockdown(P<0.05).In addition,PRDX6 knockdown resulted in increased apoptosis rate of HepG2 cells(P<0.05),decreased cell proliferation ability(P<0.05),and increased levels of cell injury markers(ALT,AST,TBIL,DBIL,TBA)in cell culture supernatants(P<0.05).Conclusion RFP increased the protein and gene expression of bile acid transporter and PRDX6 to increase in HepG2 cells.However,following knockdown of PRDX6 and treatment with RFP,the protein and gene expression levels of the bile acid transporter decreased and cell injury was aggravated,suggesting that PRDX6 played a protective role in RFP-induced adaptive response in HepG2 cells.