敲低MANF对利福平诱导HepG2细胞损伤的影响及机制

The effects and mechanism of knockdown MANF on rifampicin induced HepG2 cell injury

目的探讨敲低中脑星型胶质细胞源性神经生长因子(MANF)对利福平(RFP)诱导HepG2细胞损伤的影响及机制。方法慢病毒转染技术构建MANF敲低稳转细胞株(MANF Y25)及其对照细胞株(MANF Y07)。实验分为4组:MANF Y07+DMSO组、MANF Y07+RFP组、MANF Y25+DMSO组、MANF Y25+RFP组。给予HepG2细胞100μmol/L RFP 24 h后,Western blot和qRT-PCR检测各组细胞MANF及未折叠蛋白反应(UPR)相关基因葡萄糖调节蛋白78(GRP78)、蛋白酶R样内质网激酶(PERK)、真核翻译启动因子2α(eIF2α)、活化转录因子4(ATF4)、CCAAT/增强子结合蛋白(CHOP)、Tribbles同源蛋白3(TRIB3)、肌醇需求激酶1(IRE1)、剪接型X-盒结合蛋白1(XBP1-S)、非剪接型X-盒结合蛋白1(XBP1-U)、活化转录因子6(ATF6)的蛋白及基因表达水平;Annexin V-PE/7-AAD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谷丙转氨酶(ALT)、谷草转氨酶(AST)、碱性磷酸酶(AKP)、总胆红素(TBIL)、间接胆红素(IBIL)相对含量变化。结果在蛋白水平,RFP激活MANF、GRP78、p-eIF2α、ATF4、ATF6的蛋白表达;在基因水平,RFP诱导HepG2细胞MANF、GRP78、PERK、eIF2α、ATF4、CHOP、TRIB3的基因表达,并且MANF敲低后GRP78、p-PERK、p-eIF2α、ATF4、ATF6蛋白表达水平及上述UPR相关基因的基因表达水平进一步上调(P<0.05),MANF敲低后细胞凋亡率升高(P<0.01),细胞增殖能力降低(P<0.01),细胞培养上清液中ALT、AST、AKP、TBIL、IBIL水平升高(P<0.05),说明细胞损伤加重。结论RFP可激活UPR,敲低MANF后这种效应进一步加强,同时细胞损伤加重,表明MANF可能通过调节URP而在RFP诱导HepG2细胞损伤中发挥保护作用。

Objective To investigate the effects and mechanism of knockdown mesencephalic astrocyte-derived neurotrophic factor(MANF)on rifampicin(RFP)induced HepG2 cell injury.Methods A MANF-knockdown stable cell line(MANF Y25)and a control cell line(MANF Y07)were constructed by lentivirus transfection.After HepG2 cells were treated with 100μmol/L RFP for 24 h,the experiment was divided into MANF Y07+DMSO group,MANF Y07+RFP group,MANF Y25+DMSO group and MANF Y25+RFP group.Western blot and qRT-PCR were used to detect the protein and gene expression levels of MANF and unfolded protein response(UPR)-related genes such as glucose-regulated protein 78(GRP78),protein kinase R-like ER kinase(PERK),eukaryotic translation initiation factor 2α(eIF2α),activating transcription factor 4(ATF4),C/EBP-homologous protein(CHOP),tribbles homolog 3(TRIB3),inositol-requiring enzyme 1(IRE1),spliced X-box binding protein1-S(XBP1-S),non-spliced X-box binding protein 1-U(XBP1-U)and activating transcription factor 6(ATF6);Annexin V-PE/7-AAD double staining was used to detect the apoptosis rate of cells in each group;The changes in the cell proliferation ability were determined by the cell counting kit-8 assay;The relative contents of alanine aminotransferase(ALT),aspartate aminotransferase(AST),alkaline phosphatase(AKP),total bilirubin(TBIL)and indirect bilirubin(IBIL)in the supernatant of cell culture were detected by kits.Results At the protein level,RFP induced the protein expression of MANF,GRP78,p-eIF2α,ATF4 and ATF6;at the gene level,RFP induced the gene expression of MANF and UPR-related genes GRP78,PERK,eIF2α,ATF4,CHOP and TRIB3 in HepG2 cells.After MANF knockdown,the protein expression levels of GRP78,p-PERK,p-eIF2α,ATF4,ATF6 and the genes expression levels of UPR-related genes mentioned above were further up-regulated(P<0.05).Moreover,it was also found that after MANF knockdown,the rate of apoptosis increased(P<0.01),the cell proliferation ability decreased(P<0.01),and the levels of cell injury markers ALT,AST,AKP,TBIL and IBIL in the supernatant of cell culture increased(P<0.05).Conclusion RFP activates the UPR,which is further enhanced by MANF knockdown,and cell injury is aggravated,indicating that MANF may play a protective role in RFP-induced HepG2 cell injury by regulating the UPR.