Effects And Mechanism Of Endoplasmic Reticulum Stress In Drug-induced Liver Injury Induced By Rifampicin

Rifampicin(RFP)is a first-line anti tuberculosis drugs,which caused hepatotoxicity is an important clinical problem.The specific mechanism of RFP induced liver injury remains not clear.In our study,L02 cell and Hep G2 cell injury and its evolution induced by RFP were observed in vitro.The effects of RFP on endoplasmic reticulum stress and bile acid transporter protein expression,protein localization and gene expression were studied,and the effects of CHOP gene and Nrf2 gene silencing and overexpression on RFP induced cell injury were obseserved,and the intervention effects of 4-PBA were also studied,this is a preliminary study on the molecular mechanism of cell injury induced by RFP.1.Study on the cytotoxicity of L02 cells induced by RFPTo assess the cytotoxicity of RFP,we treated L02 cells with different concentrations of RFP(100 ?M,200 ?M,300 ?M,and 400 ?M)at different time points(12 h,24 h,36 h,48 h),to observe the morphology of L02 cells by inverted microscope,cell apoptosis was detected by FCA,the survival rate was detected by MTT,the ALT,AST,AKP,LDH and ATP level in the cell culture supernatants were detected by ELISA and micro plate culture method.Observation under an inverted microscope showed that normal L02 cells adhered to the wall and grew in a monolayer morphologically similar to epithelial cells.After treatment with RFP,the cells developed a spindle-like morphology as the concentration and treatment time increased.Furthermore,the number of adherent cells was reduced,and the number of floating cells was increased,and cell debris appeared to increase.FCA showed increases in the concentration of RFP and the time of treatment gradually elevated the L02 cell apoptosis rate.Treatment with 400 ?M RFP for 48 h resulted in the apoptosis of almost all cells.Additionally,the results of the MTT assay demonstrated that there was a reverse trend with the FCA results,as it showed that with increasing concentration of RFP and treatment time,the L02 cell survival rate gradually decreased,thus showing a dose-dependent and time-dependent manner.When the concentration exceeded 100 ?M,the cell proliferation rate was significantly inhibited.When the treatment time was > 24 h,the four concentrations of RFP had a significant effect on cell viability.The IC50 value for RFP at 24 h was approximately 548.91 ?M,and at 48 h,it was approximately 234.96 ?M.At the same time,the ALT,AST,AKP,LDH and ATP levels were significantly increased in the cell culture supernatants after treatment with 200 ?M RFP for 48 h.2.Study on the PERK-ATF4-CHOP pathway of L02 cells induced by RFPIn this study,administration of 200 ?M RFP for 48 h increased the protein expression of GRP78,PERK,and ATF4 but had no apparent effect on the protein expression of CHOP.However,100 ?M RFP for 48 h had no apparent effect on these proteins except for PERK.At the gene level,treatment with 200 ?M RFP for 48 h increased the gene expression of GRP78,PERK,ATF4 and CHOP,but 100 ?M RFP for 48 h had no significant effect.Next,we found that 200 ?M RFP for 12 h and 24 h had no apparent effect on the expression of these proteins,but at the gene level,changes began 12 h post-treatment.At the 12 h time point,RFP induced the m RNA expression of ATF4 and CHOP.At the 24 h time point,RFP induced the m RNA expression of GRP78,ATF4,and CHOP.At the 48 h time point,RFP induced the m RNA expression of GRP78,PERK,ATF4,and CHOP.Taken together,these results indicated that 200 ?M RFP activated the PERK-ATF4-CHOP pathway at both the gene and protein levels.3.The effects of CHOP gene on cell apoptosis of L02 cells induced by RFPTo determine whether RFP-induced apoptosis is CHOP-dependent,small interfereing RNA(si RNA)was used to knock down the expression of the CHOP gene.The knockdown efficiency was assessed by q RT-PCR and western blot.CHOP gene expression was reduced by approximately 70% compared with that in cells transfected with the negative control si RNA,and the CHOP protein expression showed no obvious changes.Following CHOP knockdown and co-treatment with RFP,there was a significant reduction in CHOP gene expression,but CHOP protein expression showed no significant changes.In addition,apoptosis analysis revealed that apoptosis decreased,and the MTT assay showed that the cell survival increased in these cells.Furthermore,the LDH level in the cell culture supernatants was also decreased.Interestingly,there was a significant reduction in GRP78,PERK,and ATF4 at both the gene expression level and the protein expression level.Next,L02 cells were transfected with a CHOP-overexpression plasmid.CHOP overexpression was verified by q RT-PCR and western blot.CHOP gene expression increased 243-fold compared with the expression in cells transfected with the negative control plasmid,and CHOP protein expression increased 17-fold.These results showed that the expression of CHOP increased at both the gene and protein level after transfection with the CHOP overexpression plasmid.After transfection and then treatment with RFP,there was a significant increase in CHOP gene expression and protein expression.The FCA showed cell apoptosis rate increased,and the MTT assay revealed that cell survival decreased in these samples.Additionally,the LDH level in the cell culture supernatants was also increased.At the same time,we found no significant effects on GRP78,PERK,or ATF4 protein expression or on PERK or ATF4 gene expression.However,GRP78 gene expression significantly increased after treatment with RFP.4.4-PBA inhibits the activation of PERK-ATF4-CHOP pathway of L02 cellsinduced by RFPOur results showed that 4-PBA inhibited the protein expression of GRP78,PERK and ATF4 induced by RFP,but the protein expression of CHOP,p-JNK,and JNK did not significantly change.At the gene level,4-PBA inhibited the gene expression of GRP78,PERK,ATF4 and CHOP.The results showed that 4-PBA could inhibit the PERK-ATF4-CHOP pathway induced by RFP at the protein and gene level.The staining with the anti-GRP78 antibody,anti-PERK antibody,and anti-ATF4 antibody showed light cytoplasmic expression in the control L02 cells.In contrast,the anti-GRP78 antibody,anti-PERK antibody,and anti-ATF4 antibody showed intense cytoplasmic staining in RFP-treated cells.After treatment with 4-PBA,the fluorescence intensity decreased.However,the anti-CHOP antibody staining showed no apparent change.5.4-PBA protects L02 cells from damage caused by RFPWe found that after treatment with 4-PBA,the number of apoptotic cells significantly reduced.MTT assay showed that 4-PBA increased cell survival in the RFP treatment group,and 4-PBA reduced the level of ALT,AST,AKP,LDH and ATP.These results prompt that 4-PBA has protective effect.6.Study on the cytotoxicity of Hep G2 cells induced by RFPTo assess the cell toxicity of RFP,we treated Hep G2 cells with different concentrations of RFP(100 ?M,200 ?M,300 ?M,and 400 ?M)at different time points(24 h,48 h),to observe the morphology of Hep G2 cells by inverted microscope,the survival rate was detected by MTT,the level of LDH,ALT,AST,AKP,?-GT,TBIL,DBIL,IBIL,TBA and ATP in the cell culture supernatants were detected by ELISA and micro plate culture method.Observation under an inverted microscope showed that the morphology of normal Hep G2 cells was similar to epithelial cells,and the cells were connected to each other and grown in a dense integrated chip,there are no transparent particles in the cytoplasm.With the increase of concentration and treatment time of RFP,cells developed a spindle-like morphology or uneven shape,the number of adherent cell was reduced and cells scattered in the growth,and the number of floating cells was increased and the refraction strengthened obviously.MTT showed that with increasing concentration of RFP and treatment time,the Hep G2 cell survival rate gradually decreased,the level of LDH,ALT,AST,AKP,?-GT,TBIL,DBIL,IBIL,TBA and ATP in the culture supernatants gradually increased,showing a dose-dependent and time-dependent manner.The IC50 value for RFP at 24 h was approximately 550.50 ?M,and at 48 h,it was approximately 311.01 ?M.7.Study on the expression of bile acid transporters and Nrf2 of Hep G2 cellsinduced by RFPTo study the expression of bile acid transporters and adaptive response factor Nrf2 of endoplasmic reticulum stress induced by RFP,different concentrations of RFP was administered at different time points,to observe its effect on the protein and gene expression of bile acid transporters and Nrf2.At the 24 h time point,the expression of the BSEP,MDR1,total Nrf2,and nuclear Nrf2 proteins increased to different degrees.The expression of the NTCP and cytoplasmic Nrf2 proteins decreased to different degrees.The expression of the BSEP,MRP2,NTCP,OATP2,OST?,and total Nrf2 genes also increased to different degrees.At the 48 h time point,the protein and gene expression of BSEP,MDR1,MRP2,NTCP,OATP2,OST?,and total Nrf2 increased to different degrees.Cytoplasmic Nrf2 protein decreased and nuclear Nrf2 protein increased to different degrees.8.The effects of Nrf2 gene on the expression of bile acid transporters in RFP-treatedHep G2 cells.A Nrf2-small interfering RNA(si RNA)or a Nrf2-overexpression plasmid was used to knock down or overexpress the Nrf2 gene to assess the requirement for Nrf2 in the effect of RFP on bile acid transporters expression.The knockdown or overexpression efficiency was assessed by q RT-PCR and western blotting.Nrf2 gene expression was reduced by 90% compared with that in cells transfected with the negative control-si RNA,and the total Nrf2 protein and cytoplasmic Nrf2 protein expression levels were reduced by 50%;nuclear Nrf2 protein expression was reduced by 70%.Following Nrf2 knockdown and co-treatment with 300 ?M RFP for 48 h,the expression levels of the BSEP,MDR1,MRP2,NTCP,OATP2,and OST? proteins and m RNAs were reduced to different degrees.The cell survival rate decreased,and the LDH levels in the cell culture supernatants increased.After transfection with the plasmid,the Nrf2 m RNA expression increased 50-fold compared with the expression in cells transfected with the negative control-plasmid;total and cytoplasmic Nrf2 protein expression increased 2-fold,whereas nuclear Nrf2 protein expression increased 5-fold.Following Nrf2 overexpression,the expression levels of the bile acid transporters increased.Meanwhile,the cell survival rate increased,and the LDH levels decreased.According to the above research results,this paper draws the following conclusions:(1)RFP can activate the PERK-ATF4-CHOP pathway,which closely related to the cell toxicity,and CHOP is one of the targets of RFP induced apoptosis in L02 cells;(2)4-PBA,as the inhibitor of endoplasmic reticulum stress,inhibited the protein expression and gene expression of PERK-ATF4-CHOP pathway induced by RFP,inhibited the apoptosis rate of L02 cells,increased the survival rate of cells,reduced the cell damage markers,so it has a protective effect for the cell damage induced by RFP;(3)Nrf2 mediated the adaptation increase of bile acid transporters induced by RFP.Nrf2 may be a key regulator of endoplasmic reticulum stress and bile acid transport.