Mechanism Of Oleanolic Acid Against Ochratoxin A-induced Apoptosis In HK-2 Cells

Objective: To explore the process of ochratoxin A(OTA)-induced apoptosis of human renal tubular epithelium(HK-2)cells,the effect of oleanolic acid(OA)against OTA-induced apoptosis of HK-2 cells,and the mechanism of Tumor Necrosis Factor Receptor Associated Protein 1(TRAP1)in OA against OTA-induced apoptosis signaling in HK-2 cells.Methods: 1.In this experiment,Human renal tubular epithelial(HK-2)cells were used as the experimental model.The cells were treated with fresh medium containing OTA for 24 hours,and the cell survival rate was measured using the CCK-8 method to determine the working concentration of OTA.Set control group(group C,0 ?mol/L OTA treatment for 24 h),low-dose group(group L,0.2 ?mol/L OTA treatment for 24 h),medium-dose group(group M,1 ?mol/L OTA treatment for 24 h)And the high-dose group(Group H,5 ?mol/L OTA treatment for 24 h).After the time required for OTA treatment of cells,and then Annexin V-FITC/PI double staining method was used to measure the cell apoptosis rate.Meanwhile,the expression of apoptosis-related proteins TRAP1,Lonp1,CYPD,Cyt-C,Bcl-2,Bax,Cleaved Caspase-9,Cleaved Caspase-3,GRP78,p-PERK,p-e IF2?,ATF4 and CHOP were measured by Western blot method.2.After pre-treating cells with different concentrations of OA for 2 h,the cell survival rate was measured using the CCK-8 method to determine the working concentration of OA.Set the control group(group CK,0 ?mol/L OTA treatment for 24 h),OTA group(group OT,5 ?mol/L OTA treatment for 24 h),OA control group(group OA,2 ?mol/L OA pretreatment for 2 h),OA followed by OTA group(group OO,2 ?mol/L OA pretreatment for 2 h and 5 ?mol/L OTA treatment for 24 h).After using OA and OTA to treat the cells to the required time,the CCK-8 method was used to measure the cell survival rate.Apoptosis rate was measured by Annexin V-FITC/PI double staining.In addition,the expression of apoptosis-related proteins TRAP1,Lonp1,CYPD,Cyt-C,Bcl-2,Bax,Cleaved Caspase-9,and Cleaved Caspase-3,GRP78 and CHOP were measured by Western blot.3.After transiently transfecting cells with TRAP1 siRNA for 5 hours,the inhibitory efficiency of TRAP1 siRNA on TRAP1 was determined by Western blot method.Set the control group(group CK,0 ?mol/L OTA treatment for 24 h),OTA group(group OT,5 ?mol/L OTA treatment for 24 h),OA control group(group OA,2 ?mol/L OA pretreatment for 2 h),OA followed by OTA group(group OO,2 ?mol/L OA pretreatment for 2 h and 5 ?mol/L OTA treatment for 24 h),transfection control group(group CK+,TRAP1 siRNA transfection for 5 h,0 ?mol/L OTA treatment 24 h),OTA group after transfection(group OT+,TRAP1 siRNA transfection for 5 h,5 ?mol/L OTA treatment 24 h),OA control group after transfection(group OA+,TRAP1 siRNA transfection for 5 h,2 ?mol/L OA treatment for 2 h),OA followed by OTA group after transfection(group OO+,TRAP1 siRNA transfection for 5 h,2 ?mol/L OA pretreatment for 2 h and 5 ?mol/L OTA treatment for 24 h).Cells were treated with TRAP1 siRNA,OA,and OTA to the required time,and the expression of apoptosis-related proteins CYPD,Bcl-2,Bax,GRP78 and CHOP were measured by Western blot.Results: 1.With the OTA concentration increases,the cell survival rate decreased.Compared with the group C,the apoptosis rate of the group M and H increased significantly(P<0.05).The expression of apoptosis-related proteins TRAP1,Lonp1,CYPD,Cyt-C,Bax,Cleaved Caspase-9,Cleaved Caspase-3,GRP78,p-PERK,p-eIF2?,ATF4,and CHOP was gradually increased in group L,M,and H,while Bcl-2 expression gradually decreased,especially the changes of group H related proteins were significantly different(P<0.05).2.2 ?mol/L OA pre-treatment for 2 h can significantly improve cell survival rate(P <0.05).Compared with the group OT,the group OO could significantly reduce the apoptosis rate(P<0.05).In addition,compared with the group OT,the expression of apoptosis-related proteins TRAP1,Longp1,CYPD,Cyt-C,Bax,Cleaved Caspase-9,Cleaved Caspase-3,GRP78 and CHOP in the group OO was significantly reduced(P <0.05),and the expression of Bcl-2 increased significantly(P<0.05).3.TRAP1 siRNA can significantly inhibit the expression of TRAP1(P<0.05).After instant transfection with TRAP1 siRNA for 5 hours,compared with the group CK,OT,OA and OO,the expression of apoptosis-related proteins CYPD,Bax,GRP78,and CHOP in the group CK+,OT+,OA+ and OO+ were significantly increased(P <0.05),while Bcl-2 expression decreased significantly(P<0.05).Conclusion: 1.OTA can alleviate OTA-induced decreased the viability of HK-2 cells,and inhibit OTA-induced the activation of apoptosis in the mitochondrial pathways and endoplasmic reticulum stress pathways in HK-2 cells.2.OA can antagonize the reduction of HK-2 cell viability induced by OTA,and inhibit the activation of mitochondrial and endoplasmic reticulum stress pathway apoptosis induced by OTA in HK-2 cells.3.In the process of OA antagonizing OTA-induced apoptosis of HK-2 cells,TRAP1 inhibits apoptosis-related signaling in the mitochondrial pathway and endoplasmic reticulum stress pathway.