Study On The Mechanism Of Lonp1 Involved In The Regulation Of Perfluorooctanoic Acid-induced Mitochondrial Pathway Apoptosis Of Human Hepatocyte

Objective:To investigate the mitochondrial pathway apoptotic process induced by perfluorooctanoic acid PFOA in human hepatocyte L02,and to explore the mechanism of Lonp1 involved in the regulation of this process.Methods:1.In this study,human hepatocyte L02 was used as the research object.After treating L02 cells with different concentrations of PFOA for 24 h,the survival rate of cells was determined by CCK-8 method to determine the subsequent working concentration of PFOA.Control group（group C）,low-dose group（group L）,medium-dose group（group M）and high-dose group（group H）were set.After cells were treated with different doses of PFOA for 24 h,apoptosis rate,intracellular ROS level,mitochondrial membrane potential（ΔΨm）change and intracellular Ca²⁺concentration of cells in the four groups were measured by apoptosis kit,ROS detection kit,JC-1 probe and Flu O-4 AM(Ca²⁺fluorescence probe),respectively.Finally,the expressions of Lonp1,Trap1,TXNIP,CYPD,Bax,Bak,Bim,Cyt-c,Caspase-9,Bcl-2,GRP78,CHOP,Sig-1R and IP₃R₃ were determined by Western blot.2.The cells were pretreated with 2μmol/L Lonp1 inhibitor CDDO-me for 2 h,and the inhibition of Lonp1 protein in L02 cells by CDDO-me was determined by Western blot.Control group（gruop C）,PFOA low-dose group（group L）,PFOA medium-dose group（group M）,and PFOA high-dose group（group H）were set.Inhibitor pretreatment control group（C+group）,inhibitor pretreatment+PFOA low-dose group（L+group）,inhibitor pretreatment+PFOA medium-dose group（M+group）,inhibitor pretreatment+PFOA high-dose group（H+group）.After treatment with CDDO-me and PFOA for the required time,the expressions of apoptosis-related proteins CYPD,Bax,Bcl-2,Cyt-c,GRP78,CHOP and Sig-1R were determined by Western blot.Results:1.Cell survival rate:when the concentration of PFOA was lower than 64μmol/L,the cell survival rate was not significantly different from that of the control group（P>0.05）,but the cell survival rate decreased significantly when the concentration of PFOA was higher than 64μmol/L,and the median lethal dose was obtained when the concentration of PFOA was about 256μmol/L.2.Apoptosis rate:Compared with group C,the apoptosis rate of group L was not different,while the apoptosis rate of group M and H was significantly increased（13.2%±2.2%and29.6%±5.4%respectively）（P<0.05）.3.Intracellular ROS levels:Compared with group C,ROS levels in group L were slightly increased,but there was no difference（P>0.05）;ROS levels in groups M and H were1.7±0.2 and 2.4±0.3 times higher than those in group C（P<0.05）.4.Changes of intracellular mitochondrial membrane potential:Compared with group C,the mitochondrial membrane of group L had no change（P>0.05）,while the membrane potential of groups M and H was significantly decreased（P<0.05）,with a dose dependent relationship within a certain range,by about 60.5%±7.0%in group M and 76.5%±1.3%in group H.5.Intracellular calcium ion concentration:Compared with group C,there was no difference in the intracellular Ca²⁺concentration of group L（P>0.05）,but the intracellular Ca²⁺concentration of group M and group H was significantly increased（P<0.05）,and showed a dose dependence within a certain range.The intracellular Ca²⁺concentration of group M was about 1.32±0.02 times that of the control,and that of group H was about 2.79±0.08 times that of the control.6.Expression of apoptosis-related proteins:Compared with group C,the expressions of Lonp1,Trap1,Bcl-2 and Sig-1R in groups L,M and H were gradually decreased,while the expressions of TXNIP,CYPD,Bax,Bak,Bim,Cyt-c,Caspase-9,GRP78,CHOP and IP₃R₃were gradually increased.The changes of protein expression in group H were especially significant compared with group C（P<0.05）.7.Expression of apoptosis-related proteins,Ca²⁺concentration and apoptosis rate after 2μmol/L CDDO-me pretreatment:Compared with C,L,M,and H groups,the expressions of CYPD,Bax,Cyt-C,GRP78 and CHOP were significantly increased in C+,L+,M+,and H+groups（P<0.05）,while the expressions of Bcl-2 and Sig-1R were significantly decreased（P<0.05）.Ca²⁺concentration and apoptosis rate in C+,L+,M+and H+groups were higher than those in C,L,M and H groups（P<0.05）,and increased in a dose-dependent manner with the increase of PFOA concentration.Conclusion:1.PFOA induces apoptosis in the mitochondrial pathway of L02 human hepatocytes by inducing the increase of intracellular ROS level,reducing the level ofΔΨm,disturbing the intracellular calcium ion homeostasis,promoting the expression of pro-apoptotic proteins in the mitochondrial pathway,and inhibiting the expression of anti-apoptotic proteins in the mitochondrial pathway.Finally,PFOA induced toxicity of L02human hepatocytes,and thus reduced cell survival rate.2.Inhibition of Lonp1 can increase the expression of pro-apoptotic proteins CYPD,Bax,Cyt-c,GRP78 and CHOP,and inhibit the expression of anti-apoptotic proteins Bcl-2 and Sig-1R,and accelerate the increase of intracellular Ca²⁺concentration and apoptosis rate.That is,inhibition of Lonp1 can enhance PFOA-induced apoptosis of human hepatocyte L02mitochondrial pathway,and inhibition of Lonp1 can increase the sensitivity of hepatic toxicity of PFOA.