Reactive Oxygen Species Accumulation Mediated PFOS Induced Autophagy And Apoptosis In L-02 Cells

ObjectiveThe aim is to investigate the effect of ROS in L-02 cells injury induced by PFOS,and to provide experimental evidence for further study on the hepatotoxicity of PFOS.Methods1.The MTT method was used to detect the survival rate of L-02 exposed with different concentration(0,50,100,150,200,250 ?M)of PFOS for different time(24,48 h),and to detect the survival rate of L-02 treated with 200?M PFOS for different time(6,12,24,36,48 h).The effect of PFOS on oxidative stress damage in L-02 cells was analyzed by UV spectrophotometry and fluorescence methods.2.The experiment was divided into 200 ?M PFOS treatment or by NAC,3-MA,CQ pretreated cells groups.The level of ROS in L-02 cells was detected by UV spectrophotometry,cell staining was carried out with the staining agent MDC,the rate of apoptosis were tested by Annexin-V FITC/PI dual-dyeing,Rhodamine123 fluorescent probe was used to detect the mitochondrial membrane potential in cells.3.Western Blot was used to analyze the protein levels of of Bcl-2,Bax,Cleaved-Caspase3 apoptosis related proteins and LC3,p62 autophagy related proteins.The mRNA expression of Caspase3 was detected by Q-PCR.Results1.The outcomes of MTT test showed a decrease of survival rate induced by PFOS with a time-and dose-dependent manner.The results of UV and fluorescence spectrophotometry showed that PFOS induced L-02 cells superoxide dismutase(SOD)and glutathione reductase(GSH)decreased,while the total reactive oxygen species(ROS)and malondialdehyde(MDA)levels increased.2.Compared with the control group,in 200 ?M PFOS expourse group,the levels of ROS,autophagy bubbule,apoptosis,Bax protein,Cleaved-Caspase3 protein,LC3-?protein,Caspase3 mRNA level increased significantly,the levels of MMP,Bcl-2 protein,p62 protein,Bcl-2/Bax ratio decreased significantly.3.NAC pretreated L-02 could inhibitd the level of ROS,apoptosis,Bax protein,Cleaved-Caspase3 protein expression induced by PFOS,and increased the levels of MMP,Bcl-2/Bax ratio.4.3-MA pretreated L-02 then 200 ?M PFOS compared with the 200?M PFOS exposed groups,the level of apoptosis,autophagy and bubbule,Caspase3 mRNA expression was inhibited,the levels of MMP showed increased significantly.5.CQ pretreated L-02 then 200 ?M PFOS compared with the 200 ?M PFOS expsed groups,Bax protein,Cleaved-Caspase3 protein expression was inhibited,the levels of Bcl-2 protein,LC3 protein,p62 protein,Bcl-2/Bax ratio showed increased significantly.Conclusions1.PFOS exposure decreased the cell viability of L-02 cells,and induced L-02 cells produced ROS and apoptosis.2.ROS play an important role in mthochondria damage and autophagy induced by PFOS in L-02 cells.3.Autophagy induced by PFOS was involved in apoptosis of L-02 cells,which may be related with PFOS exposure induced autophagy to produce ROS,damage mitochondria,and then promote apoptosis.