| Perfluorooctane sulfonate(PFOS)is a typical persistent organic pollutant,which has been listed as one of the 10 industrial chemical pollutants that should be given high priority globally and poses a certain threat to the ecological environment and human health.PFOS enters the human body and accumulates in the kidney and liver mainly through the hepatic and intestinal circulation.The kidney is the main excretory organ of PFOS,but PFOS damage to renal epithelial cells and the mechanism of its damage are still unclear.It has been shown that PFOS exposure can disrupt the oxidative and antioxidant balance in the body,and PFOS can induce oxidative stress injury by inhibiting antioxidant enzymes in vivo and in vitro,and PFOS can also lead to endoplasmic reticulum stress(ERS).The occurrence of ferroptosis is closely related to ROS and lipid peroxidation,and ERS plays an important role in the interaction between iron death and other types of cell death.However,it is not clear whether ERS plays a role in the mechanism of PFOS-induced injury to renal tubular epithelial cells and the role it plays in PFOS-induced ferroptosis.Objective:To clarify the damage effect of PFOS on renal tubular epithelial cells(HK-2)and explore its mechanism,in order to provide scientific basis to investigate the toxicity of PFOS and its damage to kidney.Methods:HK-2 human renal tubular epithelial cells were cultured in vitro using MEM medium containing 10%fetal bovine serum,and subsequent experiments were performed when the growth status was good and the density grew to 70%～80%.Different doses of PFOS were given for 6,12 and 24 h of incubation,and the cell survival rate was detected by CCK-8 method to determine the concentration of PFOS used in this experiment.After pretreatment with different concentrations of Fer-1 for 2 h,PFOS was given for 12 h,and the CCK-8 method was used to determine the concentration of Fer-1 used in this experiment.After pretreatment with different concentrations of 4-PBA for 2 h,PFOS was incubated for 12h and assayed by CCK-8 method to determine the concentration of 4-PB A used in this experiment.The experiment was divided into two parts,with three groups in each part.The ferroptosis part was grouped as DMSO group(incubated for 12 h using complete culture medium containing 0.1%DMSO,and the rest was left untreated),PFOS group(given 200 μM concentration of PFOS for 12 h),and PFOS+Fer-1 group(given 1 μM of Fer-1 for two hours followed by 200 μM concentration of PFOS for 12 h).The endoplasmic reticulum stress fraction was grouped as DMSO group(incubated with complete culture medium containing 0.1%DMSO for 12 h,and the rest was left untreated),PFOS group(given 200 μM concentration of PFOS for 12 h),and PFOS+4-PBA group(given 2 mM of 4-PBA for two hours followed by 200 μM concentration of PFOS for 12 h).The corresponding doses of staining interventions were performed according to the experimental subgroups.Flow cytometry and fluorescence were used to detect the expression level of ROS in each group of cells;ELISA kits were used to detect the concentration of GPX4 in each group of cells;kits were used to detect the concentration of iron ions,GSH,and MDA;Western Blot was used to detect ferroptosis-related proteins(GPX4,ACSL4,FTH1,COX-2),antioxidant-related proteins(P53,HO-1),apoptosis-related proteins(Bax,Caspase12,Caspase3),endoplasmic reticulum stress-related proteins(GRP78,IRE1,PERK,ATF6)and kidney injury-related protein(KIM-1)expression were detected using Western Blot.Statistical analysis was performed using IBM SPSS 24.0 software,and the measurement data were expressed as x±s.Correlation images were produced using GraphPad Prism 8.0.2.The analysis of One Way ANOVA method was used for comparison between multiple groups,and the LSD method(Least-significant Difference Test)was used for two-way comparison between groups.Statistical differences were considered to be present when P<0.05.Results:1.Determination of the dose of poisoningThe effect of PFOS,Fer-1 and 4-PBA on cell survival was detected using the CCK-8 method.Through the analysis of the experimental data,we selected the incubation time of 12h and the concentration of 200μM as the treatment concentration of PFOS for this experiment;1μM was selected as the concentration of Fer-1 for the subsequent experiment;and 2mM was selected as the concentration of 4-PBA for the subsequent experiment.2.Generation of ROS in each group of cellsThe intracellular ROS production was significantly higher in the PFOS group compared with the DMSO group(P<0.05);the ROS production was significantly lower in the PFOS+Fer-1 group compared with the PFOS group(P<0.05).The fluorescence intensity of ROS was significantly higher in the PFOS group compared with the DMSO group(P<0.05);the fluorescence intensity of ROS was significantly lower in the PFOS+Fer-1 group compared with the PFOS group(P<0.05).3.Expression levels of ferroptosis-related indicators in HK-2 cellsCompared with the DMSO group,ACSL4 and COX-2 protein expression levels were significantly higher(P<0.05)and GPX4 and FTH1 protein expression levels were significantly lower(P<0.05)in the PFOS group;compared with the PFOS group,ACSL4 and COX-2 protein expression levels were significantly lower(P<0.05)in the PFOS+Fer-1 group,and FTH1 protein expression levels were significantly higher in the PFOS+Fer-1 group compared with the PFOS group(P<0.05).Compared with the DMSO group,the level of GPX4 was significantly lower in the PFOS group(P<0.05)and significantly higher in the PFOS+Fer-1 group(P<0.05).The iron ion content was significantly higher in the PFOS group compared with the DMSO group(P<0.05);the iron ion content was significantly lower in the PFOS+Fer-1 group compared with the PFOS group(P<0.05).4.Lipid peroxidation levels in each group of cellsCompared with the DMSO group,the GSH content of cells in the PFOS group was significantly lower(P<0.05);compared with the PFOS group,the GSH content of cells in the PFOS+Fer-1 group was significantly higher(P<0.05).Compared with the DMSO group,the MDA content of cells in the PFOS group was significantly higher(P<0.05);compared with the PFOS group,the MDA content of cells in the PFOS+Fer-1 group was significantly lower(P<0.05).5.Expression levels of antioxidant-related proteins in HK-2 cellsCompared with the DMSO group,P53 protein expression level was significantly higher(P<0.05)and HO-1 protein expression level was significantly lower(P<0.05)in the PFOS group;compared with the PFOS group,P53 protein expression was significantly lower(P<0.05)and HO-1 protein expression level was significantly higher(P<0.05)in the PFOS+Fer-1 group.6.Levels of apoptosis in HK-2 cellsThe fluorescence intensity was significantly higher in the PFOS group compared with the DMSO group(P<0.05);the fluorescence intensity was significantly lower in the PFOS+Fer-1 group compared with the PFOS group(P<0.05).The green fluorescence in HK-2 cells exposed to PFOS was significantly enhanced compared with the DMSO group and PFOS+Fer-1 group.Bax,Caspase12 and Caspase3 protein expression levels were significantly higher in the PFOS group compared with the DMSO group(P<0.05);Bax,Caspase12 and Caspase3 protein expression were significantly lower in the PFOS+Fer-1 group compared with the PFOS group(P<0.05).7.Inhibition of endoplasmic reticulum stress-related protein expression levels after endoplasmic reticulum stressGRP78,IRE1 and PERK protein expression were significantly higher in the PFOS group compared with the DMSO group(P<0.05);GRP78 and PERK protein expression were significantly lower in the PFOS+4-PBA group compared with the PFOS group(P<0.05).8.Inhibition of endoplasmic reticulum stress-related protein expression levels after ferroptosisGRP78,IRE1 and PERK protein expression were significantly higher in the PFOS group compared with the DMSO group(P<0.05);GRP78 and PERK protein expression were significantly lower in the PFOS+4-PBA group compared with the PFOS group(P<0.05).9.The expression levels of kidney injury factors in each group of cellsCompared with the DMSO group,the KIM-1 protein expression level was significantly higher in the PFOS group(P<0.05);compared with the PFOS group,the KIM-1 protein expression level was significantly lower in the PFOS+Fer-1 group(P<0.05);compared with the PFOS group,the KIM-1 protein expression level was significantly lower in the PFOS+4-PB A group(P<0.05).Conclusions:1.PFOS can lead to oxidative stress and lipid peroxidation in HK-2 cells,while inhibiting the activation of antioxidant system,causing cell damage and apoptosis.2.PFOS can cause ferroptosis in HK-2 cells,and inhibition of ferroptosis can alleviate PFOS-induced damage in HK-2 cells.3.PFOS can cause endoplasmic reticulum stress in HK-2 cells,the mechanism of which may be mediated through the IRE1 and PERK pathways,and inhibition of endoplasmic reticulum stress can alleviate PFOS-induced damage in HK-2 cells.4.Inhibition of ferroptosis alleviated endoplasmic reticulum stress caused by PFOS,suggesting that ferroptosis may be involved in PFOS-mediated endoplasmic reticulum stress process.5.PFOS leads to apoptosis in HK-2 cells. |
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