| Perfluorooctane sulfonates(PFOS)is a kind of artificial fluorine-containing surfactant,which is commonly used in daily life,but due to its undegradability,longdistance migration ability and strong bioaccumulation,researchers have been paying increasing attention to the health risk of PFOS.Epidemiological studies have shown that exposure to perfluorinated compounds to which PFOS belong may increase the risk of cardiovascular disease(CVD)and other diseases in humans.PFOS are detected in both maternal and fetal umbilical cord blood samples and can have adverse effects on vascular endothelium.Endothelial injury is the basis of CVD,but the mechanism of endothelial injury caused by PFOS is unclear.Ferroptosis is a regulatory cell death driven by cell metabolism and iron-dependent lipid peroxidation.The role of ferroptosis in the pathogenesis of endothelial dysfunction caused by PFOS remains unclear.This study used human umbilical vein endothelial cells(HUVECs)as the object to investigate the role of ferroptosis in HUVECs induced by PFOS.Objective:To explore the effects of PFOS on the expression of oxidative stress-related proteins and other indexes.The effect of PFOS on the ferroptosis pathway in HUVECs was determined by detecting the expression of ferroptosis related proteins and other indicators.Through the intervention of ferroptosis inhibitor,the expression of endothelial damage related proteins and other indicators were detected,and the role of ferroptosis pathway in PFOS induced HUVECs injury was clarified.Methods:1.Cell culture and grouping:HUVECs were cultured in vitro in 1640 medium containing 10%fetal bovine serum.When the cell density was about 70%～80%,the normal control group was not treated.The solvent control group was treated with 0.1%DMSO for 12 h;PFOS group was treated with 180 μM PFOS for 12 h;PFOS+Fer-1 group was pretreated with 1 μM Fer-1 inhibitor for 2 h,and then treated with 180 μM PFOS for 12 h.2.CCK-8 method was used to detect the cell survival rate of HUVECs under different time and concentration exposure conditions,and to determine the optimal concentration and time of PFOS exposure.3.The ROS levels were determined by DCFH-DA fluorescent probe staining.4.The level of Lipid active oxygen(Lipid-ROS)in cells is detected by flow cytometry.5.The contents of Fe2+,MDA,GSH,GPX4,NO and PGI2 were detected by the kit.6.The cell microstructure was observed by transmission electron microscopy.7.Cell migration(healing)ability was detected by scratch test.8.CD31 levels were detected by immunofluorescence assay.9.The expressions of oxidative stress related proteins(Nrf2,Keapl),ferroptosis related proteins(ACSL4,FTH1,GPX4,P53)and endothelial damage related proteins(eNOS,CD31,MCP-1,COX-2)were detected by Western blot.10.Using IBM SPSS 24.0 software analysis data,the groups of measuring data in x±s,normal and variance of data,using the single factor analysis of variance(OneWay ANOVA)to compare the indexes of differences between different groups,LSD method is used to compare between groups of two.P<0.05 was considered to be statistically significant.Results:1.Effect of PFOS on cell survival rateWith the increase of PFOS concentration and time,the cell survival rate decreased significantly.When the reaction time was 12 h,the IC50 value of PFOS was 180 μM,and the time and concentration were used as the conditions for subsequent experiments.2.The influence of PFOS on the level of ROS and lipid-ROS in HUVECsCompared with normal control group and DMSO group,ROS production level in PFOS group was significantly increased(P<0.05).Compared with PFOS group,ROS production level of cells in PFOS+Fer-1 group was significantly decreased(P<0.05);Compared with normal control group and DMSO group,the level of cell-producing Lipid-ROS in PFOS group was significantly increased(P<0.05).Compared with PFOS group,the level of cell-producing Lipid-ROS in PFOS+Fer-1 group is significantly decreased(P<0.05).3.The effect of PFOS on the expression level of oxidative stress-related proteins in HUVECsCompared with normal control group and DMSO group,the protein expression level of Nrf2 in PFOS group was significantly increased,while the protein expression levels of Keapl was significantly decreased(P<0.05).Compared with PFOS group,the expression level of Nrf2 protein in PFOS+Fer-1 group was significantly decreased,and the expression level of Keapl protein was significantly increased(P<0.05).4.Effects of PFOS on Fe2+,MDA,GSH and GPX4 contents in HUVECsCompared with normal control group and DMSO group,PFOS significantly increased the expressions of Fe2+and MDA(P<0.05),and significantly decreased the expressions of GSH and GPX4(P<0.05).After Fer-1 treatment,the expressions of Fe2+and MDA were significantly decreased(P<0.05),while the expressions of GSH and GPX4 were significantly increased(P<0.05).5.The effect of PFOS on the microstructure of HUVECs under electron microscopeA large number of lipid droplets were observed in cells in the PFOS group compared to the normal control group,and some cells had smaller and atrophied mitochondria,reduced mitochondrial crists,and increased mitochondrial membrane density.Compared with PFOS group,lipid droplets in PFOS+Fer-1 group were relatively reduced,mitochondrial volume was larger and the atrophy degree was lower.6.Effect of PFOS on the expression levels of ferroptosis related proteins in HUVECsCompared with normal control group and DMSO group,PFOS significantly increased the expressions of ACSL4 and P53(P<0.05),and significantly decreased the expressions of GPX4 and FTH1(P<0.05).After Fer-1 treatment,the expressions of ACSL4 and P53 were significantly decreased(P<0.05),while the expressions of FTH1 and GPX4 were significantly increased(P<0.05).7.The effect of PFOS on migration(healing)ability of HUVECsAt 12h and 24h,the healing ability of PFOS group was significantly lower than that of normal control group and DMSO group(P<0.05),and the negative effect was more obvious at 24h.The healing ability of PFOS+Fer-1 group was significantly higher than that of PFOS group(P<0.05),but was also significantly lower than that of normal control group and DMSO group(P<0.05).8.The effect of PFOS on the contents of NO and PGI2 in HUVECsCompared with normal control group and DMSO group,NO in PFOS group was significantly decreased,and PGI2 was significantly increased(P<0.05),while NO in PFOS+Fer-1 group was not significantly different from that in normal cell group,and PGI2 content was significantly decreased compared with PFOS group(P<0.05).9.The effect of PFOS on the expression levels of endothelial injury related indicators in HUVECsCompared with normal control group and DMSO group,the protein expression levels of eNOS,MCP-1 and COX-2 in PFOS group were significantly increased(P<0.05),and the fluorescence intensity of CD31 was significantly higher than that in normal control group and DMSO group(P<0.05).Compared with PFOS group,the protein expression levels of eNOS,CD31,MCP-1 and COX-2 in PFOS+Fer-1 group were significantly decreased(P<0.05),and the fluorescence intensity of CD31 was significantly lower than that in PFOS group(P<0.05).Conclusion:1.PFOS can induce oxidative stress in HUVECs by activating Nrf2-Keapl pathway;2.PFOS induced ferroptosis in HUVECs by activating lipid peroxidation pathway and inhibiting Xc-GSH-GPX4 pathway;3.PFOS can induce HUVECs injury by activating oxidative stress pathway and ferroptosis pathway;4.Ferroptosis inhibitors can reduce the damage degree of HUVECs caused by PFOS,which provides a new way to avoid the toxic effect of PFOS on endothelial cells from the perspective of ferroptosis. |
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