邻苯二甲酸单(2-乙基己基)酯诱导人卵巢颗粒细胞系KGN细胞铁死亡的作用研究

Study of phthalic acid mono-2-ethylhexyl ester-induced ferroptosis in human ovarian granulosa cell line KGN cells

目的研究邻苯二甲酸单(2-乙基己基)酯(MEHP)是否诱导人卵巢颗粒细胞系KGN细胞铁死亡并影响其增殖。方法取对数增长期的人卵巢颗粒细胞系KGN细胞,给予不同浓度(0、100、200、400、800、1600μmol/L)MEHP处理24 h。通过普通光镜观察不同浓度MEHP处理后KGN细胞形态的改变;用CCK-8检测MEHP对细胞活力和增殖能力的影响;采用蛋白免疫印迹(WB)观察铁死亡相关蛋白(ACSL4、GPX4、SLC7A11、FTH1)在KGN细胞中的表达情况;使用细胞铁含量检测试剂盒、丙二醛(MDA)含量检测试剂盒、活性氧(ROS)荧光探针(DCFH-DA)及超氧化物阴离子荧光探针(DHE)分别检测对照组和400μmol/L MEHP处理组细胞的铁含量、MDA、ROS和超氧化物阴离子水平。结果(1)光镜观察显示,400μmol/L及以上MEHP处理使KGN细胞皱缩变圆,形态异常。(2)CCK-8结果显示400μmol/L及以上MEHP处理抑制细胞增殖,且具有剂量依赖性(P<0.05)。(3)100、200μmol/L MEHP处理组KGN细胞铁死亡相关蛋白表达无显著改变(P>0.05);400μmol/L MEHP处理组KGN中ACSL4蛋白表达水平显著增加(P<0.05),而GPX4、SLC7A11、FTH1蛋白表达水平显著下降(P<0.05)。(4)400μmol/L MEHP处理使得KGN细胞的铁含量、MDA水平显著上升(P<0.05),细胞ROS、超氧化物阴离子荧光染色强度明显增加。结论MEHP诱导KGN细胞铁死亡,抑制细胞增殖能力,但MEHP诱导铁死亡的具体作用机制还需进一步探讨。

Objective:To investigate whether phthalic acid mono-2-ethylhexyl ester(MEHP)induces ferroptosis in human ovarian granulosa cell line KGN cells and affects their proliferation.Methods:KGN cells at logarithmic growth stage were treated with different concentrations(0,100,200,400,800,1600μmol/L)of MEHP for 24 hours.The morphological changes of KGN cells treated with different concentrations of MEHP were observed by ordinary light microscopy.The effects of MEHP on cell viability and proliferation ability were detected by CCK-8.Western-blot was used to observe the expression of ferroptosis-related proteins(ACSL4,GPX4,SLC7A11,FTH1)in KGN cells.The iron content,malondialdehyde(MDA),reactive oxygen species(ROS)and superoxide anion levels in control and 400μmol/L MEHP-treated cells were measured by using the Cellular Iron Content Assay Kit,MDA Content Assay Kit,ROS Assay Kit and DHE,respectively.Results:Light microscopy showed that≥400μmol/L MEHP caused KGN cells to crumple and round,with abnormal morphology.CCK-8 results showed that≥400μmol/L MEHP inhibited cell proliferation in a dose-dependent manner(P<0.05).The expression of ferroptosis-related proteins in KGN cells treated with 100μmol/L and 200μmol/L MEHP lacked significantly change(P>0.05).KGN cells treated with 400μmol/L MEHP group significantly increased ACSL4 protein expression level(P<0.05),while protein expression levels of GPX4,SLC7A11 and FTH1 were significantly decreased(P<0.05).KGN cells treated with 400μmol/L MEHP caused iron content&MDA level significant increased(P<0.05),and the fluorescence staining intensity of ROS and superoxide anion in cells was evidently increased.Conclusions:MEHP induces ferroptosis in KGN cells and inhibits cell proliferation ability.However,the specific mechanisms of MEHP-induced ferroptosis need to be further explored.