邻苯二甲酸二-2-乙基己酯通过ROS/PTEN/PI3K/AKT轴诱导HD11细胞凋亡和程序性坏死

Di(2-ethylhexyl)Phthalate Induces the Apoptosis and Necroptosis of HD11 Cells through the ROS/PTEN/PI3K/AKT Axis

旨在探究邻苯二甲酸二-2-乙基己酯[di(2-ethylhexyl)phthalate,(DEHP)]暴露通过ROS/PTEN/PI3K/AKT通路诱导细胞凋亡和程序性坏死的作用机制。本试验以HD11细胞为研究对象,设置对照组(C组)、30μmol·L^(-1)DEHP组(L组)、60μmol·L^(-1)DEHP组(M组)和90μmol·L^(-1)DEHP组(H组)。用不同浓度的DEHP处理HD11细胞24 h后,采用CCK-8检测细胞存活情况,生化试剂盒检测氧化应激指标,包括活性氧(ROS)水平,丙二醛(MDA)含量与总抗氧化能力(T-AOC)水平,以及总超氧化物歧化酶(T-SOD)和谷胱甘肽过氧化物酶(GSH-PX)活性。采用AO/EB染色和流式细胞术检测凋亡率和坏死率,荧光定量RT-PCR和Western blot法检测PTEN/PI3K/AKT通路基因及凋亡与坏死相关基因的mRNA和蛋白表达,结果表明,与对照组相比,DEHP暴露可显著抑制HD11细胞的活力,且半数抑制浓度(IC_(50))为180.644μmol·L^(-1)。与对照组相比,L、M、H DEHP组AO/EB染色和流式细胞术结果显示DEHP暴露导致HD11细胞具有典型的凋亡和坏死特征。与对照组相比,L、M、H DEHP组ROS水平呈现上升趋势(P<0.01),L、M、H DEHP组MDA含量显著升高(P<0.01),而T-AOC水平显著降低(P<0.01),T-SOD和GSH-PX活性显著降低(P<0.01),提示DEHP暴露诱导HD11细胞发生氧化应激损伤。此外,与对照组相比,DEHP暴露上调了PTEN/Bax/Caspase-3/Caspase-9/RIPK1/RIPK3/MLKL的mRNA和蛋白表达(P<0.01),而PI3K/AKT/BCL-2的mRNA和蛋白表达显著降低(P<0.01),提示DEHP暴露通过PTEN/PI3K/AKT信号通路诱导HD11细胞凋亡和程序性坏死。综上所述,DEHP可以通过调控ROS/PTEN/PI3K/AKT轴诱导HD11细胞凋亡和程序性坏死,并存在明显的剂量-效应关系。

To explore the mechanism of apoptosis and necroptosis induced by di(2-ethylhexyl)phthalate(DEHP)exposure through ROS/PTEN/PI3K/AKT pathway.HD11 cells were used in this study,and control group(C group),30μmol·L^(-1)DEHP group(L group),60μmol·L^(-1)DE-HP group(M group),90μmol·L^(-1)DEHP group(H group)were set.After cells were treated with different concentrations of DEHP for 24 h,cell survival was detected by CCK-8,oxidative stress was detected by biochemical kit,included reactive oxygen species(ROS)levels,malondial-dehyde(MDA)content,total antioxidant capacity(T-AOC)levels,and total superoxide dis-mutase(T-SOD)and glutathione peroxidase(GSH-PX)activities.AO/EB staining and flow cy-tometry were used to detect the rate of apoptosis and necrosis,mRNA and protein expressions of PTEN/PI3K/AKT pathway genes and apoptosis and necroptosis related genes were detected by qRT-PCR and Western blot.The results showed that,compared with the control group,DEHP considerably reduced the viability of HD11 cells,with the median inhibitory concentration(IC_(50))being 180.644μmol·L^(-1).Besides,compared with the control group,AO/EB staining and flow cytometry validated the L,M and H DEHP group with typical characteristics of apoptosis and ne-croptosis.Additionally,compared with the control group,ROS expression levels in groups L,M and H showed an increasing trend(P<0.01),and the content of MDA were considerably in-creased(P<0.01),the T-AOC level was significantly decreased(P<0.01),and the activities of T-SOD and GSH-PX were significantly decreased(P<0.01).It was suggested that DEHP ex-posure induced oxidative stress injury of HD11 cells.In addition,compared with the control group,DEHP exposure up-regulated the mRNA and protein expression levels of PTEN/Bax/Caspase-9/Caspase-3/RIPK1/RIPK3/MLKL and down-regulated the mRNA and protein expres-sion levels of PI3K/AKT/BCL-2(P<0.01),these results suggested that DEHP exposure in-duced apoptosis and necroptosis of HD11 cells through the PTEN/PI3K/AKT signaling path-way.Overall,DEHP can induce apoptosis and necroptosis in HD11 cells by modulating the ROS/PTEN/PI3K/AKT axis,and there is a dose-effect manner.