烟草烟雾暴露的人支气管上皮细胞铁死亡情况观察及其机制

Effect of cigarette smoke exposure on ferroptosis of human bronchial epithelial cells

目的观察烟草烟雾暴露对体外培养的人支气管上皮细胞铁死亡的影响并分析其机制。方法正常人支气管上皮细胞BEAS-2B分别使用0、0.5%、1.0%和2.0%的烟草烟雾提取物(CSE)处理24 h,即为对照组、0.5%CSE组、1.0%CSE组及2.0%CSE组。采用EdU染色观察细胞增殖能力,CCK-8法观察细胞活性;比色法检测细胞Fe2+水平和还原型谷胱甘肽(GSH)水平;流式细胞术观察细胞活性氧(ROS)水平;q-PCR法及Western blotting法观察铁死亡相关因子谷胱甘肽过氧化酶4(GPx4)、铁蛋白重链1(FTH1)及环加氧酶2(Ptgs2)mRNA及蛋白;碘化丙啶染色观察细胞死亡情况;透射电镜观察细胞线粒体结构。将BEAS-2B细胞分为Fer-1组、Nec-1组、Z-VAD-FMK组及CSE组,分别给予铁死亡抑制剂Fer-1、坏死抑制剂Nec-1、凋亡抑制剂Z-VAD-FMK及不加试剂预处理后,加入2.0%CSE培养基处理24 h。采用CCK-8法观察各组细胞活性;比色法检测Fer-1组、CSE组细胞Fe2+及GSH水平,流式细胞术观察Fer-1组、CSE组细胞ROS水平;q-PCR法及Western blotting法观察Fer-1组、CSE组细胞FTH1、Ptgs2、GPx4mRNA及蛋白表达;碘化丙啶染色观察Fer-1组、CSE组细胞死亡情况。结果EdU阳性细胞数、细胞OD值对照组>0.5%CSE组>1.0%CSE组>2.0%CSE组;细胞Fe2+水平、ROS水平对照组<0.5%CSE组<1.0%CSE组<2.0%CSE组,GSH水平对照组>0.5%CSE组>1.0%CSE组>2.0%CSE组;细胞FTH1、GPx4 mRNA及蛋白对照组>0.5%CSE组>1.0%CSE组>2.0%CSE组,Ptgs2 mRNA及蛋白对照组<0.5%CSE组<1.0%CSE组<2.0%CSE组;细胞死亡率对照组<0.5%CSE组<1.0%CSE组<2.0%CSE组(P均<0.05)。透射电镜结果显示,BEAS-2B细胞经2.0%CSE处理后出现线粒体萎缩变小,线粒体双层膜密度增高,线粒体脊减少甚至消失等典型铁死亡线粒体超微结构改变。细胞OD值Fer-1组>Nec-1组、Z-VAD-FMK组、CSE组;Fer-1组细胞Fe^(2+)、ROS水平低于CSE组,GSH水平高于CSE组;Fer-1组细胞FTH1、GPx4 mRNA及蛋白高于CSE组,Ptgs2 mRNA及蛋白低于CSE组;细胞死亡率Fer-1组低于CSE组(P均<0.05)。结论烟草烟雾暴露可诱导BEAS-2B细胞发生铁死亡,该作用可能通过介导BEAS-2B细胞铁超载和氧化还原紊乱来实现。

Objective To observe the effect of cigarette smoke exposure on ferroptosis of human bronchial epithelial cells in vitro and to investigate its mechanism.Methods BEAS-2B cells were divided into 0%,0.5%,1.0%or 2.0% CSE groups,which were treated with 0%,0.5%,1.0% or 2.0% CSE(dissolved in DMEM)media for 24 h,respectively.CCK-8 assay was used to observe cell viability;EdU staining was used to detect cell proliferation ability;Fe2+and GSH levels were observed by colorimetric method;flow cytometry was used to detect ROS level;the mRNA and protein expression levels of ferroptosis-related glutathione peroxidase 4(GPx4),ferritin heavy chain 1(FTH1)and prostaglandinendoperoxide synthase 2(Ptgs2)were detected by q-PCR and Western blotting;necrotic cell death rate was indicated by PI staining;and the structure of mitochondria was observed by transmission electron microscope.Next,BEAS-2B cells were divided into the Fer-1,Nec-1,Z-VAD-FMK and 2% CSE groups,which were treated with 2% CSE in the presence or absence of Fer-1,Nec-1 or Z-VAD-FMK;cell viability was determined by CCK-8 assay;Fe2+and GSH levels were observed by colorimetric method;flow cytometry was used to detect ROS level;the expression levels of ferroptosis-related mRNAs and proteins GPx4,FTH1 and Ptgs2 were detected by q-PCR and Western blotting;and necrotic cell death rate was indicated by PI staining.Results EdU positive cells and OD values in different concentrations of CSE groups were lower than those in the control group,which in the 1.0% or 2.0% CSE groups were lower than those in the 0.5% CSE group,and those in the 2.0% CSE group were lower than those in the 1.0% CSE group(all P<0.05).Fe^(2+)and ROS levels in the 0.5%,1.0% or 2.0% CSE groups were higher than those in the control group,which in the 1.0% or 2.0% CSE groups were higher than those in the 0.5% CSE group,and those in the 2.0% CSE group were higher than those in the 1.0% CSE group(all P<0.05).GSH levels in different concentrations of CSE groups were lower than that in the control group,which in the 1.0% or 2.0% CSE groups were lower than that in the 0.5% CSE group,and that in the 2.0% CSE group was lower than that in the 1.0% CSE group(all P<0.05);the mRNA and protein expression levels of FTH1,GPx4in different concentrations of CSE groups were lower than those in the control group,which in the 1.0% or 2.0% CSE groups were lower than those in the 0.5% CSE group,and those in the 2.0% CSE group were lower than those in the 1.0% CSE group(all P<0.05);the mRNA and protein expression levels of Ptgs2 in the 0.5%,1.0% or 2.0% CSE groups were higher than those in the control group,which in the 1.0% or 2.0% CSE groups were higher than those in the 0.5% CSE group,and those in the 2.0% CSE group were higher than those in the 1.0% CSE group(all P<0.05);necrotic cell death rates in the 0.5%,1.0% or 2.0% CSE groups were higher than that in the control group,which in the 1.0% or 2.0% CSE groups was higher than that in the 0.5% CSE group,and that in the 2.0% CSE group was higher than that in the 1.0% CSE group(all P<0.05);transmission electron microscopy showed that BEAS-2B cells treated with 2.0% CSE had typical ultrastructural characteristic changes in mitochondria,including mitochondrial shrinkage,increased bilayer membrane density,reduced or even removed mitochondrial cristae.OD value in Fer-1 group was higher than those in Nec-1,Z-VADFMK and 2% CSE groups;cellular Fe2+and ROS levels in Fer-1 group were lower than those in 2% CSE group;GSH level in Fer-1 group was higher than that in 2% CSE group;the mRNA and protein expression levels of FTH1,GPx4 in Fer-1group were higher than those in 2% CSE group,however,the mRNA and protein expression levels of Ptgs2 in Fer-1 group were lower than those in 2% CSE group;cell death rate in Fer-1 group was lower than that in 2% CSE group(all P<0.05).Conclusion Cigarette smoke exposure can induce the ferroptosis of BEAS-2B cells,which might be mediated by iron overload and redox disorder in BEAS-2B cells.