摘要
目的:探讨PM2.5对人角膜上皮细胞屏障功能、氧化应激反应以及自噬的影响及其分子机制.方法:以不同浓度PM2.5处理人角膜上皮细胞(HCEC)24h,采用CCK8检测细胞增殖,荧光标记法检测细胞内活性氧(ROS)水平,ELISA试剂盒检测IL-6、IL-8和TNF-α的含量;采用跨膜电阻实验(TEER)检测细胞的TEER变化,Western blot检测Beclin1、LC3 Ⅱ/Ⅰ、Claudin1、Oc-cludin和ZO-1蛋白表达水平.再将HCEC细胞分为对照组、PM2.5组、NAC组和PM2.5+NAC组,所有组细胞培养24 h,后续进行细胞增殖水平、ROS、炎症因子、跨膜电阻值和Western blot检测.结果:与0 µg/mL比较,40 μg/mL、80 μg/mL、160 µg/mL和200 µg/mL的PM2.5处理的HCEC细胞相对增殖水平、TEER值和Claudin1、ZO-1和Occludin水平均降低(P＜0.05),上清液中IL-6、IL-8和TNF-α水平均升高(P＜0.05),HCEC细胞内ROS水平、Beclin-1和LC3 Ⅱ/Ⅰ蛋白水平均升高(P＜0.05).与PM2.5组比较,PM2.5+NAC组HCEC细胞相对增殖水平、TEER值和Claudin1、ZO-1和Occludin水平均升高(P＜0.05),上清液中IL-6、IL-8和TNF-α水平均降低(P＜0.05),HCEC细胞内ROS水平、Beclin-1和LC3 Ⅱ/Ⅰ蛋白水平均降低(P＜0.05).与NAC组比较,PM2.5+NAC组HCEC细胞相对增殖水平、TEER值和Claudin1、ZO-1和Occludin水平均降低(P＜0.05),上清液中IL-6、IL-8和TNF-α水平均升高(P＜0.05),HCEC细胞内ROS相对水平、Beclin-1和LC3 Ⅱ/Ⅰ蛋白水平均升高(P＜0.05).结论:PM2.5通过诱导炎症和氧化应激反应的产生,破坏人角膜上皮细胞的屏障功能,并促进细胞自噬.
Abstract
Objective:To investigate the effects of PM2.5 on barrier function,oxidative stress response and autophagy of human corneal epithelial cell and its molecular mechanism.Methods:Human corneal epithelial cells(HCEC)were treated with different concen-trations of PM2.5 for 24 h.The cell proliferation level was detected by CCK8.Intracellular reactive oxygen species(ROS)were detected by fluorescent labeling.The levels of IL-6,IL-8 and TNF-α were detected by ELISA kit.The changes of TEER in HCEC cells were de-tected by transmembrane resistance assay(TEER).The protein expression levels of Beclin1,LC3 Ⅱ/Ⅰ,Claudin1,Occludin and ZO-1 were detected by Western blot.Then HCEC cells were divided into control group,PM2.5 group,NAC group and PM2.5+NAC group,and the cells of all groups were cultured for 24 h.Cell proliferation level,ROS,inflammatory factors,transmembrane resistance and Western blot were subsequently detected.Results:Compared with 0 μg/mL,the relative proliferation level,TEER value,and the levels of Claudin1,ZO-1 and Occludin protein of HCEC cells treated with 40 µg/mL,80 µg/mL,160 μg/mL and 200 µg/mL PM2.5 were de-creased(P＜0.05),and the levels of IL-6,IL-8 and TNF-α in supernatant were increased(P＜0.05).The level of ROS and the protein levels of Beclin-1 and LC3 Ⅱ/Ⅰ in HCEC cells were increased(P＜0.05).Compared with the PM2.5 group,the relative proliferation level,TEER value,and the levels of Claudin1,ZO-1 and Occludin protein of HCEC cells in PM2.5+NAC group was increased(P＜0.05),the levels of IL-6,IL-8 and TNF-α in supernatant were decreased(P＜0.05),and the level of ROS and the protein levels of Beclin-1 and LC3Ⅱ/Ⅰ in HCEC cells were decreased(P＜0.05).Compared with the NAC group,the relative proliferation level,TEER value,and the lev-els of Claudin1,ZO-1 and Occludin protein of HCEC cells in PM2.5+NAC group was decreased(P＜0.05),the levels of IL-6,IL-8 and TNF-α in supernatant were increased(P＜0.05),and the level of ROS and the protein levels of Beclin-1 and LC3 Ⅱ/Ⅰ in HCEC cells were increased(P＜0.05).Conclusions:PM2.5 damages the barrier function of human corneal epithelial cells and promotes autophagy by inducing inflammation and oxidative stress responses.
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