PM2.5对角质形成细胞增殖、凋亡及对CCL20和核转录因子-κB表达的影响

Effects of PM2.5 on proliferation and apoptosis of keratinocytes and expression of CCL20 and NF-κB

目的探讨大气颗粒物(PM2.5)对皮肤角质形成细胞的生物学效应。方法角质形成细胞株(HaCaT细胞)暴露于不同浓度PM2.5混悬液(0~400μg/ml)24 h,采用CCK-8法检测不同浓度及不同暴露时间(1~24 h)的细胞存活率;Annexin V法检测细胞凋亡率;实时荧光定量聚合酶链反应(RT-PCR)、酶联免疫吸附试验(ELISA)分别检测不同浓度处理组细胞内细胞因子CCL20 mRNA的表达水平及CCL20分泌浓度;免疫印迹试验(Western blot)检测细胞中核转录因子(NF)-κB信号通路相关蛋白的表达水平。结果与对照组(0μg/ml)相比,当PM2.5浓度≥50μg/ml或刺激时间≥3 h时,各组细胞存活率均显著降低(P<0.05);随着PM2.5刺激浓度增高(PM2.5浓度≥50μg/ml),各组细胞凋亡率均显著增高(P<0.01),细胞中炎性因子CCL20的m RNA表达水平显著上调(P<0.05);随着PM2.5刺激浓度增高(PM2.5浓度≥25μg/ml),CCL20分泌水平显著上调(P<0.05);Western blot检测显示细胞中NF-κB信号通路关键蛋白p65的磷酸化水平表达上调。结论PM2.5可降低角质形成细胞存活率,促进细胞凋亡,且可能通过激活NF-κB信号通路诱导角质形成细胞内细胞因子CCL20的表达上调,进而介导多种慢性皮肤炎症反应。

Objective To investigate the biological effects of atmospheric particulate matter(PM)2.5 on keratinocytes.Methods HaCaT cells were exposed to PM2.5 suspension of different concentrations(0~400μg/ml)for 24 h.Cell viability at different concentrations and different exposure times(1~24 h)was measured by CCK-8 method,and cell apoptosis was detected by Annexin V.Real time-PCR and ELISA were used to detect the CCL20 mRNA expression and secretion levels of CCL20.The expression levels of NF-κB signaling pathway-related proteins were detected by Western blot.Results Compared with the control group(0μg/ml),when PM2.5 concentration exceeded 50μg/ml or stimulation time exceeded 3 h,the cell viability of each group decreased significantly(P<0.05).When PM2.5 concentration exceeded 50μg/ml,the cell apoptosis rate of each group increased significantly(P<0.01)and the expression levels of CCL20 mRNA increased significantly(P<0.05).When PM2.5 concentration exceeded 25μg/ml,the secretion levels of CCL20 were significantly increased(P<0.05)than those in the control group.The result of Western blot showed that with the increase of PM2.5 concentration,the level of phosphor-p65 protein which is the key protein in the NF-κB signaling pathway was up-regulated.Conlusions PM2.5 can decrease the cell viability of keratinocytes and promote cell apoptosis,and may induce the up-regulation of CCL20 in keratinocytes by activating NF-κB signaling pathway,and then mediates a variety of chronic skin inflammatory reactions.