Fu Qiuli, Lyu Danni, Zhang Lifang, Qin Zhenwei, Tang Qiaomei, Yin Houfa, Lou Xiaoming, Chen Zhijian, Yao Ke
Eye Center of the 2nd Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang Province, China; Zhejiang Provincial Key Lab of Ophthalmology, Hangzhou, Zhejiang Province, China.
Department of Environmental and Occupational Health, Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou, Zhejiang Province, China.
Environ Pollut. 2017 Aug;227:314-322. doi: 10.1016/j.envpol.2017.04.078. Epub 2017 May 4.
To investigate particulate matter (PM2.5)-induced damage to human corneal epithelial cells (HCECs) and to determine the underlying mechanisms.
HCECs were exposed to PM2.5 at a series of concentrations for various periods. Cell viability was measured by using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell proliferation was evaluated via 5-ethynyl-2'-deoxyuridine (EdU) analysis, while autophagy was determined by immunofluorescence and Western blot.
PM2.5-induced cell damage of HCECs occurred in a time- and dose-dependent manner. Decreased cell viability and proliferation as well as increased apoptosis were observed in HCECs after PM2.5 exposure for 24 h. Autophagy in HCECs was slightly inhibited in the early stage (before 4 h) of exposure but significantly activated in the late stage (after 24 h), as evidenced by a decrease in the former and increase in the latter of the expression of the autophagy-associated markers LC3B, ATG5, and BECN1. Interestingly, rapamycin, an autophagy activator, attenuated early-stage but aggravated late-stage PM2.5-induced cell damage, suggesting that the role of autophagy in HCECs may change over time during PM2.5 exposure. In addition, in the early stage, the expression of LC3B and ATG5 increased in cells co-treated with rapamycin and PM2.5 compared to rapamycin-only or PM2.5-only treated cells, suggesting that autophagy may benefit cell viability after PM2.5 exposure.
The results indicate the potential role of autophagy in the treatment of PM2.5-induced ocular corneal diseases and provide direct evidence for the cytotoxicity, possibly involving an autophagic process, of PM2.5 in HCECs.
研究细颗粒物（PM2.5）对人角膜上皮细胞（HCECs）的损伤及其潜在机制。
将HCECs暴露于一系列浓度的PM2.5中不同时长。采用3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）法检测细胞活力。通过5-乙炔基-2'-脱氧尿苷（EdU）分析评估细胞增殖，同时通过免疫荧光和蛋白质免疫印迹法检测自噬情况。
PM2.5诱导的HCECs细胞损伤呈时间和剂量依赖性。PM2.5暴露24小时后，HCECs细胞活力和增殖能力下降，凋亡增加。HCECs的自噬在暴露早期（4小时之前）略有抑制，但在后期（24小时之后）显著激活，自噬相关标志物LC3B、ATG5和BECN1的表达前者降低、后者增加证明了这一点。有趣的是，自噬激活剂雷帕霉素减轻了早期但加重了后期PM2.5诱导的细胞损伤，这表明在PM2.5暴露期间，自噬在HCECs中的作用可能随时间而变化。此外，在早期，与仅用雷帕霉素或仅用PM2.5处理的细胞相比，雷帕霉素和PM2.5共同处理的细胞中LC3B和ATG5的表达增加，这表明自噬可能对PM2.5暴露后的细胞活力有益。
结果表明自噬在治疗PM2.5诱导的眼部角膜疾病中具有潜在作用，并为PM2.5对HCECs的细胞毒性（可能涉及自噬过程）提供了直接证据。
