Division of Allergy, Asthma, and Rheumatology, Department of Pediatrics, Chang Gung Memorial Hospital, 5 Fu-Hsin Street, Kweishan, Taoyuan 33305, Taiwan.
School of Medicine, Chang Gung University College of Medicine, Taoyuan, Taiwan; Department of Pediatrics, New Taipei Municipal TuCheng Hospital, New Taipei, Taiwan; Community Medicine Research Center, Chang Gung Memorial Hospital at Keelung, Keelung, Taiwan.
Ecotoxicol Environ Saf. 2021 Oct 1;222:112484. doi: 10.1016/j.ecoenv.2021.112484. Epub 2021 Jul 6.
Exposure to particulate matter (PM) has been associated with DNA damage, but the relationships between PM, telomere length and cellular senescence remain unclear. This study aimed to investigate the effects and potential mechanisms of PM on telomere length and cellular senescence in human lung epithelial cells. Human lung epithelial A549 cells were exposed to PM for 24 h. Cell viability and cytotoxicity were measured by the WST-1 assay and the lactate dehydrogenase release, respectively. Cellular uptake of PM was observed using transmission electron microscopy. Telomere length was measured using qPCR and expressed as T/S ratio. Cell cycle progression was analyzed by flow cytometry. Expression of human telomerase reverse transcriptase (hTERT) and cell cycle regulators was measured using mRNA by qPCR and protein levels by Western blot. Cellular senescence was determined by the expression of senescence-associated β-galactosidase (SA-β-Gal) with fluorescent microscopy and flow cytometry. Exposed to PM at the concentration of 200 μg/ml decreased cell viability and increased LDH levels in culture medium. Remarkably increased uptake of PM, shortening of telomere length, induction of G0/G1 phase arrest, and increased expression of senescence hallmarks were observed after exposure to PM in A549 cells. PM exposure induced upregulation of p21 and downregulation of proliferating cell nuclear antigen (PCNA) and hTERT expression, but no significant change in p53 expression, in A549 cells. Overall, exposure to PM may downregulate hTERT and PCNA through p53-independent induction of p21 expression, leading to telomere shortening, G0/G1 arrest and the onset of cellular senescence in human lung epithelial cells.
暴露于颗粒物（PM）与 DNA 损伤有关，但 PM、端粒长度和细胞衰老之间的关系尚不清楚。本研究旨在探讨 PM 对人肺上皮细胞端粒长度和细胞衰老的影响及其潜在机制。将人肺上皮 A549 细胞暴露于 PM 中 24 小时。通过 WST-1 测定法和乳酸脱氢酶释放分别测量细胞活力和细胞毒性。使用透射电子显微镜观察 PM 的细胞摄取。使用 qPCR 测量端粒长度，并表示为 T/S 比。通过流式细胞术分析细胞周期进程。通过 qPCR 测量人类端粒酶逆转录酶（hTERT）和细胞周期调节剂的 mRNA 表达，并通过 Western blot 测量蛋白质水平。通过荧光显微镜和流式细胞术确定衰老相关β-半乳糖苷酶（SA-β-Gal）的表达来确定细胞衰老。在 200μg/ml 的 PM 浓度下暴露会降低细胞活力并增加培养基中的 LDH 水平。在 A549 细胞中暴露于 PM 后，观察到 PM 显著增加摄取，端粒长度缩短，G0/G1 期阻滞诱导以及衰老标志物的表达增加。PM 暴露诱导 A549 细胞中 p21 的上调和增殖细胞核抗原（PCNA）和 hTERT 表达的下调，但 p53 表达无明显变化。总体而言，PM 暴露可能通过 p53 非依赖性诱导 p21 表达下调 hTERT 和 PCNA，导致人肺上皮细胞中端粒缩短、G0/G1 期阻滞和细胞衰老的发生。
