Key Laboratory of Environmental Medicine Engineering of Ministry of Education, Southeast University, School of Public Health, Nanjing, Jiangsu 210009, China.
Key Laboratory of Environmental Medicine Engineering of Ministry of Education, Southeast University, School of Public Health, Nanjing, Jiangsu 210009, China.
Sci Total Environ. 2021 Mar 20;761:143290. doi: 10.1016/j.scitotenv.2020.143290. Epub 2020 Nov 2.
Autophagy is a dynamic process for waste disposal and cell equilibrium. Previous studies have demonstrated that atmospheric particulate matter (APM) induces autophagy and enhances LC3II expression in human vascular endothelial cells. However, the underlying mechanism of autophagosome accumulation in human vascular endothelial cells under the exposure to APM has not been understood. In principle, the upregulation of LC3II or autophagosomes accumulation is presumably caused by the enhancement of autophagic ability, or alternatively, by the abnormal autophagic degradation. Therefore, in the current study, autophagic ability and autophagic flux are systemically studied to decipher the exact cause of autophagosomes accumulation in human umbilical vein endothelial cells (HUVECs) in response to a standard urban particulate matter, PM SRM1648a. As a result, it was observed that after 24 h of exposure, PM SRM1648a significantly increases LC3II expression with apparent autophagosomes accumulation in HUVECs. Compared with the control group, there is a time-dependent increase in p62, a protein of autophagic substrate that can be preliminarily used to evaluate the autophagic degradation, in the PM SRM1648a-exposed HUVECs, which suggested that normal function of autophagic degradation was probably impaired. Additionally, mRFP-GFP-LC3 assay and LAMP-2/LC3B co-localization suggested that autolysosomes (fusion between autophagosomes and lysosomes) were partially inhibited in PM SRM1648a-treated HUVECs. Furthermore, LC3II turn-over assay hinted that after 24 h, LC3II upregulation is attributed to the blockage of autophagic flux instead of the enhancement of autophagic induction. Mechanistically, the blockade of autophagic flux can be explained by the detrimental effects of PM SRM1648a on lysosomal function, including lysosomal destabilization, lysosomal alkalization and hydrolase inactivation, which are involved in the blockade of fusion between autophagosomes and lysosomes, further disrupting autophagic degradation and waste disposal. These observations provide evidence that PM SRM1648a destroys the equilibrium of lysosomal stability and thus results in the dysfunction of autophagic flux, eventually contributing to endothelial cell damage.
自噬是一种用于废物处理和细胞平衡的动态过程。先前的研究表明，大气颗粒物（APM）会诱导人血管内皮细胞中的自噬，并增强 LC3II 的表达。然而，APM 暴露下人血管内皮细胞中自噬体积累的潜在机制尚不清楚。原则上，LC3II 的上调或自噬体积累的增加可能是由于自噬能力的增强，或者是由于异常的自噬降解。因此，在本研究中，我们系统地研究了自噬能力和自噬流，以解析人脐静脉内皮细胞（HUVEC）中自噬体积累的确切原因，以应对标准城市颗粒物 PM SRM1648a。结果表明，暴露 24 小时后，PM SRM1648a 可显著增加 LC3II 的表达，并在 HUVEC 中观察到明显的自噬体积累。与对照组相比，PM SRM1648a 暴露的 HUVEC 中自噬底物 p62 的表达呈时间依赖性增加，p62 可初步用于评估自噬降解，这表明自噬降解的正常功能可能受损。此外，mRFP-GFP-LC3 测定和 LAMP-2/LC3B 共定位表明，PM SRM1648a 处理的 HUVEC 中自溶酶体（自噬体与溶酶体融合）部分受到抑制。此外，LC3II 周转率测定表明，暴露 24 小时后，LC3II 的上调归因于自噬流的阻断，而不是自噬诱导的增强。从机制上讲，自噬流的阻断可以用 PM SRM1648a 对溶酶体功能的有害影响来解释，包括溶酶体不稳定、溶酶体碱化和水解酶失活，这些都参与了自噬体与溶酶体融合的阻断，进一步破坏了自噬降解和废物处理。这些观察结果提供了证据表明，PM SRM1648a 破坏了溶酶体稳定性的平衡，从而导致自噬流的功能障碍，最终导致内皮细胞损伤。
