Department of Neurology, Ren Ji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Department of Neurology, Nanjing First Hospital, Nanjing Medical University, Nanjing, China.
CNS Neurosci Ther. 2022 Jul;28(7):1045-1058. doi: 10.1111/cns.13837. Epub 2022 Apr 10.
Accumulating evidence has suggested that airborne fine particulate matter (PM2.5) exposure is associated with an increased risk of ischemic stroke. However, the underlying mechanisms have not been fully elucidated. In this study, we aim to investigate the role and mechanisms of NLRP3 inflammasome and pyroptosis in ischemic stroke after PM2.5 exposure.
The BV-2 and HMC-3 microglial cell lines were established and subjected to oxygen-glucose deprivation and reoxygenation (OGD/R) with or without PM2.5 exposure. We used the CCK-8 assay to explore the effects of PM2.5 on cell viability of BV-2 and HMC-3 cells. Then, the effects of PM2.5 exposure on NLRP3 inflammasome and pyroptosis following OGD/R were detected by western blotting, ELISA, and the confocal immunofluorescence staining. Afterwards, NLRP3 was knocked down to further validate the effects of PM2.5 on cell viability, NLRP3 inflammasome activation, and pyroptosis after OGD/R in HMC-3 cells. Finally, the intracellular reactive oxygen species (ROS) was measured and the ROS inhibitor N-acetyl-L-cysteine (NAC) was used to investigate whether ROS was required for PM2.5-induced NLRP3 inflammasome activation and pyroptosis under ischemic conditions.
We found that PM2.5 exposure decreased the viability of BV-2 and HMC-3 cells in a dose- and time-dependent manner under ischemic conditions. Furthermore, PM2.5 exposure aggravated NLRP3 inflammasome activation and pyroptosis after OGD/R, as indicated by an increased expression of NLRP3, ASC, pro-caspase-1, Caspase-1, GSDMD, and GSDMD-N; increased production of IL-1β and IL-18; and enhanced Caspase-1 activity and SYTOX green uptake. However, shRNA NLRP3 treatment attenuated the effects of PM2.5 on cell viability, NLRP3 inflammasome activation, and pyroptosis. Moreover, we observed that PM2.5 exposure increased the production of intracellular ROS following OGD/R, while inhibiting ROS production with NAC partially attenuated PM2.5-induced NLRP3 inflammasome activation and pyroptosis under ischemic conditions.
These results suggested that PM2.5 exposure triggered the activation of NLRP3 inflammasome and pyroptosis under ischemic conditions, which may be mediated by increased ROS production after ischemic stroke. These findings may provide a more enhanced understanding of the interplay between PM2.5 and neuroinflammation and cell death, and reveal a novel mechanism of PM2.5-mediated toxic effects after ischemic stroke.
越来越多的证据表明，空气中的细颗粒物（PM2.5）暴露与缺血性中风的风险增加有关。然而，其潜在机制尚未完全阐明。在这项研究中，我们旨在探讨 NLRP3 炎性体和细胞焦亡在 PM2.5 暴露后缺血性中风中的作用和机制。
建立 BV-2 和 HMC-3 小胶质细胞系，并进行氧葡萄糖剥夺和复氧（OGD/R），同时或不进行 PM2.5 暴露。我们使用 CCK-8 测定法来研究 PM2.5 对 BV-2 和 HMC-3 细胞活力的影响。然后，通过 Western blot、ELISA 和共聚焦免疫荧光染色检测 PM2.5 暴露对 OGD/R 后 NLRP3 炎性体和细胞焦亡的影响。随后，敲低 NLRP3 以进一步验证 PM2.5 对 OGD/R 后 HMC-3 细胞活力、NLRP3 炎性体激活和细胞焦亡的影响。最后，测量细胞内活性氧（ROS），并使用 ROS 抑制剂 N-乙酰-L-半胱氨酸（NAC）来研究在缺血条件下 ROS 是否是 PM2.5 诱导的 NLRP3 炎性体激活和细胞焦亡所必需的。
我们发现，在缺血条件下，PM2.5 暴露以剂量和时间依赖的方式降低了 BV-2 和 HMC-3 细胞的活力。此外，PM2.5 暴露加重了 OGD/R 后 NLRP3 炎性体的激活和细胞焦亡，表现为 NLRP3、ASC、前胱天蛋白酶-1、胱天蛋白酶-1、GSDMD 和 GSDMD-N 的表达增加；IL-1β和 IL-18 的产生增加；以及 Caspase-1 活性和 SYTOX 绿色摄取增强。然而，shRNA NLRP3 处理减弱了 PM2.5 对细胞活力、NLRP3 炎性体激活和细胞焦亡的影响。此外，我们观察到 PM2.5 暴露在 OGD/R 后增加了细胞内 ROS 的产生，而用 NAC 抑制 ROS 产生部分减弱了缺血条件下 PM2.5 诱导的 NLRP3 炎性体激活和细胞焦亡。
这些结果表明，PM2.5 暴露在缺血条件下引发 NLRP3 炎性体和细胞焦亡的激活，这可能是缺血性中风后 ROS 产生增加所介导的。这些发现可能提供了对 PM2.5 与神经炎症和细胞死亡之间相互作用的更深入理解，并揭示了 PM2.5 介导的缺血性中风后毒性作用的新机制。
