Department of Pediatrics, The Third Xiangya Hospital of Central South University, Changsha, People's Republic of China.
Department of Neurology, The Third Xiangya Hospital of Central South University, Changsha, People's Republic of China.
Environ Toxicol. 2019 Jun;34(6):699-707. doi: 10.1002/tox.22736. Epub 2019 Mar 5.
The insecticide exposure has been linked to Parkinson's disease (PD). In the present study, we used a most widely used cell line in study of PD, the SH-SY5Y cells, to investigate mechanisms of chlorpyrifos (CPF) induced cell toxicity and the possible roles of cell pyroptosis and oxidative stress in SH-SY5Y cells, as well as role of miR-181/SIRT1/PGC-1α/Nrf2 signaling pathway in this process.
SH-SY5Y cells were treated with different concentrations of CPF. Cell viability was measured using CCK-8 assay. Cell pyroptosis was determined by immunofluorescence of caspase-1 and TUNEL assay. The miR-181 (has-miR-181-5p) level was determined by qRT-PCR. Expression of SIRT1, PGC-1α, Nrf2, and pyroptosis related proteins NLRP3, caspase-1, IL-1β, and IL-18 was determined by both qRT-PCR and Western blotting.
Cell viability was found to be decreased with the increased CPF concentrations. The pyroptosis related proteins, ROS levels, as well as level of caspase-1 and the TUNEL positive cells were all significantly up-regulated by CPF. Meanwhile, expression of miR-181 and pyroptosis proteins was also enhanced, while the SIRT1/PGC-1α/Nrf2 signaling was inhibited by CPF. Knockdown of Nrf2 significantly up-regulated the expression of pyroptosis related proteins, ROS level, caspase-1, and the TUNEL positive cells, while over-expression of Nrf2 resulted in opposite results. The expression of PGC-1α and Nrf2 was significantly down-regulated when SIRT1 was inhibited, while over-expressed SIRT1 led to increased PGC-1α and Nrf2 levels. Besides, miR-181 promoted the CPF induced activation of pyroptosis and oxidative stress, as well as down-regulated SIRT1/PGC-1α/Nrf2 signaling, while inhibition of miR-181 led to opposite results.
Chlorpyrifos could inhibit cell proliferation, activate cell pyroptosis and increase susceptibility on oxidative stress-induced toxicity by elevating miR-181 through down-regulation of the SIRT1/PGC-1α/Nrf2 pathway in human neuroblastoma SH-SY5Y cells. This study might give deeper insights for mechanisms of CPF induced toxicity and might give some novel research targets for PD treatment.
杀虫剂的接触已与帕金森病（PD）有关。在本研究中，我们使用了研究 PD 中最常用的细胞系，即 SH-SY5Y 细胞，以研究毒死蜱（CPF）诱导的细胞毒性的机制，以及细胞焦亡和氧化应激在 SH-SY5Y 细胞中的可能作用，以及 miR-181/SIRT1/PGC-1α/Nrf2 信号通路在此过程中的作用。
用不同浓度的 CPF 处理 SH-SY5Y 细胞。用 CCK-8 法测定细胞活力。用 caspase-1 的免疫荧光和 TUNEL 测定法测定细胞焦亡。用 qRT-PCR 测定 miR-181（has-miR-181-5p）水平。用 qRT-PCR 和 Western blot 测定 SIRT1、PGC-1α、Nrf2 和焦亡相关蛋白 NLRP3、caspase-1、IL-1β 和 IL-18 的表达。
发现细胞活力随 CPF 浓度的增加而降低。CPF 显著上调焦亡相关蛋白、ROS 水平以及 caspase-1 和 TUNEL 阳性细胞数。同时，miR-181 和焦亡蛋白的表达也增强，而 CPF 抑制了 SIRT1/PGC-1α/Nrf2 信号通路。Nrf2 敲低显著上调焦亡相关蛋白、ROS 水平、caspase-1 和 TUNEL 阳性细胞数，而过表达 Nrf2 则产生相反的结果。当 SIRT1 被抑制时，PGC-1α 和 Nrf2 的表达显著下调，而过表达 SIRT1 则导致 PGC-1α 和 Nrf2 水平升高。此外，miR-181 促进 CPF 诱导的细胞焦亡和氧化应激的激活，并下调 SIRT1/PGC-1α/Nrf2 信号通路，而抑制 miR-181 则产生相反的结果。
毒死蜱可能通过下调 SIRT1/PGC-1α/Nrf2 通路，通过上调 miR-181，抑制人神经母细胞瘤 SH-SY5Y 细胞的增殖，激活细胞焦亡并增加氧化应激诱导的毒性易感性。本研究可能为 CPF 诱导的毒性机制提供更深入的了解，并为 PD 治疗提供新的研究靶点。
