BACKGROUND Parkinson's disease (PD) is characterized by the selective death of dopaminergic neurons in the substantia nigra. Recently, NLRP3 inflammasome and pyroptosis were found to be associated with PD. Cyclosporine A (CsA), an immunosuppressant, reduces neuronal death in PD. However, CsA could hardly pass through the blood-brain barrier (BBB) and high dose is associated with severe side effects and toxicity. N-methyl-4-isoleucine-cyclosporine (NIM811) is a CsA derivate that can pass through the BBB. However, little is known about its effect on PD. OBJECTIVE The objectives of this study were to explore the mechanism of rotenone-induced cell damage and to examine the protective effects of NIM811 on the neurotoxicity of a Parkinson-like in vitro model induced by rotenone. METHODS Murine hippocampal HT22 cells were cultured with the mitochondrial complex I inhibitor rotenone, a widely used pesticide that has been used for many years as a tool to induce a PD model in vitro and in vivo and proven to be reproducible. NIM811 was added to the culture media 3 h prior to the rotenone incubation. Cell viability was determined by resazurin assay, reactive oxygen species (ROS) production by dihydroethidine (DHE), and mitochondrial membrane potential by tetramethyl rhodamine methyl ester (TMRM). TUNEL and caspase-1 immunofluorescent double staining was used to detect pyroptosis. NLRP3, caspase-1, pro-caspase-1, GSDMD, and interleukin-18 (IL-18) were measured using Western blotting after 24 h of rotenone incubation. The reactivity of interleukin-1β (IL-1β) was determined by ELISA. RESULTS Our results demonstrated that rotenone caused more than 40% of cell death, increased ROS production, and reduced mitochondrial membrane potential, while NIM811 reversed these alterations. Immunofluorescent double staining showed that rotenone increased the percentage of caspase-1 and TUNEL double-labelled cells, an indication of pyroptosis, after 24 h of incubation. The protein expression of NLRP3, caspase-1, pro-caspase-1, GSDMD, IL-18, and IL-1β was significantly increased after 24 h of rotenone incubation. NIM811 suppressed rotenone-induced pyroptosis and downregulated the protein expression of NLRP3, caspase-1, pro-caspase-1, GSDMD, IL-1β, and IL-18. CONCLUSION These results provide evidence that rotenone activates the NLRP3 inflammomere and induces pyroptosis. NIM811 protects the cell from rotenone-induced damage and inhibits NLRP3 inflammasome and pyroptosis. NIM811 might serve as a potential therapeutic drug in the treatment of PD.

中文翻译：

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NIM811 在鱼藤酮暴露细胞中抑制 NLRP3 炎症小体、细胞焦亡和细胞死亡作为帕金森病的体外模型

背景帕金森病(PD)的特征在于黑质中多巴胺能神经元的选择性死亡。最近，发现 NLRP3 炎症小体和细胞焦亡与 PD 相关。环孢素 A (CsA) 是一种免疫抑制剂，可减少 PD 中的神经元死亡。然而，CsA 几乎不能通过血脑屏障 (BBB)，并且高剂量与严重的副作用和毒性有关。N-甲基-4-异亮氨酸-环孢菌素 (NIM811) 是一种 CsA 衍生物，可以通过 BBB。然而，关于它对 PD 的影响知之甚少。目的本研究的目的是探讨鱼藤酮诱导细胞损伤的机制，并检查NIM811对鱼藤酮诱导的帕金森样体外模型神经毒性的保护作用。方法 小鼠海马 HT22 细胞与线粒体复合物 I 抑制剂鱼藤酮一起培养，鱼藤酮是一种广泛使用的杀虫剂，多年来一直被用作在体外和体内诱导 PD 模型的工具，并被证明是可重复的。在鱼藤酮孵育前 3 小时将 NIM811 添加到培养基中。通过刃天青测定法、二氢乙啶 (DHE) 产生的活性氧 (ROS) 和四甲基罗丹明甲酯 (TMRM) 产生的线粒体膜电位来确定细胞活力。TUNEL和caspase-1免疫荧光双染色用于检测细胞焦亡。在鱼藤酮孵育 24 小时后，使用蛋白质印迹法测量 NLRP3、caspase-1、pro-caspase-1、GSDMD 和白细胞介素-18（IL-18）。白细胞介素-1β (IL-1β) 的反应性通过 ELISA 测定。结果 我们的结果表明，鱼藤酮导致超过 40% 的细胞死亡，增加 ROS 的产生，并降低线粒体膜电位，而 NIM811 逆转了这些改变。免疫荧光双染色显示，在孵育 24 小时后，鱼藤酮增加了 caspase-1 和 TUNEL 双标记细胞的百分比，这是细胞焦亡的迹象。鱼藤酮孵育 24 小时后，NLRP3、caspase-1、pro-caspase-1、GSDMD、IL-18 和 IL-1β 的蛋白表达显着增加。NIM811 抑制鱼藤酮诱导的细胞焦亡并下调 NLRP3、caspase-1、pro-caspase-1、GSDMD、IL-1β 和 IL-18 的蛋白质表达。结论这些结果提供了鱼藤酮激活NLRP3炎性粒并诱导细胞焦亡的证据。NIM811 保护细胞免受鱼藤酮诱导的损伤并抑制 NLRP3 炎症小体和细胞焦亡。NIM811 可能作为治疗 PD 的潜在治疗药物。