Suppression of NLRP3 Inflammasome, Pyroptosis, and Cell Death by NIM811 in Rotenone-Exposed Cells as an in vitro Model of Parkinson's Disease

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.