Cypermethrin-activated P2X7 induces mouse TM4 cell pyroptosis through the NLRP3/Caspase-1/GSDMD pathway

PURPOSE: Cypermethrin (CYP), a common pyrethroid insecticide, has potential male reproductive toxicity, but its mechanism in mouse testicular Sertoli cells (TM4, key for spermatogenesis) remains unclear. Thus, this study aimed to explore CYP-induced pyroptosis in TM4 cells and clarify if CYP acts via activating the purinergic P2X7 receptor and regulating the NLRP3/Caspase-1/GSDMD signaling pathway. In vitro, a CYP-induced TM4 injury model was established, with multiple assays applied: CCK-8 for cell viability, LDH assay for membrane damage, NO assay for oxidative stress, TEM for cell ultrastructure, WB/PCR for expression of NLRP3, Caspase-1, ASC, GSDMD and P2X7 (protein/mRNA), immunofluorescence for Caspase-1/P2X7 localization, TUNEL for cell death, ELISA for IL-1β/IL-18, ATP kit for intracellular ATP, and PBFI-AM for intracellular K⁺. Additionally, NLRP3 inhibitor (MCC950) and P2X7 antagonist (A-740003) were separately added to verify their effects on CYP-induced pyroptosis and confirm the mechanism. RESULTS: Compared with the control group, TM4 cells exposed to 40 μM and 80 μM CYP showed reduced cell viability, increased LDH release, decreased NO release, and TUNEL results showed that with the increase of cypermethrin concentration, the number of positive cells gradually increased; meanwhile, the protein and mRNA expression of pyroptosis effector gene GSDMD, as well as that of pyroptosis-related genes NLRP3, Caspase-1 and ASC, were upregulated, accompanied by increased release of IL-1β and IL-18, enhanced ATP depletion, reduced intracellular K⁺ content, and increased protein and mRNA expression of P2X7 receptor. Notably, after treatment with NLRP3 inhibitor (MCC950) or purinergic P2X7 receptor antagonist (A-740003), the degree of cell pyroptosis was alleviated. CONCLUSION: Cypermethrin can induce pyroptosis in TM4 cells. Its mechanism may involve the activation of the P2X7 receptor and the induction of pyroptosis in TM4 cells through the upregulation of the NLRP3/Caspase-1/GSDMD pathway.