TRPM4 modulates endothelial inflammation and pyroptosis via the HSP60-NF-κB axis

Background: Atherosclerosis is a chronic inflammatory condition of the arterial wall in which endothelial dysfunction serves as a key driver of disease progression. Endothelial inflammation and pyroptosis are major contributors in this context; therefore, targeting these processes may confer therapeutic benefits. Transient receptor potential cation channel subfamily M member 4 (TRPM4) is a voltage-sensitive, non-selective cation channel belonging to the transient receptor potential family. Although TRPM4 contributes to the regulation of vascular endothelium, its precise role in endothelial inflammation remains poorly understood. Accordingly, this study aims to elucidate the function and molecular mechanisms of TRPM4 in vascular endothelial inflammation and pyroptosis. Methods: model of endothelial inflammation and pyroptosis was established by stimulating human umbilical vein endothelial cells (HUVECs) with tumor necrosis factor-α (TNF-α). The TRPM4-specific inhibitor 9-Phenanthrol (9-Phe) was applied to assess TRPM4 involvement. Expression levels of TRPM4, inflammatory adhesion molecules (VCAM-1, ICAM-1), pyroptosis-related proteins (NLRP3, cleaved caspase-1, GSDMD, IL-1β, IL-18), and key transcriptional regulators were quantified via quantitative real-time PCR (qRT-PCR) and Western blot. Intracellular calcium flux was measured using Fluo-4 AM. We used co-immunoprecipitation to assess the interaction between HSP60 and the IKKα/β complex, and Immunofluorescence to visualize nuclear translocation of phosphorylated NF-κB p65. Results: TNF-α stimulation significantly upregulated the expression of TRPM4. Administration of the TRPM4 inhibitor 9-Phe attenuated this increase. Treatment with 9-Phe also reduced the TNF-α-induced elevation of adhesion molecules VCAM-1 and ICAM-1. It further decreased the expression of pyroptosis-related markers, including NLRP3, caspase-1, GSDMD, IL-1β, and IL-18. In addition, 9-Phe markedly reduced the TNF-α-driven nuclear translocation of phosphorylated NF-κB p65. HSP60 knockdown intensified TNF-α-induced inflammation and pyroptosis. Mechanistic analysis showed that TRPM4 inhibition reduced the interaction between HSP60 and the IKKα/β complex. Conclusion: TRPM4 plays a critical role in TNF-α-induced endothelial inflammation and pyroptosis, and its inhibition attenuates these pathological changes. Mechanistic findings indicate that TRPM4 promotes the interaction between HSP60 and IKKα/β, thereby activating the NF-κB pathway. Collectively, these results identify the TRPM4-HSP60-NF-κB axis as a central regulator of endothelial inflammation and pyroptosis, and suggest its potential as a therapeutic target for atherosclerosis.