Membrane Raft Redox Signaling Mediates Trimethylamine N-Oxide-Induced NLRP3 Inflammasome Activation and Endothelial Dysfunction.

Trimethylamine-N-oxide (TMAO), a gut microbiota-derived metabolite, is a known risk factor for cardiovascular disease. We previously showed that TMAO induces NLRP3 inflammasome activation and contributes to endothelial dysfunction. However, the upstream mechanisms linking TMAO to inflammasome activation and barrier injury remain unclear. In the present study, we examined whether membrane raft (MR) redox signaling contributes to TMAO-induced NLRP3 inflammasome activation and endothelial dysfunction. MR clustering, colocalization of MRs with NADPH oxidase subunits, inflammasome formation, and junction protein expression were assessed by Western blot analysis. RT-qPCR was performed to further assess the mRNA expression of junctional genes. Superoxide production was measured by electron spin resonance (ESR), caspase-1 activity was determined using a biochemical assay kit, IL-1β production was measured by ELISA, and endothelial permeability was evaluated using an FITC-dextran assay. TMAO increased MR clustering in endothelial cells in a dose-dependent manner. TMAO also enhanced the colocalization of MRs with p47phox and gp91phox, indicating the formation of an MR-associated redox signaling axis. In addition, TMAO increased superoxide production; promoted NLRP3 inflammasome formation; elevated caspase-1 activity and IL-1β production; reduced the expression of ZO-2, ZO-1, VE cadherin and occludin; and increased endothelial permeability. Pretreatment with the MR disruptor MCD, the NADPH oxidase inhibitor DPI, or the caspase-1 inhibitor WEHD significantly attenuated these TMAO-induced effects. These findings demonstrate that TMAO-mediated endothelial injury is dependent on the MR-associated redox signaling axis. TMAO promotes MR-associated redox signaling, leading to NADPH oxidase activation, superoxide generation, NLRP3 inflammasome activation, disruption of endothelial junction proteins, and barrier dysfunction. Targeting MR-associated signaling pathways may offer a novel therapeutic strategy to mitigate TMAO-induced vascular inflammation and endothelial dysfunction.