P2X7 RECEPTOR INVOLVEMENT IN THE PRO-INFLAMMATORY EFFECTS INDUCED BY TRIMETHYLAMINE N-OXIDE (TMAO) IN HUVECS

Cardiovascular diseases remain the leading cause of death worldwide. Trimethylamine N-oxide (TMAO) is a metabolite produced by the gut microbiota from dietary precursors such as choline and L-carnitine. Elevated TMAO levels have been associated with the activation of pro-inflammatory pathways and the development of atherosclerosis, suggesting an important role in cardiovascular pathophysiology. Studies have shown that TMAO induces NLRP3 inflammasome activation in endothelial cells, promoting vascular inflammation and endothelial dysfunction. The purinergic receptor P2X7, activated by high extracellular ATP concentrations (1-5 mM), promotes K⁺ efflux and NLRP3 inflammasome activation. This complex activates caspase-1, leading to the cleavage of pro-IL-1β and gasdermin D, promoting inflammatory cytokine release and pyroptosis. Therefore, this pathway has been implicated in several inflammatory diseases, including atherosclerosis. To investigate the contribution of P2X7 signaling to TMAO-induced inflammation, HUVECs (Human Umbilical Vein Endothelial Cells) were cultured in high-glucose DMEM supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic solution. After reaching 80% confluence, cells were treated with TMAO (100, 300, and 600 μM) in the presence or absence of the P2X7 antagonists Brilliant Blue G (BBG, 10 μM) and Lu AF27139 (LuAF, 150 nM). P2X7-mediated permeabilization was evaluated using ethidium bromide fluorescence in the presence of ATP (2 mM). TMAO treatment (50 and 100 μM) increased cellular permeabilization, an effect reduced by BBG. Western blot analysis revealed increased levels of cleaved caspase-1 and gasdermin N in TMAO-treated cells at all evaluated concentrations, while P2X7 antagonists attenuated these effects mainly at higher TMAO concentrations. Additionally, ELISA analysis demonstrated increased IL-1β release following TMAO treatment, which was reduced by BBG or LuAF. The obtained results provide initial evidence that P2X7 receptor-mediated signaling may contribute to the pro-inflammatory effects induced by TMAO in HUVECs. Further analyses are required to confirm the underlying mechanism involved.