Hirudin inhibit the formation of NLRP3 inflammasome in cardiomyocytes via suppressing oxidative stress and activating mitophagy

Context: Cardiomyocyte hypertrophy due to hemodynamic overload eventually leads to heart failure. Hirudin has been widely used in the treatment of cardiovascular diseases and NLRP3 inflammasome was proven to induce cardiomyocyte pyroptosis. However, the mechanism by which it inhibits cardiomyocyte hypertrophy remains unclear.Objective: To explore the mechanism of hirudin inhibiting cardiomyocyte hypertrophy based on NLRP3 inflammasome activation and mitophagy.Materials & methods: 1 mu M AngII was used for cardiac hypertrophy modeling in H9C2 cells, and cell viability was quantified by CCK-8 assay to screen the appropriate action concentrations of hirudin. After that, we cultured AngII induced-H9C2 cells for 24 h with 0, 0.3, 0.6, and 1.2 mM hirudin, respectively. Next, we marked H9C2 cells with phalloidine and observed them using fluorescence microscope. IL-1 beta, IL-18, IL-6, TNF-alpha, ANP, BNP, beta-MHC, and mtDNA were analyzed by qRT-PCR; ROS were quantified by Flow cytometry; SOD, MDA, and GSH-Px were detected by ELISA; and proteins including NLRP3, ASC, caspase-1, pro-caspase-1, IL-1 beta, IL-18, PINK-1, Parkin, beclin-1, LC3-I, LC3-II, p62, were quantified by western blotting.Results: It was discovered that hirudin reduced the superficial area of AngII-induced H9C2 cells and inhibited the AngII-induced up-regulation of ANP, BNP, and beta-MHC. Besides, hirudin down-regulated the expressions of NLRP3 inflammasome-related cytokines, containing IL-1 beta, IL-18, IL -6, TNF-alpha. It also down-regulated the expression of mtDNA and ROS, decreased the expression levels of NLRP3 inflammasome activation related proteins, including NLRP3, ASC, caspase-1, pro-