Abstract: Objective To observe the effect of inhibiting mitochondrial oxidative stress and NLRP3 inflammasomes on high glucose (HG)-induced pyroptosis and ferroptosis in H9C2 cardiac muscle cells and explore the possible interactions between mitochondrial reactive oxygen species (ROS) and inflammasomes. Methods H9C2 cells exposed to high glucose (35 mmol/L) were treated with the mitochondrial antioxidant mitoquinone (MitoQ), the NLRP3 antagonist MCC950, or both MCC950 and rotenone (a mitochondrial electron transport antagonist), and the cell viability was measured with CCK-8 assay. The cellular and mitochondrial ROS levels were measured using CellRox and Mitosox fluorescent probes, respectively. The cellular NLRP3 inflammasome level was detected with immunofluorescence assay, and the expressions of the key proteins related with pyroptosis and ferroptosis were determined with Western blotting. Results HG exposure significantly lowered the viability of H9C2 cells (P<0.01), reduced the expression of GPX4 protein (a key protein related with ferroptosis) (P<0.01), and increased the fluorescence intensities of NLRP3 (P<0.01) and ROS (at both the cellular and mitochondrial levels, P<0.01) and the protein expressions of NLRP3 and GSDMD-NT (P<0.01). Treatment with either MitoQ or MCC950 significantly increased the viability of HG-exposed cells (P<0.01), increased GPX4 expression (P<0.01), and reduced the fluorescence intensities of NLRP3 (P<0.01) and cellular and mitochondrial ROS (P<0.01) and the protein expressions of NLRP3 and GSDMD-NT (P<0.05). Compared with MCC950 treatment, treatment with both MCC950 and rotenone significantly reduced the viability of HG-exposed cells (P<0.01), lowered GPX4 expression (P<0.01), and increased the fluorescence intensities of ROS and NLRP3 (P<0.01) and the protein levels of NLRP3 and GSDMD-NT (P<0.05). Conclusion MitoQ inhibits mitochondrial ROS production to reduce HG-induced NLRP3 inflammasome activation and thus suppress pyroptosis and ferroptosis of cardiac muscle cells. There may be an interaction between mitochondrial ROS and NLRP3 inflammasomes.

Key words: high glucose, mitochondrial reactive oxygen species, NLRP3 inflammasome, pyroptosis, ferroptosis