Fig. 5

Exogenous 8-OHdG decreases IL-1β expression, GSDMD-NT levels, and pyroptosis in DOX-treated H9c2 cells. A–C H9c2 cells were treated with 1 μM DOX and 100 μg/mL or 250 μg/mL 8-OHdG for 24 h. A Caspase-1 activity in cells as determined by a pyroptosis/caspase-1 assay kit. Levels of active caspase-1 in the cells after treatment with DOX and 8-OHdG. After treatment with 1 μM DOX and 100 or 250 μg/mL 8-OHdG, cells were stained with FAM-YVAD-FMK, and caspase-1 activity was measured with a fluorescence microplate reader. B Western blot analysis of GSDMD, pro-IL-1β, IL-1β, and β-actin protein levels in DOX- and 8-OHdG-treated H9c2 cells. C 8-OHdG inhibited DOX-induced caspase-1 activity in H9c2 cells. Scale bar, 20 μm. After fixation, cells stained with FAM-YVAD-FMK were observed with a confocal microscope (left). Bar graph showing the quantification of active caspase-1 positive cells. 8-OHdG treatment decreased caspase-1 activity in DOX-treated cells (right). D Schematic diagram of the proposed mechanism for the effect of 8-OHdG in DOX-exposed cardiomyocytes. Exogenous 8-OHdG decreases the expression of NOX1/2/4, TLR2/4, and NF-κB in DOX-treated cells, which eventually leads to reduced NLRP3 inflammasome, IL-1β, and GSDMD-NT levels. Furthermore, the decrease in the levels of pyroptosis-related factors leads to the decreased expression of myocarditis-related markers, such as ANP and BNP. CTL, control; DOX, doxorubicin; 8-OHdG, 8-hydroxydeoxyguanosine; GSDMD-NT, gasdermin D N-terminal cleavage product; IL, interleukin; TLR, toll-like receptor; NOX, NADPH oxidase; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; TNF, tumor necrosis factor; NLRP, NLR family pyrin domain containing. ^****P < 0.0001 versus control; ^##P < 0.01 and ^###P < 0.001 versus DOX-treated groups