Exogenous 8-hydroxydeoxyguanosine attenuates doxorubicin-induced cardiotoxicity by decreasing pyroptosis in H9c2 cardiomyocytes

Fig. 1. Viability and cytotoxicity of doxorubicin- and 8-hydroxydeoxyguanosine-treated H9c2 cells. An MTT assay was used to examine H9c2 cell viability after treatment with various concentrations of DOX (A) and 8-OHdG (B) for 24, 48, and 72 h. C, After H9c2 cells were treated with DOX (1 μM) and 8-OHdG (50, 100, 250, and 500 μg/mL) for 24 h, cell proliferation and cytotoxicity were measured using MTT (C) and LDH (D) assays, respectively. DOX, doxorubicin; 8-OHdG, 8-hydroxydeoxyguanosine. **P < 0.01, ***P < 0.001, and ****P < 0.0001 versus control; #P < 0.05, ##P < 0.01, ###P < 0.001, and ####P < 0.001 versus DOX

Fig. 2. Exogenous 8-OHdG decreases the expression of cardiotoxicity-related markers in DOX-treated H9c2 cells. H9c2 cells were treated with 1 μM DOX and 100 or 250 μg/mL 8-OHdG for 24 h. Quantitative real-time PCR for ANP, BNP, GATA4, and GATA6 in DOX and 8-OHdG treated H9c2 cells. CTL, control; DOX, doxorubicin; 8-OHdG, 8-hydroxydeoxyguanosine; ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide. ***P < 0.001 and ****P < 0.0001 versus control; #P < 0.05, ##P < 0.01, and ###P < 0.001 versus DOX

Fig. 3. Exogenous 8-OHdG decreases NOX1/2/4 expression and NF-κB phosphorylation, and increases the reduced glutathione/oxidized glutathione ratio in DOX-treated H9c2 cells. A Rac1 activation assay. After H9c2 cells were treated with DOX (1 μM) and 8-OHdG (100 or 250 μg/mL) for 1 h, cell lysates were precipitated by p21-activated protein kinase (PAK) p21-binding domain (PBD) agarose beads and immunoblotted by Rac1 specific monoclonal antibody. B–D H9c2 cells were treated with 1 μM DOX and 100 μg/mL or 250 μg/mL 8-OHdG for 24 h. B–C Western blot analysis of the NOX1/2/4, p65, and phosphor-p65 protein levels of in DOX- and 8-OHdG-treated H9c2 cells. D GSH/GSSG ratio was determined using a glutathione assay kit. CTL, control; DOX, doxorubicin; 8-OHdG, 8-hydroxydeoxyguanosine; NOX, NADPH oxidase; GSH/GSSG, reduced glutathione/oxidized glutathione ratio. *P < 0.05 versus control; #P < 0.05 and ##P < 0.01 versus DOX-treated groups

Fig. 4. Exogenous 8-OHdG decreases the expression of NLRP3, ASC, and caspase-1 in DOX-treated H9c2 cells. A-B H9c2 cells were treated with 1 μM DOX and 100 μg/mL or 250 μg/mL 8-OHdG for 24 h. A Quantitative real-time PCR for TNF-α, TLR2, TLR4, and NLRP3 in DOX- and 8-OHdG-treated H9c2 cells. B Western blot analysis ASC and caspase-1 protein levels in DOX- and 8-OHdG-treated H9c2 cells. CTL, control; DOX, doxorubicin; 8-OHdG, 8-hydroxydeoxyguanosine; TNF, tumor necrosis factor; TLR, toll-like receptor; NLRP, NLR family pyrin domain containing; ASC, apoptosis-associated speck-like protein containing a CARD c-terminal caspase recruitment domain. **P < 0.01 and ****P < 0.0001 versus control; #P < 0.05, ##P < 0.01, ###P < 0.001, and ####P < 0.0001 versus DOX-treated groups

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…