Wogonin Alleviates Cisplatin-induced Cardiotoxicity in Mice Via Inhibiting Gasdermin D-mediated Pyroptosis

Abstract

Cardiotoxicity has been well documented as a side effect of cisplatin (CDDP) treatment. The inflammatory response plays a crucial role in the pathological process of CDDP-induced cardiotoxicity. Wogonin is a natural flavonoid compound that possesses cardioprotective and anti-inflammatory qualities. Knowledge of the pharmacological effect and mechanism of wogonin could reveal an efficient way to identify therapeutic strategies. In this study, the potential of wogonin to antagonize CDDP-induced cardiotoxicity was evaluated in C57BL/6 mice in vivo and in H9c2 cells in vitro. The results showed that wogonin protected against CDDP-induced cardiac dysfunction, myocardial injury, and pyroptosis in vivo. Using a Gasdermin D expression plasmid, we revealed that wogonin dramatically reduced CDDP-induced pyroptosis by modulating the Gasdermin D protein in H9c2 cells. In conclusion, wogonin has great potential in attenuating CDDP-induced cardiotoxicity. In addition, greater emphasis should be placed on the antipyroptotic effects of wogonin for the treatment of other diseases.

FIGURE 1.

Wogonin prevents CDDP-induced cardiac dysfunction in vivo. A, Chemical structure of wogonin. B, Echocardiographic data showing the effects of wogonin on cardiac dysfunction induced by CDDP. C, Representative images of the M-mode of the left ventricle (n = 6 in each group; *, vs. the Ctrl group; #, vs. the CDDP group; #P < 0.05, ** and ##P < 0.01, ***P < 0.001).

FIGURE 2.

Wogonin protects against CDDP-induced myocardial injury in vivo. A, Representative images of H&E staining (×400 magnification). B, Representative images of TUNEL staining (Green: TUNEL-positive cells, DAPI: nucleus; ×400 magnification). C, Levels of serum CK-MB in CDDP-induced mice and mice treated with wogonin. D, LDH levels in the heart tissue of CDDP-induced mice and mice treated with wogonin. E, LDH levels in serum (n = 6 in each group; *, vs. the Ctrl group; #, vs. the CDDP group; ##P < 0.01, *** and ###P < 0.001).

FIGURE 3.

Wogonin alleviates CDDP-induced myocardial pyroptosis in vivo. A, mRNA levels of NLRP3 in heart tissues. B and C, Cell lysates were collected and subjected to Caspase-1 or Caspase-11 enzymatic assays. Ac-YVAD-pNA is the substrate of caspase in the assay kit. D, Western blot analysis of caspase1 and GSDMD expression in heart tissues. Densitometric analysis is shown in the right panel. E, Representative immunohistochemical images of GSDMD (brown) in heart tissues. F–G, mRNA levels of IL-18 and IL-1β in the heart tissues of mice were determined by RT-qPCR. H, Representative immunohistochemical images of IL-1β (brown) in heart tissues (n = 6 in each group; *, vs. the Ctrl group; #, vs. the CDDP group; #P < 0.05, ##P < 0.01, ***P < 0.001).

FIGURE 4.

The antipyroptotic effects of wogonin involve modulating GSDMD. A, H9c2 cells were transfected with GSDMD-expressing plasmid (plsGSDMD) or empty plasmid (negative control, NC). Western blotting was performed to assess the protein levels of GSDMD. Densitometric analysis is shown in lower panel. B, H9c2 cells were treated with CDDP at the indicated concentrations for 72 hours, and then cell viability was analyzed by CCK-8 assays. H9c2 cells were pretreated with wogonin (20 μM) for 1 hour and then incubated with CDDP (5 μM) for the indicated times. C–D, After exposure to CDDP for 24 hours, IL-18 and IL-1β release from GSDMD-overexpressing H9c2 cells in the presence or absence of wogonin was measured by ELISA. E, After exposure to CDDP for 24 hours, LDH release from GSDMD-overexpressing H9c2 cells in the presence or absence of wogonin was measured by an assay kit. F, Schematic illustration of the role of wogonin in GSDMD-mediated pyroptosis in CDDP-induced myocardial injury (n = 3 in each group; *, vs. the Ctrl group; #, vs. the CDDP group; $, vs. the CDDP + wogonin group; * and $P < 0.05, ** and ##P < 0.01, *** and ###P < 0.001).