DDX3X deficiency alleviates LPS‑induced H9c2 cardiomyocytes pyroptosis by suppressing activation of NLRP3 inflammasome

Figures

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  Figure 1. DDX3X expression in LPS-treated cardiomyocytes. (A) Western blot analysis was performed to detect DDX3X protein expression in H9C2 cardiomyocytes treated with different concentrations of LPS (0, 100, 500 and 1,000 ng/ml) for 24 h. Representative blots (n=3). (B) Reverse transcription-quantitative PCR analysis was performed to detect DDX3X mRNA expression in H9C2 cardiomyocytes treated with different concentrations of LPS (0, 100, 500 and 1,000 ng/ml) for 24 h. Data are presented as the mean ± SEM (n=3). (C) Western blot analysis was performed to detect DDX3X protein expression in H9C2 cardiomyocytes stimulated with LPS (1,000 ng/ml) for 0, 6, 12 and 24 h. Representative blots (n=3). **P<0.01 and ***P<0.001. LPS, lipopolysaccharide.

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  Figure 2. LPS induces pyroptosis by increasing intracellular reactive oxygen species levels and activating NLRP3 inflammasome. Reverse transcription-quantitative PCR analysis was performed to detect the mRNA expression levels of (A) NLRP3 and (B) IL-1β in H9C2 cardiomyocytes treated with different concentrations of LPS (0, 100, 500 and 1,000 ng/ml) for 24 h. Data are presented as the mean ± SEM (n=4). (C) Western blot analysis was performed to detect the protein expression levels of NLRP3 and pro-IL-1β in H9C2 cardiomyocytes treated with different concentrations of LPS (0, 100, 500 and 1,000 ng/ml) for 24 h. Representative blots (n=3). (D) Western blot analysis was performed to detect NLRP3 protein expression in H9C2 cardiomyocytes stimulated with LPS (1,000 ng/ml) for 0, 6, 12 and 24 h. Representative blots (n=3). (E) The DCFH-DA assay was performed to detect ROS production in H9C2 cardiomyocytes treated with LPS (1,000 ng/ml) for 6 h. Bar, 50 µm (n=3). (F) Western blot analysis was performed to detect intracellular protein levels of pro-caspase-1 and caspase-1 p20, and the level of the cleaved IL-1β in the supernatant of culture medium in H9C2 cardiomyocytes treated with different…

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  Figure 3. Ddx3x knockdown blocks NLRP3 inflammasome activation in LPS-stimulated H9C2 cardiomyocytes. (A) Western blotting confirmed the efficiency of Ddx3x knockdown. Representative blots (n=3). (B) RT-qPCR analysis confirmed the efficiency of Ddx3x knockdown. Data are presented as the mean ± SEM (n=3). (C) Western blot analysis was performed to detect the protein expression levels of DDX3X, NLRP3, caspase-1 cleavage and cleaved IL-1β in the supernatant of culture medium in H9C2 cardiomyocytes treated with or without LPS (1,000 ng/ml) for 24 h following siRNA-mediated knockdown of Ddx3x. Data are presented as the mean ± SEM (n=3). (D) RT-qPCR analysis was performed to detect the mRNA expression levels of DDX3X, NLRP3 and IL-1β in H9C2 cardiomyocytes treated with or without LPS (1,000 ng/ml) for 24 h following siRNA-mediated knockdown of Ddx3x. Data are presented as the mean ± SEM (n=3). (E) Caspase-1 activity was detected in H9C2 cardiomyocytes treated with or without LPS (1,000 ng/ml) for 24 h following siRNA-mediated knockdown of Ddx3x. Data are presented as the mean ± SEM (n=3). *P<0.05, **P<0.01 and ***P<0.001. NLRP3, NOD-like receptor protein 3; LPS, lipopolysaccharide; RT-qPCR,…

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  Figure 4. Ddx3x knockdown inhibits NOD-like receptor protein 3 inflammasome activation in LPS-treated cardiomyocytes. (A) Cell Counting Kit-8 assay was performed to detect the viability of H9C2 cells. Data are presented as the mean ± SEM (n=4). (B) LDH release was measured via the LDH activity assay. Data are presented as the mean ± SEM (n=4). (C) The PI staining assay was performed to assess the status of pyroptotic cell death. Bar, 100 µm (n=3). *P<0.05, **P<0.01 and ***P<0.001. LPS, lipopolysaccharide; LDH, lactate dehydrogenase; PI, propidium iodide; si, small interfering; ns, not significant.

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  Figure 5. A putative scheme illustrating the mechanism by which DDX3X contributes to pyroptosis of cardiomyocytes in LPS-induced cardiac injury. LPS binding to TLR4 promotes the upregulation of DDX3X, NLRP3 and IL-1β. DDX3X directly binds to the NACHT domain of NLRP3, contributing to the NLRP3 inflammasome assembly and the activation of caspase-1. Activated caspase-1 induces the maturation of IL-1β and shears GSDMD. Subsequently, the cleaved N-terminal domain of GSDMD translocates to the plasma membrane and forms pores, facilitating the extracellular secretion of IL-1β into the circulation system and triggers pyroptosis. LPS, lipopolysaccharide; TLR4, Toll-like receptor 4; NLRP3, NOD-like receptor protein 3; GSDMD, gasdermin D.