Kaempferitrin Attenuates Lipopolysaccharide‐Induced Cardiac Dysfunction Through Suppression of the NF‐κB/NLRP3 Signaling Pathway

Figures

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  Figure 1. Kaempferitrin inhibits LPS‐induced cardiac inflammation and improves myocardial injury and cardiac function in mice. (A) The structure of Kaempferitrin. (B) The brief experimental setup of the in vivo experiment. After intraperitoneal injection of Kaempferitrin for 2 h, mice were then exposed to LPS (10 mg/kg) for 12 h. (C) Representative transthoracic echocardiography M‐model images from each group. (D) EF% and FS% of the mice in each group. (E) Serum levels of CK‐MB and LDH in mice from each group. (F) Representative images of HE staining for heart tissue. Scale bar = 50 μm. (G) Representative images of TUNEL staining for heart tissue. Scale bar = 50 μm. N = 6. (One‐way analysis of variance [ANOVA] followed by Bonferroni post hoc test for comparing multiple groups. *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant).

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  Figure 2. Kae inhibited LPS‐induced cardiomyocyte inflammation and the activation of NF‐κB/NLRP3/pyroptosis pathway in vitro. (A–C) Transcriptome sequencing of myocardial tissue from LPS group and LPS + Kae group. It showed a volcano plot (A), GSEA analysis of NF‐κB pathway (B), and a heat map of genes involved in pyroptosis and NF‐κB signaling pathway (C), N = 3. (D) CCK8 assay evaluating the appropriate concentration of Kaempferitrin Kae). (E) The brief experimental setup of the in vitro experiment. After maltreatment with Kaempferitrin for 2 h, cardiomyocytes were then exposed to LPS (1 μg/L) for 12 h. (F) The relative RNA levels of Il‐1β, Il‐6, and Tnf‐α in H9c2 cells. (G) The relative protein levels of NLRP3, p65, p‐p65, Ik‐Bα, p‐Ik‐Bα and GAPDH in H9c2 cell were detected using western blot assay. (H) The densitometric quantification of NLRP3, p‐Ik‐Bα/Ik‐Bα and p‐P65/P65 in H9c2 cells. (I) The relative mRNA levels of Il‐1β, Il‐6, and Tnf‐α in AC16 cells. (J) The relative protein levels of NLRP3, p65, p‐p65, Ik‐Bα, p‐Ik‐Bα, and GAPDH in AC16 cell were detected using western blot assay. (K) Densitometric quantification of NLRP3, p‐Ik‐Bα/Ik‐Bα, and p‐P65/P65 in AC16 cells. (L, M) The…

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  Figure 3. Kaempferitrin may ameliorate LPS‐induced cardiac injury by inhibiting pyroptosis via the NF‐κB/NLRP3 pathway in vivo. (A) The relative protein levels of NLRP3, P65, p‐P65, IkBα, p‐IkBα, and GAPDH in the heart tissue were detected using western blot assay. (B)The relative protein levels of GSDMD, Cleaved‐caspase1, and GAPDH in the heart tissue were detected using western blot assay. C‐E, Densitometric quantification of NLRP3 (C), p‐IkBα/IkBα (D), and PP65/P65 (E). (F, G) Densitometric quantification of Cleaved‐GSDMD and Cleaved‐caspase 1. (H, I) Levels of IL‐1β (H), IL‐18 (I) in heart tissue detected by ELISA assay. N = 6. (One‐way analysis of variance [ANOVA] followed by Bonferroni post hoc test for comparing multiple groups. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant).

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  Figure 4. Kaempferitrin improves LPS‐induced myocardial inflammatory damage by inhibiting the NF‐κB/NLRP3/pyroptosis pathway. After pretreatment with Kaempferitrin, VX765 or Kaempferitrin+VX765 for 2 h, cardiomyocytes were then exposed to LPS (1 μg/mL) for 12 h. (A) The relative protein levels of GSDMD, Cleaved‐caspase1 and GAPDH in H9c2 cell were detected using western blot assay. The densitometric quantification of Cleaved‐GSDMD (B), and Cleaved‐Caspase1 (C) in H9c2 cells. (D) The relative mRNA levels of Il‐1β, Il‐6, and Tnf‐α in H9c2 cells. (E) The relative protein levels of GSDMD, Cleaved‐caspase1, and GAPDH in AC16 cell were detected using western blot assay. The densitometric quantification of Cleaved‐GSDMD (F) and Cleaved‐Caspase1 (G) in AC16 cells (H). N = 3. (One‐way analysis of variance [ANOVA] followed by Bonferroni post hoc test for comparing multiple groups. *p < 0.05, **p < 0.01, ns = not significant).

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  Figure 5. NLRP3 is required for Kae to defend against cardiotoxicity induced by LPS in vivo. The WT mice and NLRP3 knockout mice were exposed to LPS (10 mg/kg) for 12 h. (A) Representative transthoracic echocardiography M‐model images from each group. (B) EF% and (C) FS% of the mice in each group. (D, E) Serum levels of CK‐MB and LDH in mice from each group. (F) Representative images of HE staining and TUNEL staining for heart tissue. Scale bar = 50 μm. N = 6. (G) The relative protein levels of Cleaved‐GSDMD, Cleaved‐caspase1, and GAPDH in the heart tissue were detected using western blot assay. Densitometric quantification of C‐GSDMD (H) and C‐Caspase 1 (I) were shown. N = 6. (One‐way analysis of variance [ANOVA] followed by Bonferroni post hoc test for comparing multiple groups. *p < 0.05, **p < 0.01, **** p < 0.0001, ns = not significant).

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  Figure 6. The graphic abstract shows that Kae could alleviate myocardial damage associated with sepsis via the NF‐κB/NLRP3/pyroptosis pathway.