Prunetin ameliorates myocardial pyroptosis after myocardial infarction by inhibiting the TNF-α/p38 MAPK/ERK pathway

Figures

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  Figure 1. PRU ameliorates cardiac function and fibrosis in mice with MI. (A) Experimental flowchart of mouse experiments. (B) Representative echocardiographic images of each group and statistical graphs of LVEF and LVFS, n = 3. (C) Quantitative analysis of serum NT-proBNP levels, n = 3. (D) Representative images of heart sections stained with HE and Masson, and quantitative analysis of cardiac fibrosis area (%), scale bar = 500 µm (upper), scale bar = 100 µm (lower), n = 3. Data are presented as mean ± SD from independent biological replicates. For panels (B–D), which passed the normality assumption, one-way ANOVA followed by Tukey's post-hoc test was applied. Compared with the Sham group, **P < 0.01, *P < 0.05; compared with the MI group, ##P < 0.01, #P < 0.05; compared with the MI + PRU group.

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  Figure 2. Multi-technology screening and verification of potential targets of PRU for MI therapy. (A) Chemical structure of PRU. (B) Venn diagram of PRU targets and MI-related targets. (C) PPI network. (D) Visualization of core gene PPI network. (E) GO functional enrichment analysis. (F) KEGG pathway enrichment analysis. (G) Molecular docking of PRU with targets. (H) MD simulation of PRU and TNF-α. (I) SPR assay for binding affinity between PRU and TNF-α.

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  Figure 3. PRU improves cardiac function in MI mice by inhibiting TNF-α function. (A) Schematic flowchart of in vivo experiments. (B) Representative echocardiographic images from each experimental group. (C) Quantitative analysis of LVEF and LVFS, n = 5. (D,E) HE and Masson staining show representative images of cardiac tissue and quantitative analysis of cardiac fibrosis area (%), scale bar = 1,000 µm (upper), scale bar = 200 µm (lower), n = 5. Data are presented as mean ± SD from independent biological replicates. For panels (B–D), which passed the normality assumption, one-way ANOVA followed by Tukey's post-hoc test was applied. Compared with the Sham group, **P < 0.01, *P < 0.05; compared with the MI group, ##P < 0.01, #P < 0.05; compared with the MI + PRU group, &&P < 0.01, &P < 0.05.

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  Figure 4. Effects of PRU on TNF-α pathway proteins and oxidative stress markers in infarcted myocardium. (A) mRNA expression levels of TNF-α, IL-6, and IL-1β in cardiac tissue, n = 3. (B,C) Expression of TNF-α, p-p38, and p-ERK proteins in cardiac tissue measured and quantified by WB, n = 3. (D) Serum NT-proBNP levels in MI mice, n = 5. (E–G) Levels of SOD, MDA, and LDH in mice cardiac tissue, n = 5. Data are presented as mean ± SD from independent biological replicates. For panels (A,C,D,F,G), which passed the normality assumption, one-way ANOVA followed by Tukey's post-hoc test was applied. For panel (E), which violated the normality assumption, the Kruskal–Wallis test followed by Dunn's post-hoc test was used for multiple comparisons. Compared with the Sham group, **P < 0.01, *P < 0.05; compared with the MI group, ##P < 0.01, #P < 0.05; compared with the MI + PRU group, &&P < 0.01, &P < 0.05.

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  Figure 5. RNA-Seq analysis of PRU-regulated MI mice. (A) Component analysis chart. (B) Volcano plot of differential genes. (C) Heatmap of differential genes. (D) Gene enrichment analysis. (E,F) GO and KEGG analysis of differential genes.

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  Figure 6. PRU inhibits cardiomyocyte pyroptosis. (A–D) Expression of NLRP3, Cleaved IL-1β, and GSDMD-N/GSDMD-F proteins in cardiac tissues measured and quantified by WB, n = 3. Data are presented as mean ± SD from independent biological replicates. For panels (B,D), which passed the normality assumption, one-way ANOVA followed by Tukey's post-hoc test was applied. For panel (C), which violated the normality assumption, the Kruskal–Wallis test followed by Dunn's post-hoc test was used for multiple comparisons. Compared with the Sham group, **P < 0.01, *P < 0.05; compared with the MI group, #P < 0.05, ##P < 0.01; compared with the MI + PRU group, &&P < 0.01, &P < 0.05.

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  Figure 7. PRU ameliorates OGD-induced injury in cardiac organoids by inhibiting TNF-α. (A) Schematic diagram of the experimental timeline for cardiac organoid treatment. (B) Viability of cardiac organoids in response to treatment with PRU and R848, n = 6. (C) Concentrations of NT-proBNP in cardiac organoid culture supernatants measured by ELISA, n = 6. (D–F) Levels of LDH, MDA and SOD in the supernatant of cardiac organoids, n = 6. (G) Representative images of cardiac organoids stained with HE and Masson, along with quantitative analysis of cardiac fibrosis area (%), scale bar = 1,000 µm, n = 3. Data are presented as mean ± SD from independent biological replicates. For panels (B,C,F,G), which passed the normality assumption, one-way ANOVA followed by Tukey's post-hoc test was applied. For panels (D,E), which violated the normality assumption, the Kruskal–Wallis test followed by Dunn's post-hoc test was used for multiple comparisons. Compared with the Control group, **P < 0.01, *P < 0.05; compared with the OGD group, ##P < 0.01, #P < 0.05; compared with the OGD + PRU group, &&P < 0.01, &P < 0.05.

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  Figure 8. Mechanism underlying the protective effect of PRU against OGD-induced injury in cardiac organoids. (A,B) Expression and quantitative analysis of TNF-α, p-p38, and p-ERK in cardiac organoids, scale bar = 100 µm, n = 3. (C,D) Expression and quantitative analysis of GSDMD in cardiac organoids, scale bar = 100 µm, n = 3. (E) The levels of TNF-α and IL-1β in the supernatant of cardiac organoids were determined by ELISA, n = 6. Data are presented as mean ± SD from independent biological replicates. For panels (B,E), which passed the normality assumption, one-way ANOVA followed by Tukey's post-hoc test was applied. For panel (D), which violated the normality assumption, the Kruskal–Wallis test followed by Dunn's post-hoc test was used for multiple comparisons. Compared with the Control group, **P < 0.01, *P < 0.05; compared with the OGD group, ##P < 0.01, #P < 0.05; compared with the OGD + PRU group, &&P < 0.01, &P < 0.05.