Plant‐Derived Polyphenol Salvianolic Acid B Attenuates Cardiomyocyte Pyroptosis by Modulating the MMP12 – NLRP3 Signaling Axis

Figures

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  FIGURE 1. Establishment of HG‐induced cardiomyocyte injury model and cytotoxicity assessment of Sal B (A) Natural source and chemical structure of Sal B (B) Determination of the optimal concentration of Sal B in AC16 cardiomyocytes (C–E) Establishment of HG‐induced injury model in AC16 cardiomyocytes; arrow indicates a representative TUNEL‐positive cell. Data are shown as mean ± standard deviation. (*p < 0.05, **p < 0.01, ***p < 0.001 vs. control group. n = 3). DAPI, 4′,6‐diamidino‐2‐phenylindole; HG, high glucose; Sal B, salvianolic acid B.

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  FIGURE 2. Sal B attenuates HG‐induced injury in AC16 cardiomyocytes. (A) Cell Counting Kit‐8 assay assessing cell viability. (B) Enzyme‐linked immunosorbent assay quantification of proinflammatory cytokines IL‐1β, IL‐18, and TNF‐α in cell culture supernatants. (C) Flow cytometric measurement of intracellular reactive oxygen species. (D) Biochemical quantification of oxidative stress markers MDA and SOD, and the cytotoxicity marker LDH. (E) TUNEL fluorescence staining for quantifying DNA fragmentation as an indicator of cell death in AC16 cardiomyocytes; arrow indicates a representative TUNEL‐positive cell. Data are shown as mean ± standard deviation. (*p < 0.05, **p < 0.01, ***p < 0.001 vs. control group; ### p < 0.001 vs. HG group. n = 3). DAPI, 4′,6‐diamidino‐2‐phenylindole; HG, high glucose; IL, interleukin; LDH, lactate dehydrogenase; MDA, malondialdehyde; Sal B, salvianolic acid B; SOD, superoxide dismutase; TNF, tumor necrosis factor.

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  FIGURE 3. Sal B attenuates HG‐induced cardiomyocyte injury via modulating NLRP3 inflammasome‐associated pyroptotic signaling. (A) Immunofluorescence detection of caspase‐1 and NLRP3. (B) Reverse transcription quantitative polymerase chain reaction profiling of mRNA expressions: NLRP3, ASC, CASP1 (Caspase‐1), and GSDMD. (C) Protein expressions via western blot: NLRP3, ASC, pro‐caspase‐1, cleaved‐caspase‐1, GSDMD, and GSDMD‐N. Data are shown as mean ± standard deviation. (*p < 0.05, **p < 0.01 ***p < 0.001 vs. control group; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. HG group. n = 3). ASC, apoptosis‐associated speck‐like protein containing a CARD; GSDMD, gasdermin D; GSDMD‐N, active N‐terminal fragment of GSDMD; HG, high glucose; NLRP3, nucleotide‐binding domain, leucine‐rich–containing family, pyrin domain–containing‐3; ns, not significant; Sal B, salvianolic acid B.

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  FIGURE 4. Bioinformatic identification of MMP12 as a candidate molecular mediator associated with MCM. (A) Limma volcano plot of DEGs in the GSE205891 dataset. (B) Limma volcano plot of DEGs in the GSE76811 dataset. (C) Weighted gene co‐expression network analysis. (D) Dynamic clustering dendrogram. (E) Relationship between consensus modules and samples. (F) Cross‐module gene significance. (G) Turquoise module representing MCM gene significance. (H) Venn diagram showing intersection of DEGs, turquoise‐colored module genes, and UniProt gene set identifying one candidate gene. (I) ROC curve evaluating the discriminatory performance of MMP12 in the discovery dataset. (J) Spearman correlation coefficient plot. AUC, area under the curve; DEGs, differentially expressed genes; MCM, metabolic cardiomyopathy; MMP12, matrix metalloproteinase 12; ROC, receiver operating characteristic.

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  FIGURE 5. Molecular docking analysis of Sal B with MMP12. (A) Overall binding pose of Sal B (depicted in blue) within the active‐site cavity of the MMP12 protein (rendered as a gray molecular surface). (B) Coordination geometry between the catalytic Zn2+ ion (shown as a pink sphere) and the three conserved histidine residues, His218, His222, and His228 (indicated by green dashed lines; bond lengths reported in Å). (C) Hydrogen‐bonding and polar interactions between Sal B and functionally relevant residues of MMP12 (represented by yellow dashed lines; distances labeled in Å). (D) Electrostatic surface potential map of MMP12, highlighting the charge distribution across the Sal B binding region.

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  FIGURE 6. Establishment and validation of MMP12 overexpression in AC16 cardiomyocytes. (A) Reverse transcription quantitative polymerase chain reaction analysis of MMP12 mRNA expression levels. (B) Quantification of MMP12 protein via western blot. Data are shown as mean ± standard deviation. (***p < 0.001 vs. Blank group; ### p < 0.001 vs. Vector group. n = 3). MMP12, matrix metalloproteinase 12; OE, overexpression.

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  FIGURE 7. MMP12 overexpression attenuates the cytoprotective effects of Sal B in HG‐injured cardiomyocytes. (A) TUNEL staining analysis quantifying DNA fragmentation as an indicator of cell death in AC16 cardiomyocytes. (B) Levels of IL‐1β, IL‐18, and TNF‐α. (C) Western blot analysis and quantification of protein levels of NLRP3, ASC, pro‐caspase‐1, cleaved‐caspase‐1, GSDMD, and GSDMD‐N. Data are shown as mean ± standard deviation. (There are three groups: HG, HG + Sal B, and HG + Sal B + MMP12 OE. *p < 0.05, **p < 0.01, ***p < 0.001 vs. HG group; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. HG + Sal B group. n = 3). ASC, apoptosis‐associated speck‐like protein containing a CARD; DAPI, 4′,6‐diamidino‐2‐phenylindole; GSDMD, gasdermin D; GSDMD‐N, active N‐terminal fragment of GSDMD; HG, high glucose; IL, interleukin; MMP12, matrix metalloproteinase 12; NLRP3, nucleotide‐binding domain, leucine‐rich–containing family, pyrin domain–containing‐3; ns, not significant; OE, overexpression; Sal B, salvianolic acid B; TNF, tumor necrosis factor.