Bone Regeneration Drug BMP-7 Mitigates Ponatinib-Induced Cardiotoxicity via Inhibition of Pyroptosis and Modulation of TGF-β/SMAD Signaling Pathway

Figures

• 

  Figure 1. Study design schematic overview. Adult C57BL/6J mice received either saline, PON, or PON+BMP-7 according to the weekly dosing schedule. On D19, mice were euthanized, and hearts were collected for molecular and histological analyses.

• 

  Figure 2. BMP-7 treatment reduces PON-induced monocyte infiltration and M1 macrophage polarization. PON increases monocyte infiltration (CD14, (A)) and M1 macrophage polarization (iNOS, (E)), which are reduced by BMP-7. CD14 and iNOS are shown in red (a, f, k). Cardiomyocytes are labeled in green with Src-α-actin (b, g, l), and nuclei are stained blue with DAPI (c, h, m). Merged images (d, i, n) and corresponding magnified views (e, j, o) highlight the areas marked by dashed boxes. Scale bar = 100 μm. (B,F) IHC quantification (n = 7/group). (C,G) Representative blots and densitometry (n = 6–7/group, A.U.). (D,H) TNF-α and IL-6 gene expression (n = 5–7/group). Results are shown as mean ± SEM. * p < 0.05, ** p < 0.01, and **** p < 0.0001.

• 

  Figure 3. BMP-7 treatment inhibits PON-induced HMGB1-TLR4 signaling and pyroptosis initiation. (A,D) Representative photomicrographs of HMGB1 and TLR4 IHC staining. HMGB1 and TLR4 are shown in red (a, f, k). Cardiomyocytes are labeled in green with Src-α-actin (b, g, l), and nuclei are stained blue with DAPI (c, h, m). Merged images (d, i, n) and magnified panels (e, j, o) provide enhanced clarity and facilitate accurate identification of marker distribution within the cardiac tissue. Scale bar = 100 μm. (B,E) IHC quantitative analysis (n = 7/group). (C,F) Immunoblots and protein quantification of HMGB1 (n = 6–7/group, A.U.) and TLR4 (n = 6/group, A.U.). Results are shown as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

• 

  Figure 4. BMP-7 treatment ameliorates PON-induced pyroptotic cell death. (A,E,I) PON increases NLRP3, caspase-1, and GSDMD expression, which is reduced by BMP-7. NLRP3, caspase-1, and GSDMD are shown in red (a, f, k). Cardiomyocytes are labeled in green with Src-α-actin (b, g, l), and nuclei are stained blue with DAPI (c, h, m). Merged images (d, i, n) and corresponding magnified views (e, j, o) highlight the areas marked by dashed boxes. Scale bar = 100 μm. (B,F,J) IHC staining quantification (n = 7/group). (C,G,K) Relative mRNA expression (n = 5–6/group). (D,H,L) Western blots and densitometric analysis (n = 6–8/group, A.U.). Results are shown as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

• 

  Figure 5. BMP-7 attenuates PON-induced expression of pyroptosis-associated cytokines. (A,E) IHC staining of IL-1β and IL-18 in cardiac sections. IL-1β and IL-18 are shown in red (a, f, k). Cardiomyocytes are labeled in green with Src-α-actin (b, g, l), and nuclei are stained blue with DAPI (c, h, m). Merged images (d, i, n) and enlarged images (e, j, o) enable precise localization and clearer visualization of the marker expression within the cardiac tissue. Scale bar = 100 μm. (B,F) IHC quantification (n = 7/group). (C,G) Relative gene expression levels of IL-1β (n = 6–7/group) and IL-18 (n = 5–6/group). (D,H) Representative blots and protein quantification of IL-1β (n = 6–7/group, A.U.) and IL-18 (n = 6/group, A.U.). Results are shown as mean ± SEM. * p < 0.05, ** p < 0.01, and **** p < 0.0001.

• 

  Figure 6. BMP-7 treatment enhances M2 macrophage polarization. (A,D,G) Representative immunofluorescence images showing CD206, arginase-1, and IL-10 stain. Red fluorescence indicates CD206, arginase-1, and IL-10 expression (a, f, k). Cardiomyocytes are visualized in green using Src-α-actin labeling (b, g, l), while nuclei are counterstained blue with DAPI (c, h, m). Merged images are shown in panels (d, i, n), and enlarged views (e, j, o) provide detail of the regions outlined by dashed boxes. Scale bar = 100 μm. (B,E,H) IHC quantitative analysis (n = 7/group). (C,F) Western blots and densitometric analysis for CD206 and arginase-1 (n = 6–7/group, A.U.). (I) Relative IL-10 mRNA expression (n = 5/group). Results are shown as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

• 

  Figure 7. BMP-7 treatment inhibits PON-activated TGF-β1/SMAD2/3 signaling pathway. (A–C) Immunoblots and protein quantitative analysis of TGF-β1 (n = 6/group), pSMAD2 (n = 6/group), and pSMAD3 (n = 6–7/group). Data are expressed as A.U. and shown as mean ± SEM. * p < 0.05 and ** p < 0.01.