Salvianolic Acid B Preserves Myocardial Viability and Extends the Preservation Window of Mouse Hearts During Static Cold Storage

Figures

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  Figure 1. SalB mitigates cold ischemia-induced injury and bolsters antioxidant capacity in transplanted mouse hearts. (a–c) Quantification of cardiac enzyme leakage (BNP, CK-MB, and LDH) in the coronary effluent via ELISA (n = 6). (d–f) RT-qPCR analysis of myocardial pro-inflammatory cytokine mRNA expression (IL-1β and TNF-α) following 4 h, 8 h, and 10 h of cold storage (n = 6). Statistical significance was determined using one-way ANOVA followed by Tamhane’s T2 post hoc test. ns, relatively not significant (p > 0.05); * p < 0.05; ** p < 0.01; *** p < 0.001.

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  Figure 2. (a) Representative Western blots and (d) quantitative analysis of CX43 protein expression in myocardial tissues (n = 3). (b,c) Assessment of myocardial antioxidant capacity based on SOD activity and MDA content (n = 6). (e) Representative H&E staining images demonstrating myocardial tissue architecture across groups, scale bar = 20 μm (original magnification, 400×). Data are presented as mean ± SD. Statistical significance was determined using one-way ANOVA followed by Tamhane’s T2 post hoc test. ns, relatively not significant (p > 0.05); * p < 0.05; ** p < 0.01; *** p < 0.001.

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  Figure 3. SalB exerts an anti-apoptotic effect during prolonged cold storage. (a) Representative TUNEL staining images assessing cardiomyocyte apoptosis. Brown nuclei indicate apoptotic cells, while blue-purple indicates normal cell nuclei. Scale bar = 50 μm (original magnification, 400×). (b) Representative Western blots and (c) corresponding quantitative analysis of apoptosis-related proteins (Bcl-2, Bax, and Cleaved Caspase-3) in myocardial tissues (n = 3). Data are presented as mean ± SD. ns, relatively not significant (p > 0.05); * p < 0.05; ** p < 0.01; *** p < 0.001 vs. respective groups.

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  Figure 4. SalB enhances autophagic flux in cold-stored mouse hearts. (a) Representative Western blots and (b) quantitative analysis of autophagy markers (LC3 II/I, p62, ATG5, and Beclin-1) (n = 3). (c) Representative immunofluorescence images of LC3 expression (green) with DAPI nuclear counterstaining (blue). Scale bar = 50 μm (original magnification, 400×). (d) Quantification of relative LC3 fluorescence intensity (n = 3). Data are presented as mean ± SD. ns, relatively not significant (p > 0.05); * p < 0.05; ** p < 0.01; *** p < 0.001.

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  Figure 5. The cardioprotective effect of SalB is partially mediated via counteracting 3-MA-induced autophagic inhibition. (a,b) Western blot analysis and corresponding quantification of autophagy-related proteins following 3-MA pretreatment (n = 3). (c,d) Immunofluorescence detection and quantitative analysis of LC3 expression (n = 3). Scale bar = 50 μm (original magnification, 400×). (e–g) ELISA quantification of myocardial enzyme leakage (BNP, CK-MB, and LDH) demonstrating the functional reversal of 3-MA-induced injury by SalB (n = 6). Data are presented as mean ± SD. Statistical significance was assessed using one-way ANOVA with Tamhane’s T2 test., p > 0.05; ** p < 0.01; *** p < 0.001.

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  Figure 6. Schematic illustration of the experimental design, subject preparation, and group allocation strategy. (Created with BioGDP.com [34]).