Yixinshu attenuates myocardial infarction via SHP1/JAK2/STAT3-mediated regulation of mitochondrial function and apoptosis

Figures

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  Fig. 1. YXS Improved Heart Failure Induced by Myocardial Infarction in Mice. A Schematic diagram of the experimental protocol and treatment schedule. B Kaplan–Meier curves. C Representative M-mode echocardiographic images of mice. Quantification of echocardiographic parameters, namely EF (D), and FS (E) (n = 10). F Representative macroscopic images of hearts. G The ratios of HW/TL (n = 7). H Representative Masson’s trichrome staining images of heart tissues (scale bar = 500 µm or 20 µm). Fibrotic area of mice hearts (n = 3). I WGA staining images of heart tissues (scale bar = 10 µm). J Quantification of the cross-sectional area of cardiomyocytes in mice. K Venn diagram of YXS and coronary heart disease. L GO functional analysis. M KEGG functional analysis. Data were shown as mean ± SD. *p < 0.05, **p < 0.01 vs. the sham group; #p < 0.05, ##p < 0.01 vs. the MI group

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  Fig. 2. YXS promoted STAT3 phosphorylation and suppresses cardiomyocyte apoptosis. A Representative transmission electron microscopy images of heart tissues (scale bar = 5 μm or 1 μm). B Ratio of damaged mitochondrial (n = 6). C, D Immunoblotting to test the levels of proteins in mice hearts (n = 5). E Protein–protein interaction network of common targets of YXS and coronary heart disease. F, G mRNA levels of BCL2 and STAT3 in mice hearts determined with qRT-PCR (n = 3). H Immunoblotting to test the levels of proteins in mice hearts (n = 5). I Protein–protein interaction network of STAT3. J The SHP1/JAK2/STAT3 signaling pathway. K Co-IP experiments in hearts of mice. Lysates were immunoprecipitated with anti-SHP1 and blotted with JAK2 antibody and STAT3antibody. L Immunoblotting to test the levels of proteins in mice hearts (n = 6). M mRNA levels of SHP1 in mice hearts determined with qRT-PCR. Data were shown as mean ± SD. *p < 0.05, **p < 0.01 vs. the sham group; #p < 0.05, ##p < 0.01 vs. the MI group

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  Fig. 3. DHT as the Active Component of YXS. A BPCs of each sample group under positive ion mode. B BPCs of each sample group under negative ion mode. C Classification and proportion of blood-absorbed compounds from YXS across chemical categories. D–G Danshensu, Dihydrotanshinone-I (DHT), Ginsenoside, Ginsenoside-Rg6 were capable of binding with SHP1. H The binding affinity of SHP1 with DHT was determined using an SPR assay. I Representative Western blot and quantification of STAT3, p-STAT3, p-JAK2, JAK2 and SHP1 (n = 3–5). J Co-IP experiments in Primary neonatal rat cardiomyocytes. Lysates were immunoprecipitated with anti-SHP1 antibody and blotted with ubiquitin antibody and SHP1 antibody. Data were shown as mean ± SD. *p < 0.05, **p < 0.01 vs. sham or control group; #p < 0.05, ##p < 0.01 vs. MI or OGD group

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  Fig. 4. DHT inhibited apoptosis and enhanced mitochondrial activity in primary cardiomyocytes. A Representative Western blot and quantification of Cleaved-Caspase3, BAX and BCL2 (n = 3–4). B TUNEL staining (scale bar = 50 μm). C Apoptosis of cardiomyocytes was assessed by flow cytometry (n = 5). D, E Mitochondrial membrane potential was assessed using JC-10 staining (scale bar = 25 μm) (n = 3). F Co-localization of active mitochondria and apoptotic signals (scale bar = 25 μm). G OCR was measured by Seahorse to evaluate mitochondrial bioenergetics. And Quantitative analysis of OCR. n = 3. Data were shown as mean ± SD. *p < 0.05, **p < 0.01 vs. sham or control group; #p < 0.05, ##p < 0.01 vs. MI or OGD group

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  Fig. 5. The anti-apoptotic effect of DHT is dependent on SHP1 downregulation. A Adenoviral transduction of primary cardiomyocytes. B mRNA levels of SHP1 in primary cardiomyocytes (scale bar = 100 μm) (n = 4). C, D Representative Western blot images and corresponding quantification of protein expression (n = 3–5). E Mitochondrial membrane potential was detected by Mito-Tracker Red CMXRos assay (scale bar = 50 μm) (n = 3). F TUNEL staining (scale bar = 50 μm). G Apoptosis of cardiomyocytes was assessed by flow cytometry (n = 3). Data were shown as mean ± SD. *p < 0.05, **p < 0.01

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  Fig. 6. SHP1 overexpression abrogates the cardioprotective effects of DHT. A Representative M-mode echocardiographic images of mice. Quantification of echocardiographic parameters, namely EF, and FS (n = 6). B Representative Masson’s trichrome staining images of heart tissues (scale bar = 500 µm). C Fibrotic area of mice hearts (n = 3). D Representative Western blot images and corresponding quantification of protein expression (n = 3–5). Data were shown as mean ± SD. *p < 0.05, **p < 0.01

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  Fig. 7. The potential mechanism of YXS by which it regulates myocardial apoptosis and mitochondrial homeostasis