Astragaloside IV Ameliorates Myocardial Infarction Induced Apoptosis and Restores Cardiac Function

FIGURE 1. Effect of astragaloside IV (AS-IV) on high glucose/high fat and hypoxia-induced apoptosis in H9c2 myocytes. To mimic the pathological condition with high glucose and high fat (HG/HF), H9c2 myocytes were cultured in HG/HF medium for 12 h followed by hypoxia for 2 h. Then H9c2 myocytes were treated with phosphate-buffered saline (PBS) or AS-IV solution (10 or 50 ng/ml). HG/HF medium was maintained during AS-IV treatment. Cell apoptosis and proliferation were examined 48 h later. (A) Cell apoptosis was detected by flow cytometry after staining with propidium iodide (PI) and Annexin V-FITC. Data represent means ± standard deviation (SD). ***p < 0.001, versus PBS group, n = 3 biologically independent samples. (B) Fluorescence images of live/dead H9c2 myocytes after AS-IV treatment. Cell viability was detected using LIVE/DEADTM Viability/Cytotoxicity Kit. Live and dead cells were stained as green and red. Scale bar = 50 μm. (C) Cell viability was examined via Cell Count Kit-8 assay. Data represent means ± SD. **p < 0.01; ***p < 0.001, versus the PBS group, n = 3 biologically independent samples.

FIGURE 2. Effect of AS-IV on the protein expression in mitogen-activated protein kinase (MAPK) signaling pathway post HG/HF and hypoxia treatment. (A) Representative images of Western blot of MAPKs-p38 enzymes (p38), p-p38, c-Jun NH2-terminal kinases (JNK), p-JNK, extracellular signals-regulated kinases (ERK), and p-ERK. The protein expressions of p38 (B), p-p38 (C), JNK (D), p-JNK (E), ERK (F), and p-ERK (G) were quantified by Image J software. H9c2 myocytes were cultured in HG/HF medium for 12 h followed by hypoxia for 2 h. Then H9c2 myocytes were treated with PBS, MAPK inhibitors (U0126, 25 nM; SB20358, 25 nM; SP600125, 25 nM), AS-IV (50 ng/ml), and the cell apoptosis was examined 48 h later. (H) Cell viability of H9c2 myocytes. Data represent means ± SD. *p < 0.05; **p < 0.01; ***p < 0.001, versus the PBS group, normalized to GAPDH level, n = 3 biologically independent samples.

FIGURE 3. Effect of AS-IV on cardiac function in post-MI diabetic mice. (A) Experimental protocol. C57BL/6J mice were fed high-fat diet for 4 weeks prior to overnight fasting. Mice were then given 100 μl solution of streptozotocin (50 mg/kg body weight/day) for two consecutive days. After confirming the characteristic of diabetes mellitus, myocardial infarction models were established, and the mice were orally administrated with AS-IV (10 or 50 mg/kg/day) for 2 weeks. (B) Detection of fasting plasma glucose (FPG) with or without high-fat diet and streptozotocin administration. Data represent means ± SD. ***p < 0.001, n = 6 biologically independent mice. (C) Representative M-mode echocardiographic images from four respective mouse groups. Cardiac function was assessed using echocardiography at the 2 weeks post-myocardial infarction (MI). (D) Ejection fraction. (E) Fractional shortening. (F) LVIDs, left ventricular internal dimensions during systole (end-systolic diameter). Data represent means ± SD. *p < 0.05; **p < 0.01; ***p < 0.001, versus the PBS group, n = 6 biologically independent mice.

FIGURE 4. Effects of AS-IV on cardiac fibrosis and inflammation in post-MI diabetic mice. (A) Representative Masson’s trichrome-stained myocardial sections in hearts. Blue, scar tissue; red, viable myocardium. (B) Quantitation of infarct size in the four indicated experimental groups. Data represent means ± SD. **p < 0.05; **p < 0.01. n = 6 biologically independent mice. Quantitative analysis of mRNA expression of proinflammatory cytokines and chemokines, including TNF-α (C), IL-1β (D), IL-6 (E), and MCP-1 (F), in the border zone of infarct at 1 week post-MI. mRNA expression normalized to GAPDH expression of the same group. Data represent means ± SD. *p < 0.05; **p < 0.01; ***p < 0.001, n = 6 biologically independent mice.

FIGURE 5. Effect of AS-IV treatment on the activation of MAPK signaling pathway in diabetic MI hearts. (A) Representative images of Western blot of p38, p-p38, JNK, p-JNK, ERK, and p-ERK. The protein expressions of p38 (B), p-p38 (C), JNK (D), p-JNK (E), ERK (F), and p-ERK (G) were quantified by Image J software. Data represent means ± SD. ns, no significant difference, *p < 0.05; **p < 0.01; ***p < 0.001, versus PBS group, normalized to GAPDH level, n = 3 biologically independent samples.