Cardioprotective effects of salvianolic Acid a on myocardial ischemia-reperfusion injury in vivo and in vitro

Abstract

Salvianolic acid A (SAA), one of the major active components of Danshen that is a traditional Chinese medicine, has been reported to possess protective effect in cardiac diseases and antioxidative activity. This study aims to investigate the cardioprotection of SAA in vivo and in vitro using the model of myocardial ischemia-reperfusion in rat and hydrogen peroxide (H(2)O(2))-induced H9c2 rat cardiomyoblasts apoptosis. It was found that SAA significantly limited infarct size of ischemic myocardium when given immediately prior to reperfusion. SAA also significantly suppressed cellular injury and apoptotic cell death. Additionally, the results of western blot and phospho-specific antibody microarray analysis showed that SAA could up-regulate Bcl-2 expression and increase the phosphorylation of proteins such as Akt, p42/p44 extracellular signal-related kinases (Erk1/2), and their related effectors. The phosphorylation of those points was related to suppress apoptosis. In summary, SAA possesses marked protective effect on myocardial ischemia-reperfusion injury, which is related to its ability to reduce myocardial cell apoptosis and damage induced by oxidative stress. The protection is achieved via up-regulation of Bcl-2 expression and affecting protein phosphorylation. These findings indicate that SAA may be of value in cardioprotection during myocardial ischemia-reperfusion injury, which provide pharmacological evidence for clinical application.

Figure 1

Chemical structure of salvianolic acid A (SAA). Molecular formula: C₂₆H₂₂O₁₀; Molecular weight: 495.45.

Figure 2

Effect of salvianolic acid A (SAA) on infarct size following cardiac ischemia-reperfusion. Rats were subjected to 90 min of ischemia and 24 h reperfusion. SAA (5, 10, 20, and 40 mg/kg) was intravenously administered 20 min before and 1 h after reperfusion using syringe pump, respectively. At the end of the reperfusion, the heart was excised and the ischemic zone was visualized using Evans blue dye. Necrotic tissue was evaluated by triphenyltetrazolium staining. Data are represented as mean ± SD of 12 animals for each group. *P < .05, **P < .01 versus vehicle. I/R: ischemia-reperfusion.

Figure 3

Salvianolic acid A (SAA) inhibits H₂O₂-induced deficiency of cell viability. H9c2 cells were pretreated with or without SAA (0.64–10⁴ nM) for 24 h prior to incubation of cells with H₂O₂ for 1 h. Data are represented as means ± SD of three independent experiments. ^## P < .01 versus control; *P < .05, **P < .01 versus H₂O₂.

Figure 4

Effect of salvianolic acid A (SAA) on SOD activity in H9c2 cells treated with H₂O₂. H9c2 cells were pretreated with or without SAA for 24 h prior to incubation of cells with H₂O₂ for 1 h. SOD activity was measured according to the corresponding commercial kits. Data are represented as mean ± SD of three independent experiments. ^## P < .01 versus normal control; *P < .05, **P < .01 versus H₂O₂.

Figure 5

Salvianolic acid A (SAA) protects H9c2 cells from H₂O₂-induced apoptosis. FCM analysis with Annexin-V binding. H9c2 cells were pretreated with or without SAA for 24 h prior to incubation of cells with H₂O₂ for 1 h. Cells in region A4 represent early apoptotic cells. Data are expressed as mean ± SD of three independent experiments. ^## P < .01 versus control; *P < .05, **P < .01 versus H₂O₂. PI: propidium iodide.

Figure 6

Effect of salvianolic acid A (SAA) on expression of Bcl-2 and Bax in H9c2 cells treated with H₂O₂. H9c2 cells were pretreated with or without SAA for 24 h prior to incubation of cells with H₂O₂ for 1 h. Thereafter, proteins were extracted and the protein levels of Bcl-2 and Bax were determined by Western blot analysis. Lane 1: control; lane 2: H₂O₂ (120 μM); lane 3: H₂O₂ (120 μM) + SAA 0.64 nM; lane 4: H₂O₂ (120 μM) + SAA 3.2 nM; lane 5: H₂O₂ (120 μM) + SAA 16 nM. Data are expressed as mean ± SD of three independent experiments. ^## P < .01 versus control; *P < .05, **P < .01 versus H₂O₂.