Myocardial infarction is a major cause of death and disability worldwide [1]. Indeed, the use of thrombolytic therapy or primary percutaneous coronary intervention is the most effective strategy for reducing the size of a myocardial infarct and improving the clinical outcome. Unfortunately, restoring blood flow to the ischemic myocardium can also induce injury. It was coined the term of myocardial ischemia-reperfusion (I/R) injury [2]. How to protect against myocardial I/R injury has become a hot topic. The function of apoptosis in cardiovascular diseases is gaining recognition. Recent studies demonstrated that oxidative stress and I/R injury not only caused myocardial necrosis but also induced cardiomyocyte apoptosis [3]. Apoptosis is controlled by a series of physiological processes that mediate the signaling pathway; blocking the signaling pathway helps prevent myocardial apoptosis [3]. Procyanidins are the major group of polyphenols in the human diet because of their widespread occurrence in fruits, beans, cocoa-based products, wine, and beer [4, 5]. In recent years, the study of the physiological and pharmacological effects of procyanidins has been increasing. In the cardiovascular diseases, procyanidins provide several benefits, such as vasodilatation, antioxidation, improvement of endothelial function, anti-inflammatory and so on [6]. However, the effect of procyanidin on apoptosis has not been reported in myocardial infarction model. Therefore, the present study was designed to evaluate the effects of procyanidin on I/R and explore its potential mechanism.

The underlying pathophysiological mechanisms of I/R injury have not been fully elucidated [10]. It has been suggested that cell apoptosis, oxidative stress and calcium overload during the first minutes of reflow might be involved [10, 11]. Apoptosis is a process of normal cell death that maintains tissue homeostasis, but excessive apoptosis or its dysregulation can lead to various pathological processes, such as myocardial I/R injury [12]. Myocardial apoptosis accelerates the development of necrosis which might determine the degree of myocardial injury. How to reduce myocardial apoptosis is one of the hot spots in myocardial preservation. Oxidative stress is closely associated with cell apoptosis [13]. Reactive oxygen species ROS, including superoxide anion radicals, hydrogen peroxide, and hydroxyl radical, are produced from normal cellular metabolism process and some external factors such as exposure to agents known to cause oxidative stress. Physiological levels of ROS play an important role in intracellular signal transduction, follicle development, ovulation, and gene expression, while excessive ROS production leads to oxidative stress, which damages intracellular DNA, biomembrane lipids, proteins, and other macromolecules [14]. Accumulating evidence shows that the increased production of ROS has been related to vascular diseases, such as atherosclerosis, hypertension, diabetes vasculopathy, and restenosis. This increase seems to contribute to endothelial dysfunction, which is an early step of atherosclerosis [15]. At the beginning of myocardial I/R, a transient release of ROS is promptly generated which is responsible for a further reperfusion injury [16]. A great deal of research has showed that ROS generation may result in apoptosis during reperfusion injury, and reduced ROS generation can suppress reperfusion injury via activation of pathways [17].

Lysiak et al. [18] have investigated ROS in the mouse testis after I/R was mediated via a mitochondrial Caspase-9-dependent pathway involving the upstream mediators Caspase-2 and Bax. Shibata et al. [19] have demonstrated apoptosis in the neurodegenerative diseases was correlated with the activation of a ROS-p53-Caspase-9-mediated pathway. The ability of p53 to induce programmed cell death, or apoptosis, of cells exposed to environmental or oncogenic stress constituted a major pathway whereby p53 exerts its tumor suppressor function [20]. Liu et al. have found that removing ROS production significantly reduced p53, which exerting a positive feedback on ROS production [21]. P53 gene was an activator of cell apoptosis and inhibiting p53 gene significantly reduced cell apoptosis [22]. P53 can be transferred from the cytoplasm to the mitochondrial surface, ultimately leading to the endogenous mitochondrial dysfunction [23]. Endogenous mitochondrial dysfunction pathway was considered to be one of the major mechanisms responsible for I/R-induced cell death in ischemic heart [24].

Antiapoptotic members such as Bcl-2 family members play essential roles in apoptosis caused by all kinds of stimulus signals. Bcl-2 and Bax play important pathophysiological roles in the protection and acceleration, respectively, of myocyte apoptosis after ischemia and/or reperfusion [25]. Bcl-2 prevents death that occurs through the intrinsic apoptotic pathway by preventing the loss of outer mitochondrial membrane integrity. Bax, a member of the bcl-family, homodimerizes and forms heterodimers with bcl-2 protein, reducing its anti-apoptotic effect. The balance of Bcl-2 and Bax in apoptotic cells is directly related to the regulation of apoptosis: the increase in Bax levels promotes cell apoptosis, whereas Bcl-2 increases the inhibition of cell apoptosis. The Bcl-2/Bax ratio determines the viability of cells after apoptotic stimulation [26]. When the Bcl-2/Bax ratio increases, the apoptosis rate is inhibited. And the decreased Bcl-2/Bax ratio leads to apoptosis. Caspases-activated mitochondrial pathway is a definitely regulatory mechanism of apoptosis. In the Caspases-activated mitochondrial pathway, the Caspases family is responsible for important apoptosis regulation. Caspases-3 is the most important apoptotic performer. Bcl-2 blocks the activation of upstream Caspases by interfering with the cytochrome c release and inhibits cell apoptosis [27]. Increases in p53 and Bax expressions may lead to mitochondrial depolarization and cytochrome c release [25]. Cytochrome c and apoptosis protease activation factor 1 (Apaf-1) and procaspase-9 binded and formed apoptotic bodies and then further activated procaspase-9 into Caspase-9, resulting in the downstream activation of executioner Caspases (Caspase-3, 6, 7) to augment apoptosis [28, 29]. Our results indicate, compared with the ischemic group, the low dose of procyanidin and the high dose of procyanidin groups decreased p53, Caspase-9, Caspase-3 and Bax expressions and increased Bcl-2 protein expression as well as Bcl-2/Bax ratio, especially in high dose of procyanidin group. Although we have identified some mechanisms of procyanidin-mediated cardioprotection, these are not necessarily the only, or even the most important, effects. It still requires a great number of animal experiments in order to verify the mechanism of procyanidin-mediated cardioprotection.

Our study also demonstrates that procyanidin effectively protects cardiac myocytes against I/R injury. Compared with the ischemic group, the low dose of procyanidin and the high dose of procyanidin groups decreased the number of apoptotic cells, serum CK-MB level. Thereby we proposed that procyanidin protected against reperfusion injury-induced cell apoptosis with a dose-dependent.

In summary, our study shows that procyanidin ameliorated reperfusion injury by exerting an anti-apoptotic effect on rats. This beneficial effect is partially dependant on decreased ROS, p53, Caspase-9, Caspase-3 and Bax expressions, increased Bcl-2 expression and Bcl-2/Bax ratio. The present study provides experimental evidence to merit further exploration of the possible use of procyanidin, as a therapeutic approach in the treatment of I/R injury on rats. It has only begun with a prologue for a new research and still requires a great number of experiments in order to verify the novel therapeutic approaches to ischemic injury.