Treatment with Sheng-Mai-San Reduces Myocardial Infarct Size Through Activation of Protein Kinase C and Opening of Mitochondrial KATP Channel

American Journal of Chinese Medicine,  Spring, 2001  by Ningyuan Wang,  Shinya Minatoguchi,  Yoshihiro Uno,  Masazumi Arai,  Kazuaki Hashimoto,  Yasuko Hashimoto,  Kazushige Yamaguchi,  Kazunori Fukuda,  Seigo Akao,  Hisayoshi Fujiwara

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Abstract: Sheng-Mei-San (SMS), a traditional Chinese formulation comprising Radix Ginseng, Radix Ophiopogonis and Fructus Schisandrae, has long been used for more than 700 years for patients with coronary heart disease. We attempted to clarify 1) whether SMS reduces myocardial infarct size, and 2) whether the infarct size-reducing effect of SMS is related to activation of protein kinase C and the opening of the mitochondrial KATP channels in Japanese white rabbits without collateral circulation. The results indicate that three days treatment but not acute treatment with SMS reduces myocardial infarct size through activation of protein kinase C and opening of the mitochondrial KATP channels.

Sheng-Mai-San (SMS), a traditional Chinese formulation comprising Radix Ginseng, Radix Ophiopogonis and Fructus Schisandrae, has long been used for treatment of loss of essence-energy and excessive body fluid, and is especially prescribed for patients with coronary artery disease (Chen and Li, 1993). Recently, it has been reported that SMS is protective against myocardial infarction (Dong and Chen, 1983) and that SMS attenuated the ultrastructural damage of myocytes during ischemia and reperfusion (Yao et al., 1988). Furthermore, it has been reported that SMS was found to be effective in protecting the heart against isoproterenol-induced myocardial injury and ischemia-reperfusion injury in isolated rat hearts (Li et al., 1996). However, it has not yet been elucidated whether SMS reduces myocardial infarct size in an in vivo model of ischemia and reperfusion. Therefore, the aim of the present study was to clarify 1) whether SMS reduces myocardial infarct size, and 2) whether the infarct size-reducing effect of SMS is related to activation of protein kinase C and the opening of the mitochondrial KATP channels in an in vivo model of rabbits without collateral circulation.

Materials and Methods

Animal

Male Japanese white rabbits (Chubu-Kagaku-Shizai Co., Nagoya, Japan) each weighing 2 kg were used. None of the rabbits had any clinically evident infections.

In this study, all rabbits received humane care in accordance with the Guide for the Care and Use of Laboratory Animals, published by the U.S. National Institute of Health (NIH publication 8523, revised 1985). The study protocol was approved by the Ethical Committee of Gifu University School of Medicine, Gifu, Japan.

Surgical Preparation

Rabbits were anesthetized with sodium pentobarbital (30-40 mg/kg, i.v.) and additional doses were given when required throughout the experiment. They were intubated and ventilated with room air supplemented with a low flow of oxygen by a mechanical ventilator (tidal volume, 20-30 ml; respiratory rate, 20-30/min; model SN-480-5; Shinano, Tokyo, Japan). Serial blood gas analysis was performed, and ventilatory parameters were adjusted to keep the arterial blood gas within the physiologic range. Surgery was performed under sterile conditions. The right carotid artery and jugular vein were cannulated to monitor peripheral arterial pressure and to administer drugs or saline and to take blood samples. Then rabbits were given heparin (500 U/kg). A thoracotomy was performed in the third intercostal space, and the heart was exposed after excising the pericardium. A 4-0 silk suture on a small curved needle was passed through the myocardium beneath the middle segment of the large arterial branch coursing down the middle of the anterolateral surface of the left ventricle. A small vinyl tube was passed into both ends of the suture, and the coronary branch was occluded by pulling the snare, which was then fixed by clamping the tube with a mosquito hemostat. Myocardial ischemia was confirmed by regional cyanosis and electrocardiographic change. Reperfusion was confirmed by myocardial blush over the risk area after releasing the snare. After the initial preparation but before coronary occlusion, the animals were assigned randomly to each group. All rabbits were allowed to rest for 20 min after completion of the surgical preparation before the start of the protocol. All rabbits underwent a 30-min occlusion of an anterolateral branch of the coronary artery followed by a 48-h reperfusion.

Protocol

Figure 1 summarizes the experimental protocol. The animals were subjected to 30 min of ischemia and 48 hour of reperfusion. The control group (n = 10) was injected with saline 30 min before the 30 min ischemia. The SMS1-1 group (n = 7) was given bolus i.v. 1.67 ml/kg of SMS (Radix Ginseng 0.1 g/ml, Radix Ophiopogonis 0.321 g/ml and Fructus Schisandrae 0.156 g/ml) 30 min before ischemia. The SMS 1-2 group was given 3.34 ml/kg of SMS. The SMS2 group (n = 9) was given SMS (1.67 ml/kg/day) for 3 days before ischemia. The SMS2+ST group (n = 8) was given staurosporine (ST, an inhibitor of protein kinase C, 50 [micro]g/kg, i.v.) 10 min before ischemia in the presence of SMS as in the SMS2 group. The SMS2+5-HD group (n = 8) was given 5-hydroxydecanoate (5-HD, an inhibitor of the mitochondrial KATP channel, 5 mg/kg, i.v.) 10 min before ischemia in the presence of SMS as in the SMS2 group. The ST group (n = 7) and 5-HD group (n = 7) were given ST and 5-HD 10 min before ischemia respectively. Hemodynamic parameters (mean blood pressure, heart rate) were monitored throughout the experiment. After the experiment, the chest was closed and the rabbits were allowed to recover from anesthesia for 2 days. The animals were killed after 48 hr of reperfusion.