Bloodletting Acupuncture at Jing-Well Points Alleviates Myocardial Injury in Acute Altitude Hypoxic Rats by Activating HIF-1 α/BNIP3 Signaling-Mediated Mitochondrial Autophagyand Decreasing Oxidative Stress

Objective: To explore the protective effect and possible mechanisms of bloodletting acupuncture at Jing-well points(BAJP) pre-treatment on acute hypobaric hypoxia(AHH)-induced myocardium injury rat. Methods:Seventy-five rats were randomly divided into 5 groups by a random number table: a control group(n=15), a model group(n=15), a BAJP group(n=15), a BAJP+3-methyladenine(3-MA) group(n=15), and a BANA(bloodletting at nonacupoint;tail bleeding, n=15) group. Except for the control group, the AHH rat model was established in the other groups, and the corresponding treatment methods were adopted. Enzyme-linked immunosorbent assay(ELISA) was used to detect creatine kinase isoenzyme MB(CK-MB) and cardiac troponins I(CTn I) levels in serum and superoxide dismutase(SOD) and malondialdehyde(MDA) levels in myocardial tissue. Hematoxylin-eosin(HE)staining was used to observe myocardial injury, and terminal deoxynucleotidyltransferase-mediated d UTP-biotin nick end labeling(TUNEL) staining was used to observe cell apoptosis. Transmission electron microscopy detection was used to observe mitochondrial damage and autophagosomes in the myocardium. The mitochondrial membrane potential of the myocardium was analyzed with the fluorescent dye JC-1. Mitochondrial respiratory chain complex(complex Ⅰ, Ⅲ, and Ⅳ) activities and ATPase in the myocardium were detected by mitochondrial respiratory chain complex assay kits. Western blot analysis was used to detect the autophagy index and hypoxia inducible factor-1α(HIF-1α)/Bcl-2 and adenovirus E1B 19k Da-interacting protein 3(BNIP3) signaling. Results:BAJP reduced myocardial injury and inhibited myocardial cell apoptosis in AHH rats. BAJP pretreatment decreased MDA levels and increased SOD levels in AHH rats(all P<0.01). Moreover, BAJP pretreatment increased the mitochondrial membrane potential(P<0.01), mitochondrial respiratory chain complex(complexes Ⅰ, Ⅲ, and Ⅳ)activities(P<0.01), and mitochondrial ATPase activity in AHH rats(P<0.05). The results from electron microscopy demonstrated that BAJP pretreatment improved mitochondrial swelling and increased the autophagosome number in the myocardium of AHH rats. In addition, BAJP pretreatment activated the HIF-1α/BNIP3pathway and autophagy. Finally, the results of using 3-MA to inhibit autophagy in BAJP-treated AHH rats showed that suppression of autophagy attenuated the treatment effects of BAJP in AHH rats, further proving that autophagy constitutes a potential target for BAJP treatment of AHH. Conclusion: BAJP is an effective treatment for AHH-induced myocardial injury, and the mechanism might involve increasing HIF-1α/BNIP3 signaling-mediated autophagy and decreasing oxidative stress.

Synergic effects of NO and oxygen free radicals in the injury of ischemia-reperfused myocardium——ESR studies on NO free radicals generated from ischemia-reperfused myocardium

The ESR signal of NO bound to hemoglobin was detected during the ischemia-reperfusion of myocardium with low temperature ESR technique, and the synergic effects of NO and oxygen free radicals in the injury of the process were studied with this technique. Oxygen free radicals and NO bound to β-subunit of hemoglobin (β-NO complex) could be detected simultaneously in the ischemia-reperfused myocardium. Those signals could not be detected from the normal myocardium even in the presence of L-arginme. However, those signals could be detected and were dose-dependent with L-arginine in the ischemia-reperfused myocardiums and the signal could be suppressed with the inhibitor of NO synthetase, NG-nitro-L-arginine methylester (NAME). Measurement of the activities of lactate dehydrogenase (LDH) and creatine kinase (CK) in the coronary artery effluent of ischemia-reperfused heart showed that L-arginine at lower concentration (<1 mmol/L) could protect the heart from the ischemia-reperfusion injury but at higher concentration aggravate the injury. Addition of NAME to the reperfusion solution could also protect the myocardium. Addition of xanthine (X) ,'xanthine oxidase (XO) or Fe2+/H2O2 to the reperfusion solution increased the production of NO and oxygen free radicals and the ischemia-reperfused injury simultaneously. Addition of superoxide dismutase (SOD) and catalase decreased the production of NO and oxygen free radicals and the ischemia-reperfusion injury.

INCREASED VULNERABILITY OF HYPERTROPHIED MYOCARDIUM TO ISCHEMIA AND REPERFUSION INJURY RELATION TO CARDIAC RENIN-ANGIOTENSIN SYSTEM

Hearts of pressure-overload hypertrophy show an increased activation of intracardiac renin-angiotensin system which may contribute to ischemia and reperfusion injury. The purpose of this study is to evaluate whether the hypertrophied myocardium is more vulnerable to ischemia and reperfusion injury and to find out its relation to the cardiac renin-angiotensin system. Hypertrophied rat hearts induced by abdominal aortic banding for 6 weeks were subjected to 2 hours of hypothermic ischemic arrest followed by 30 minutes of reperfusion, and their cardiac function recovery was compared with that of sham-operated normal control hearts. The cardiac renin activity and angiotensin II content before ischemia and after reperfusion were determined. It was found that both the pre-ischemic renin activity and angiotensin II level were higher in hypertrophied myocardium than those in the control: ischemia and reperfusion injury increased both renin activity and angiotensin II content in the two groups, but the renin activity and angiotensin II level were further elevated after reperfusion in the hypertrophied hearts than those in the control hearts. Meanwhile, the cardiac function recovery after 30 minutes reperfusion in the hypertrophied hearts was poorer than that in the control. Correlation analysis revealed that there was a negative correlation between the cardiac output recovery and the myocardial angiotensin II content (r=-0.841), P<0.001), It is concluded that ischemia and reperfusion injury can activate cardiac renin-angiotensin system in isolated rat heart, which may be responsible for the increased susceptibility of the hypertrophied myocardium to ischemia and reperfusion injury.

Protective effect of Shenfu injection on myocardial mitochondria injured by ischemia-reperfusion in rabbits

The main active components of Shenfu injection (SFI), an extract of traditional Chinese herbs, are ginsenoside and higenamine. Ginsenoside can improve ischemic myocardium metabolism, scavenge free radicals, protect myocardial ultrastructure and reduce Ca^(2+) overload. Higenamine can enhance heart contractility, improve coronary circulation and decrease the effect of acute myocardial ischemia. SFI was found to have had some cardiac protective effect during cardiopulmonary bypass.^(1,2) SFI was added into St. Thomas crystal cardioplegic solution in this study to investigate the protective effect of SFI on ischemic-reperfused rabbit heart and to observe the influence on mitochondrial oxygen free radical (OFR), Ca^(2+) and mitochondrial ultrastructure.