| Objective:Acupuncture, an ideal treatment for myocardial ischemia, has a good efficiency in curing that disease, however, its mechanism in the treatment is still be undefined. This study aims to contrast the effect and regulation of cytikine such as thromboxane B2( TXB2), prostaglandin 1α(PGF1α),hypoxia-inducible factor(HIF-1α), nitric oxide(NO) in pre and post treatment via exploring the curative function of electroacupuncture at Neiguan as well as observe the different structure of myocardial cells before and after acupuncture treatment, meanwhile, a breakthrough in traditional study of sodium, potassium and calcium ion channels during the process of myocardial ischemia is obtained to reveal the mechanism of electroacupuncture for myocardial ischemia at the angle of acid-sensing ion channels.Materials and methods:Part one:50 healthy SPF grade male SD rats were selected, weight 230±50g,provided by Liaoning Changsheng Biotechnology Co,Ltd. The rats were randomly divided into groups of 10, consisting normal group(A), model group(B), Neiguan [PC6] group(C), Lieque [LU7] group(D), random acupoint group(E). The rats in each group were weighted before injectingdrugs, they were continuously administered with 85mg/kg once daily with intravenous isoprenaline(ISO), then the myocardial ischemia rats in Neiguan [PC6] group(C), Lieque [LU7] group(D), random acupoint group(E) were given electro-acupuncture treatment. After one week, SD rats in each group were weighted, and 3.5% sodium pentobarbital were injected intraperitoneally, then separated left ventricular myocardial tissue of anesthetized rats, and put it in 4% paraformaldehyde, observed the morphology change of myocardial cell with hematoxylin-eosin(HE)staining light and apoptosis fluorescence expression of myocardial cell by TUNEL method. After separation of left ventricular myocardial tissue,drew abdominal aorta blood of rats in each group, put it into EP tube added by anticoagulant, and took the serum after centrifugal then detected concentration of TXB2 〠PGF1α with the method of double antibody sandwich elisa.Part two:50 healthy SPF grade male SD rats were selected, weight 230±50g,provided by Liaoning Changsheng Biotechnology Co,Ltd. The rats were randomly divided into groups of 10, consisting normal group(A), model group(B), Neiguan [PC6] group(C), Lieque [LU7] group(D), random acupoint group(E). The rats in each group were weighted before injecting drugs, they were continuously administered with 85mg/kg once daily with intravenous isoprenaline(ISO), then the myocardial ischemia rats inNeiguan [PC6] group(C), Lieque [LU7] group(D), random acupoint group(E) were given electro-acupuncture treatment. After one week, SD rats in each group were weighted, and 3.5% sodium pentobarbital were injected intraperitoneally, drew abdominal aorta blood of anesthetized rats in each group, put it into EP tube added by anticoagulant, and took the serum after centrifugal, then detected concentration of HIF-1α with the method of ELISA, meanwhile, detected absorbance and NO’s concentration of rats in each group.Part three:50 healthy SPF grade male SD rats were selected, weight 230±50g,provided by Liaoning Changsheng Biotechnology Co,Ltd. The rats were randomly divided into groups of 10, consisting normal group(A), model group(B), Neiguan [PC6] group(C), Lieque [LU7] group(D), random acupoint group(E). The rats in each group were weighted before injecting drugs, they were continuously administered with 85mg/kg once daily with intravenous isoprenaline(ISO), then the myocardial ischemia rats in Neiguan [PC6] group(C), Lieque [LU7] group(D), random acupoint group(E) were given electro-acupuncture treatment. After one week, SD rats in each group were weighted, and 3.5% sodium pentobarbital were injected intraperitoneally, then separated left ventricular myocardium, put it into EP tube, then placed in-70℃ before use. Western blot analysis and RT-PCR were performed to check for the protein and genomicexpression of ASIC2 and ASIC3. The genomic expression was quantified by 2^(-ΔCtof). Drew abdominal aorta blood after separation of myocardium, put it into EP tube added by anticoagulant, and took the serum after centrifugal, then detected concentration of CK-MB, and LDH after ELISA.All the measurement data were expressed by mean ± standard deviation( x ±s), analyzed by SPSS 13.0 statistical software, one-way analysis of variance(ANOVA) were applied in group comparison and least significant difference(LSD) were applied in multiple comparison.When p<0.05 or p<0.01, means that it has significant difference.Results:Part one:1. By observing the morphology change of myocardial cells in each group via electron microscope(400), after HE staining, myocardial tissues of rats in ISO model group are necrosis with different degrees, the center part in necrotic myocardial cells are disintegrated and disappeared,and have lots of inflammatory cells infiltration, meanwhile, myocardial cells are at the status of vacuolar and granular degeneration with different degrees, interstitial edema is obviously, severe cases are emerged minimal necrosis, in which lymphocytic infiltration can be seen. Vacuolar degeneration of myocardial cells in Neiguan group is decreased,myocardial cells have less inflammatory infiltration and are arrangedneatly. There is no significant difference in Lieque group and random acupoint group.After paraffin section of myocardial tissues, five high power light microscope vision(×400) are selected for each slice, 30 visions are used to record the number of positive and myocardial cells, the apoptosis condition of myocardial cells in each group are reflected by apoptosis index(AI), after one week electroacupuncture, AI of rats in each group are decreased, contrast with model group, it has a significant difference(p<0.05), AI in Neiguan group are more decreased(p<0.01). After contrast in LSD method, there is a significant difference between random acupoint group and Neiguan group(p<0.05).2. Observing the myocardial tissues of rats in ISO model group via fluorescence microscope with TUNEL method finds that there are more positive apoptotic cells with brown-yellow staining nuclear in normal group, while positive apoptotic cells with brown-yellow staining nuclear in Neiguan group are decreased. There are no significant difference both in Lieque group and random acupoint group.3. Contrast with normal group, the serum concentration of TXB2 is increased and PGF1α is decreased(p<0.05). After one week electroacupuncture, the serum concentration of TXB2 in each group are decreased, PGF1α in each group are increased, contrast with model group,there is a significant difference(p<0.05), change of TXB2 and PGF1α’sconcentration in Neiguan group are more significant(p<0.01). After contrast among all the groups with LSD method, concentration of TXB2 and PGF1α in each group have significant differences(p<0.05).Part two:1. Contrast with normal group, HIF-1α’s concentration of rats in model group is increased(p<0.05). After one week electroacupuncture,HIF-1α’s concentration of rats in each group are decreased, contrast with model group, there is a significant difference(p<0.05), change of HIF-1α’s concentration in Neiguan group is more significantly(p<0.01).There is a significant difference between random acupoint group and Neiguan group(p<0.05).2. Contrast with normal group, concentration of NO in model group is increased(p<0.05). After one week electroacupuncture, concentration of NO in each group are decreased, contrast with model group, there is a significant difference(p<0.05), that in Neiguan group is changed significantly(p<0.01). Contrast between random acupoint group and Neiguan group, there is a significant difference(p<0.05).Part three:1. Contrast with normal group, the serum concentration of creatine kinase isoenzyme(CK-MB) and lactate dehydrogenase(LDH) are increased(p<0.01). After one week electroacupuncture, concentration of CK-MB and LDH in each group are decreased, contrast with model group,there is a significant difference(p<0.05 or p<0.01), contrast Neiguan group with other groups, concentration of CK-MB and LDH in each group are decreased, there is a significant difference(p<0.05 or p<0.01).2. Contrast with normal group, expression content of ASIC2, ASIC3 in model group are significantly increased(p<0.01). After one week electroacupuncture, the expression content in each group are decreased,contrast with model group, there is a significant difference(p<0.05 or p<0.01), contrast with other groups, that in Neiguan group are decreased,there is a significant difference(p<0.05 or p<0.01).3. Contrast with normal group, expression content of ASIC2, ASIC3 in model group are significantly increased(p<0.01). After one week electroacupuncture, that in each group are decreased, contrast with model group, there is a significant difference(p<0.05 or P<0.01), contrast with other groups, expression content of ASIC2, ASIC3 in Neiguan group are decreased, there is a significant difference(p<0.05 or p<0.01).Conclusion:1. Electroacupuncture at Neiguan can improve the injury and apoptosis of myocardial ischemia rats’ myocardial cells, decrease the serum concentration of TXB2, increase the concentration of PGF1α,inhibit the openness of ASICS.2. Electroacupuncture at Neiguan can decrease the serum concentration of HIF-1α and content of NO, inhibit the openness ofASICS.3. Electroacupuncture at Neiguan can decrease the serum concentration of CK-MB and LDH.4. Electroacupuncture at Neiguan can decrease expression content of ASIC2, ASIC3, so that inhibit the openness of ASIC and improve injury of myocardial cells.
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