| Aim:With myocardial ischemia rats as for the study of carrier, we have observed the amount change of purinergic substances at PC6 and PC3, PC2 located on the pericardium meridian under different conditions and explored the protective effect of acupuncture at PC6 via A2b receptor signalling on the cardiac tissue, thus providing the scientific supports for acupoint specificity of selecting acupoints on the involved meridian.Methods:1、We assigned rats into healthy and model conditions, each condition again divided into three groups:PC6-PC3, PC6-LI11, PC6-PC2 groups. Thus we had total six groups. All the rats were fixed under anesthesia and were acupunctured at PC6 acupoint by using 1 cun needle. At the beginning of insertion, we twirled, thrusted and lifted the needle for 1 minute, then repeated these manipulations every 5 minutes, which lasting for 30 minutes only once. In PC6-PC3 group, we had acupunctured at PC6 and sampled tissue fluids of PC6 and PC3 by microdialysis technique; in PC6-LI11 group, we acupunctured at PC6 and sampled PC6 and LI11; in PC6-PC2 group, we punctured at PC6 and sampled PC6 and PC2. Then the collected tissue fluids were analyzed for amount changes of four different purinergic substances, which including adenosine triphosphate, adenosine diphosphate, adenosine monophosphate and adenosine by using High Performance Liquid Chromatography. Simultaneously, we also took electrocardiogram for each rats. In this way, we could explore the change characteristics of purinergic substances at location of PC6, PC3, PC2 and LI11 under different conditions.2、We had assigned rats into control, fake operation, operation groups. The rats in operation group had been taken left anterior descending ligation then randomly divided into model and acupuncture groups. Thus we had total four groups in this session. At the third day after the operation, the rats in acupuncture group were fixed without anesthesia and acupunctured at bilateral PC6 with 1 cun needles, which connected with Han’s acupoint nerve stimulator(HANS LH200). We were choosing needle model and dilatational wave 2/100Hz, then appling current intensity of 0.5～ 1mA. Under this stimulation, the muscle of rats would slightly shaked. The acupuncture duration was 30 minutes each day, which was lasting for 5 days. Meanwhile, the rats in control, fake operation and model groups were also fixed with the same methods without anesthesia for 5 days. Then we had applied physiological recording instrument, hematoxylin-eosin staining, TdT-mediated dUTP nick end labeling methods to observe the change of EKG, cardiac pathomorphology and the average apoptosis rate in four groups, which to explicit the protective effect of acupuncture at selected acupoint on the involved meridian. Then we had used immunohistochemistry and western blot techniques to explore the specific proteins expression in the signalling pathway of cardiac A2b receptor, including HIF-1α、CRE、 CREB、cullin-1、NEDD8、PER2, trying to reveal the possible biological mechanism of acupuncture at selected acupoint on the involved meridian for treating myocardial ischemia.Results:1、The research on characteristics of purinergic signal changes under different conditions(1) The effect of PC6 acupuncture on HRV of rats:Before acupuncture, the HRV(LF/HF) of rats in model group were higher than healthy group(P<0.05). In healthy state, acupuncture at PC6 had no influence on HRV, while acupuncture at PC6 could significantly reduce the value of HRV(P<0.05) in myocardial ischemia state.(2) The effect of PC6 acupuncture on purinergic substances at PC6 area:In healthy state, when PC6 was acupunctured, the concentrations of ATP and ADP at PC6 area were increased significantly(P<0.05), while the ADO was decreased greatly after acupuncture (P<0.05).In myocardial ischemia state, during acupuncture at PC6 point, the concentrations of ATP, AMP and ADO were raised greatly(P<0.05), then ATP and ADO were decreased significantly after acupuncture manipulation(P<0.05).In ischemia state, during acupuncture at PC6 point, the raising rates of ADO were significantly higher than in healthy state(P<0.05). After acupuncture, the decreasing rate of ADO was also higher than in healthy state(P<0.05).(3) The effect of PC6 acupuncture on purinergic substances at PC3 and PC2:In healthy state, when PC6 was acupunctured, no change had been occurred at PC3 and only ATP at PC2 area was increased significantly(.P<0.05). However, in ischemia state, the concentrations of ATP、ADP and ADO at PC3 area were increased significantly(P<0.05), while the ATP、ADO had been decreased greatly after acupuncture manipulation(P<0.05). At the same time, the concentration of ATP, ADP and ADO at PC2 were raised obviously(P<0.05), while the ADP and ADO were greatly declined after acupuncture(P<0.05).In ishchemia state, the content of ADO at PC3 area was lower than in healthy state(P<0.05). After acupuncture at PC6, the declining rate of ATP at PC3 was greatly higher than in healthy state(P<0.05) and no change had been found at PC2 area.(4) The effect of PC6 acupuncture on purinergic substances at LI 11 area:In healthy state, when PC6 was acupunctured, the concentrations of ADO at LI 11 area was declined significantly(P<0.05), while in ischemia state, theATP、ADO and AMP at LI11 was declined after acupuncture(P<0.05). Which is more, no differences have been found at LI11 area.2、The research on protective effect of acupuncture at selected acupoint on the pericardium meridian on myocardial ischemia rats via cardiac A2b receptor(1) The regulation effect of acupuncture at PC6 on EKG of myocardial ischemia rats:The EKG analysis had showed that the ST segments in model and acupuncture group were both raised significantly at ligation time(P<0.05), while after five days the QRS waves were also deformity in model group and waves in acupuncture group were much similar to normal EKG. However, the ST segments had no statistical differences in four groups eight days later(P>0.05).(2) The effect of acupuncture at PC6 on physiological morphological change and average apoptosis rate of myocardial ischemia rats:The HE staining declared that the cardiac myocytes in control and fake operation group were complete, while the cytoplasm, nucleus and striples were clearly visible. In model group, multiple cardiac myocytes were necrosis, while the inflammatory cells were infiltration and connective tissue were hyperplasia. At the same time, most cardiac myocytes in acupuncture group were complete, and the cytoplasm, nucleus and stripes were almost visible. Little inflammatory cells were infiltration. In TUNEL staining, the average apoptosis rates in model and acupuncture group were significantly higher than the control and fake operation groups(P<0.05), while the rate in acupuncture group was significantly lower than model group(P<0.05).(3) The regulation effect of acupuncture at PC6 on cardiac A2b receptor expression of myocardial ischemia rats:In immunohistochemistry test, the average optical density in model group was significantly higher than control and fake operation group(P<0.05), while the average optical density in acupuncture group was lower than model group(P<0.05).(4) The regulation effect of acupuncture at PC6 on the signal pathway of cardiac A2b receptor of myocardial ischemia rats:By western blot technique, the concentrations of protein HIF-1α, CRE, CREB and PER2 were significantly high in model group when compared with control and fake operation group(P<0.05). The content of protein HIF-1α, CREB and PER2 in acupuncture group were greatly lower than model group(P<0.05). While the concentrations of protein CRE had no statistical differences between acupuncture and model group. However, the concentrations of protein cullin-1 and NEDD8 had no differences in four groups(P>0.05).Conclusions:1、Represented by purinergic substances, the activation and transmission of acupuncture signal is actually possessing the characteristics of space responsibility and time dynamicity. The space responsibility means that the acupuncture could activated purinergic substances in PC6 local area and also let it high at remote acupoints on the involved pericardium meridian, while the time dynamicity is the sharp raise and sharp down trend of purinergic substances before and after acupuncture. Which is more, it is not that all the purinergic substances are taking part in acupuncture signal. This substantial differences are greatly influenced by body functional states.2、The possible mechanism of acupuncture at selected acupoint on the involved pericardium meridian:Five days’ acupuncture manipulation could effectively restrain the hypoxia tolerance signalling pathway mediated by cardiac A2b receptor in myocardial ischemia rats, which result in suppression of anaerobic glycolysis in cardiac tissue, relief of the accumulation of metabolic and alleviation of inflammatory and apoptosis in cardio myocytes. |
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