| Background:Manifestations of ischemic cardiomyopathy include myocardial ischemia and myocardial infarction. The improvement of living standards and the pressures of social competition result in an increasing incidence, which showing a trend of younger age.How to reduce mortality and improve myocardial ischemia after myocardial infarction has became the hot spots. In recent years, electro-acupuncture (EA) treatment on ischemic cardiomyopathy has shown its own unique effect at home and abroad. In this adjustment process, the hypothalamus and the rostral ventrolateral medulla (RVLM) are considered as key areas for central cardiovascular control, which may be involved. But its mechanism is still not very clear. In1998, researchers discovered a novel hypothalamic neuropeptide called orexin, which has diverse functions in appetite, metabolism, and sleep-wakefulness regulation. Meanwhile, cardiovascular regulation of orexin gets more and more attention. Recent studies have confirmed that central reactive oxygen species (ROS) is involved in the cardiovascular effects via sympathoexcitatory pathway. Well then, whether central orexin is involved in the adjustment process of electro-acupuncture on myocardial ischemia, and whether the PVN and the RVLM participate in regulating the cardiac function when EA was applied and whether ROS mediate orexin to regulating myocardial ischemia. These mechanisms require further study.Objectives:This present study is to establish acute myocardial ischemia model in rats and observe that electroacupuncture (EA) at "Neiguan"(Pe6) acupoint could regulate the cardiac function of acute myocardial ischemia (AMI) rats.we detect the expression of OXA in hypothalamus and OX1R in RVLM. Meanwhile we investigate the the influence of the inhibitors of OXA and NADPH oxidase, the superoxide dismutase mimicry on the cardiovascular effects of OXA in RVLM and test the content change of amino acid transmitters in RVLM. Thus, we test the hypothesis that center ROS mediate the cardiovascular effects of OXA, and further investigate the central cardiovascular mechanisms of EA on AMI rats. Methods:AMI model was established by occlusion of the left anterior descending branch of the coronary artery. Bilateral "Neiguan" points (PC6) was used as the acupoint and bilateral "lieque" points as control. Heart rate(HR^mean artery pressure(MAP) and intraventricular pressure(IVP) were monitored, so as to observe the effects of EA on AMI rats. The infarcted area was shown by tetrazolium chloride (TTC) staining and HE staining was used to observe the micro-structure of infracted myocardium.The echocardiography showed the heart wall structure. Program of heart rate variability (HRV) recorded cardiac autonomic nervous system function. Plasma norepinephrine (NE) and epinephrine (E) were measured by enzyme-linked immunosorbent assay (Elisa). Immunohistochemistry (IHC), real-time PCR and western blot analysis were used to detect the expression OXA in HPO and OX1R in RVLM of every group. Superoxide production was detected by using lucigenin-enhanced chemilumnescence assay. Confocal laser microscopy combined with the double-label immunofluorescence was used to observe the colocalization and close contact between OX1R and NADPH subunit. By means of microinjection, we tested the central cardiovascular effects of OXA and the influence of the inhibitors of OXA and NADPH oxidase, the superoxide dismutase mimicry on the cardiovascular effects. In addition, Micro-dialysis combined with high performance liquid charomatography (HPLC) was applied to test the amino acid transmitters in RVLM of every group.Results:(1) Establishment of AMI model:Successful of duplicating AMI model was depended on S-T segment elevating immediately in ECG typical lead II and the myocardium presenting pale and poor weakening movement. TTC and HE staining showed the infarcted myocardial tissue, the echocardiography showed that left ventricular end-diastolic dimension (LVDd) and left ventricular end-systolic dimension (LVDs) obviously increased, but interventricular septum thickness became thin.(2) Changs of cardiac functional indices:Post-ligation5days, HR increased, other cardiac functional decreased, including LVSP, LVEDP,-dp/dt,+dp/dt (P<0.01,n=6). After EA at bilateral "neiguan" acupoints treatment, HR decreased, other cardiac functional were improved (P<0.01,n=6). However, compared with bilateral "neiguan" acupoints, EA at bilateral "lieque" acupoints did not improved cardiac function of AMI rats (P<0.05or P<0.01,n=6)(3) HRV and assay of NE and E:HRV analysis of AMI rats showed that LF/HF ratio increased and the ratio decreased after EA treatment (P<0.05,n=6). EA could reduce the concentrations of NE and E in the serum compared with the AMI (P<0.05,n=6).(4) IHC showed that in AMI rats, the OXA immunoreactive neurons in hypothalamus and OX1R immunoreactive neurons in RVLM were signigicantly up-regulated, which was suggested by increased ROD and the number of the positive neurons (P<0.05, n=6). EAat bilateral "Neiguan" acupoints can down-regulate the expression of OXA immunoreactive neurons and OX1R immunoreactive neurons, respectively, compared with that of AMI rats. Conversely, EA at "Lieque" did not significantly decrease the expression of OXA in the PVN and the expression of OX1R the RVLM of AMI rats, compared with EA at bilateral "neiguan" acupoints.(5) Western blot analysis showed that the expression of OX1R in RVLM of AMI rats was significantly increased, compared with control rats (P<0.01,n=4). After EA treatment the expression of OX1R in RVLM decreased, which, however, had no significant statistical difference.(6) Realtime-PCR showed PPO mRNA expression was higher in the hypothalamus of AMI, compared with those of the sham control rats (P<0.01,n=7). After EA treatment at "neiguan" acupoints, PPO mRNA expression decreased (P<0.05,n=7).(7) Confocal laser microscopy showed the colocalization and close contact between OX1R and NADPH subunit. NADPH memberance-bound subunit gp91phox or cytosolic subunit p47phox in RVLM were double-labeled with OX1R respectively.(8) Lucigenin-enhanced chemilumnescence assay showed superoxide production in RVLM of AMI rats was significantly increased, compared with control rats (P＜0.05, n=7). With EA treatment, they decreased (P＜0.05, n=7). Intracerebroventricular injection of OXA (100mol) could increase superoxide production in RVLM of AMI rats, while SB (1OOpmol)、Tempol (50nmol) or Apo (5nmol) could block the effects of OXA (P＜0.05or P<0.01,n=4). (9) Unilateral microinjection of OXA(100pmol) into RVLM in every group(control group、AMI group and AMI+EA group) induced significant elevation of parameters of the cardiac function (HR、MAP、+dp/dtmax and-dp/dtmax), and the increased extent was more remarkable in AMI rats. Injection of SB、Tempol and Apocynin in RVLM could block the effects of OXA (P＜0.05or P＜0.01, n=7)(10) Micro-dialysis combined with HPLC showed the excitatory amino acid (EAA), glutamate (Glu), increased in the dialysate during the first and second periods of10min after the microinjection of OXA into RVLM, while level of inhibitory amino acid(IAA), glycine (Gly) and taurine (Tau), decreased in the cerebrospinal fluid in RVLM (P＜0.05, n=6). Compared with microinjection of OXA alone, Tempol (50nmol)、Apocynin (5nmol) microinjected5min before OXA could abolish the effects OXA, leading to the decrease of Glu and an little increase of Gly and Tau (P<0.05,n=6).Conclusions:By the method of ligating left anterior descending (LAD) coronary artery could successfully duplicate the rat model of AMI.Cardiac function parameters of the model were abnormal and accompanied augmented sympathetic activity. EA at "neiguan" acupoints on AMI rats could improve the cardiac function and decrease sympathetic activity. The mechanism was related to up-regulated expression of OXA in HPO and OX1R in RVLM. Moreover superoxide production in RVLM of AMI rats was significantly increased and OX1R was colocalization with NADPH subunit in RVLM. And injection of Tempol and Apocynin in RVLM could block the cardiovascular responses of exogenous OXA. Furthermore, after the microinjection of OXA into RVLM, level of EAA increased and level of IAA decreased. Injection of Tempol and Apocynin in RVLM could block the effects of exogenous OXA. All of these suggest one of the mechanisms of EA improving AMI:EA treatment can down-regulate expression of OXA and OX1R, then activity of NADPH subunit decreased and the release of superoxide production reduced, furthermore, level of EAA decreased and level of IAA increased. In shot, the over-excited sympathetic outflow reduce and sympathetic activity close to normal, then myocardium gets protections and cardiac function gets improved. |
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