| Background:Cardiovascular disease represents the world’s largest killers, which affects adult and aged individuals in both Western and Eastern societies. In particular, substantial morbidity and mortality result from ischemic cardiovascular disease. How to reduce mortality and improve myocardial ischemia has become one of the hot spots. In recent years, electroacupuncture (EA) treatment on ischemic cardiomyopathy has shown its own unique effects at home and abroad. The rostral ventrolateral medulla (RVLM) is a key area for regulation of cardiovascular activities. This area contains many neurotransmitters/neuromodulators, such as angiotensin-(1-7), carbon monoxide, adenosine (ADO) and nitric oxide (NO), which are thought to be important in cardiovascular control. NO is acted as a neural transmitter and is involved in central cardiovascular regulation, but the detailed mechanism is not so clear and may be more complex in the central nervous system (CNS) than that in the periphery. Recent studies have confirmed that central ADO and adenosine A2A receptor (A2AR) are involved in the cardiovascular effects by inhibitory effects on sympathetic outflow. Well then, whether medullary NO/NOS system is involved in the therapeutic effect of EA on myocardial ischemia, and whether adenosine mediates NO/NOS to regulating myocardial ischemia. These mechanisms require further study.Objectives and hypothesis:The present study was performed on acute myocardial ischemia (AMI) model of ligating the left anterior descending coronary artery in rats. To investigate the effect of EA on cardiac function in AMI rats, we observed the expressions of NOS in the RVLM. Meanwhile, we investigated the crosstalk of NO/NOS and ADO/A2AR, which subsequently inhibited acetylcholine (ACh)-M1 receptor (M1R) activation, in order to further study the mechanisms underlying the EA. Thus, we tested the hypothesis that the central NO-dependent sympathetic inhibition was mediated through ADO/A2AR and ACh-M1R in the RVLM, and further investigated the central cardiovascular mechanisms of EA on AMI rats. Methods:AMI model was duplicated by ligation of the left anterior descending coronary artery. Bilateral "Neiguan" (PC 6) acupoints were used as the acupoint and bilateral "lieque" (LU 7) acupoint as the control. Mean artery pressure (MAP) and heart rate (HR) were monitored, so as to observe the effects of EA on AMI rats. The ischemic area and the micro-structure of ischemic myocardium were detected by tetrazolium chloride (TTC) and hematoxylin-eosin (HE) staining. The heart wall structure was shown by the echocardiography. Plasma epinephrine (E) and norepinephrine (NE) were measured by enzyme-linked immunosorbent assay (ELISA). Program of heart rate variability (HRV) and baroreflex sensitivity (BRS) were used for reflecting changes of cardiac autonomic nervous tones. Immunohistochemistry (IHC) and western blot analysis were used to detect the expression of nNOS and iNOS in the RVLM. L-Arg, L-NAME, SCH58261, AG and 7-NI were respectively microinjected into the RVLM. ADO and ACh were measured by ELISA. A2AR and MIR were measured by western blot.Results:1. Preparation of AMI modelSuccess of duplicating AMI model was depended on S-T segment elevating immediately in ECG typical lead II, the myocardium presenting pale and poor weakening movement. And the ischemic myocardium remained pale in color by TTC staining. HE staining showed that AMI cardiac muscle fibers expressed obviously acidophilic alteration. Moreover, 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. All the results demonstrated the success of duplicating AMI model.2. Assay of E and NE and HRV, BRS analysisThe concentrations of E and NE in the serum of AMI rats increased significantly. They were decreased in the EA PC6 group compared with that in the AMI group (P<0.05, n=6). Analysis of HRV and BRS demonstrated that the lower frequency components (LF) and the higher frequency components (HF) of AMI rats decreased, whereas the ratio of LF/HF increased. LF/HF of AMI rats decreased after EA PC6 treatment (P<0.05, n=6). Conversely, EA at the bilateral Lieque acupoints did not alter all parameters of HRVand BRS.3. The expression of nNOS and iNOS positive neurons in the RVLM of each group(1). The numbers of nNOS immunoreactive neurons in the RVLM of AMI group were significantly increased compared with that of the sham operated group (P<0.05, n=6). The number of iNOS immunoreactive neurons in the RVLM of AMI rats was significantly decreased compared with that of the sham operated group (P<0.05, n=6). EA at bilateral "Neiguan" acupoints could decrease the expression of nNOS immunoreactive neurons and increase the expression of iNOS immunoreactive neurons in the RVLM of AMI rats, respectively. Conversely, EA at "Lieque" did not significantly decrease the expression of nNOS in the RVLM and increase the expression of iNOS in the RVLM of AMI rats.(2). Western blot analysis showed that the expressions of nNOS in the RVLM of AMI rats were significantly increased and iNOS was significantly decreased, compared with those of sham operated rats (P<0.05, n=6). After EA PC6 treatment the expressions of nNOS in the RVLM of AMI rats decreased and iNOS in the RVLM of AMI rats increased.4. The NO content and NOS activity in the RVLM of each groupMethod of nitrate reductase was used to detect the NO content and NOS activity. NO content in the RVLM of AMI group was significantly increased compared with that of the sham operated group (P<0.05, n=8). NOS activity was significantly enhanced, while iNOS activity was significantly attenuated. After EA PC6 treatment, NO content was markedly decreased, and the activities of NOS and iNOS were significantly enhanced.5. The effect of L-Arg, L-NAME, SCH58261, AG and 7-NI in the RVLM of normal rats on MAP, HR and power spectral analysis of HRV.L-Arginine (L-Arg, NO donor) was microinjected into the RVLM, and the MAP, HR and LF/HF of the HRV decreased. Whereas pre-treatment of a competitive antagonist of A2AR, SCH58261, the above inhibitory effects of L-Arg were attenuated. Western blot and ELISA showed that following L-Arg microinjection, ADO and A2AR levels increased, whereas ACh and ACh MIR levels decreased significantly in the RVLM along with the cardiovascular responses. And again, the decrease of ACh was abolished by pre-treatment of SCH58261. Moreover, microinjection of NG-nitro-L-Arginine methyl ester (L-NAME, a nonselective nitric oxide synthase (NOS) inhibitor), into the RVLM induced a significant increase in MAP, HR and sympathetic activity shown from the HRV (all of the LF, HF and LF/HF ratio were increased). Aminoguanidine (AG), a competitive antagonist of iNOS, produced increases in MAP, HR and LF/HF.7-nitroindazole sodium salt (7-NI), an antagonist of nNOS, almost produced decreases in MAP, HR and LF/HF.6. The effect of L-Arg, L-NAME, SCH58261, AG and 7-NI in the RVLM of sham, AMI and AMI+EA PC6 rats on MAP, HR and power spectral analysis of HRVMicroinjection of L-NAME into the RVLM in AMI+EA PC6 group induced significant elevation of MAP and HR. The cardiovascular effect of EA was blocked by L-NAME. Microinjection of L-Arg, SCH58261+L-Arg, AG and 7-NI into RVLM in each group (Sham group, AMI group and AMI+EA PC6 group) induced respectively changes of MAP and HR. L-Arg induced the decrease of MAP, HR and HRV, and the decreased extent was more remarkable in AMI rats. This inhibitory effect was attenuated by pre-treatment of SCH58261 in each group. AG produced increases in MAP, HR and LF/HF in all group.7-NI produced decreases in MAP, HR and LF/HF in all group.Conclusions:1. By the method of ligating left anterior descending coronary artery could successfully duplicate the rat model of AMI. Sympathetic outflows of AMI rats increased.2. EA at "neiguan" acupoints on AMI rats could decrease sympathetic outflow. The mechanism was related to the upregulation of nNOS and downregulation of iNOS in the RVLM.3. Our results supported that the central NO/NOS system modulated cardiovascular activities via activation of A2AR, which subsequently inhibited ACh-M1R activation.4. All of these suggested one of the mechanisms of EA PC6 improving AMI. EA PC6 treatment could modulate cardiovascular activity though the central NO/NOS system, which modulated cardiovascular activities via activation of A2AR, which subsequently inhibited ACh-M1R activation. |
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