Acupuncture At Neiguan Acupoint Regulates NO/NOS System And Correlated Neurotransmitters In The Medulla Oblongata Of Acute Myocardial Ischemia Rat

Objectives:We observed that electroacupuncture(EA) at "Neiguan"(Pe 6) acupoint could regulate the cardiac function of acute myocardial ischemia(AMI) rats and the expression of inducible nitric oxide(iNOS) synthase and neuronal nitric oxide synthase(nNOS) in the RVLM.We tested NO/NOS central roles in cardiovascular activities in AMI rats,as well as studied the interaction between the NO/NOS system and amino acid neurotransmitters in regulating cardiovascular functions in the RVLM, so as to investigate the cardiovascular effects and central mechanisms of EA on AMI rats.Methods:Experiment was performed on male Wistar rats and AMI model was established by occlusion of the left anterior descending branch of the coronary artery; electro-acupuncture bilateral "Neiguan" acupoints on AMI rats was performed and the functional indices were observed on rats;The infarcted area was shown by tetrazolium chloride(TTC) staining and HE staining was used to observe the micro-structure of infracted myocardium.Plasma norepinephrine(NE) and brain natriuretic peptide(BNP) were measured by radioimmunoassay(RIA);Real time-polymerase chain reaction(Real time-PCR) and immunohistochemistry(IHC) methods were used to test the iNOS and nNOS expressions in the RVLM;In addition,we tested the central cardiovascular effect of NO/NOS in the RVLM by micro-injection.Micro-dialysis combining high performance liquid chromatography (HPLC) method was exploited,by which we tested the amino acid transmitters in the samples in AMI and acupunctured rats.Results:(1) Successful of duplicating AMI model was depended on S-T segment elevating immediately in ECG typical leadⅡand the myocardium presenting pale and poor weakening movement.Post-ligation 5 days,TTC and HE staining showed the infarcted myocardial tissue.NE and BNP concentrations were higher in serum compared with those of the sham control.(2) Electroacupunctue "Neiguan" for 20 min daily and for successively for 5 days could reduce the infarcted area and concentrations of NE and BNP in the serum compared with the AMI;(3) In AMI+EA group,HR,MAP were decreased,other cardiac functional indices were improved,including decreases in LVSP,-dp/dt,+dp/dt and LVEDP;(4) Prior to application of EA,if microinjection of L-NAME(NOS nonselective inhibitor,15 nmol/100 nl) or AG(iNOS selective inhibitor,250 pmol/100 nl),7-NI (nNOS selective inhibitor,5 pmol/100 nl) into the RVLM,respectively,except the HR and MAP,the improving effect of EA on cardiac function could be abolished or partially blocked.(5) Realtime-PCR showed nNOS mRNA expression was higher while iNOS was lower in the RVLM of AMI as compared with those of the sham operated group;(6) IHC showed that both nNOS and iNOS-like immunoreactivities (IR) were distributed in the medulla oblongata,nNOS and iNOS IR mainly exist in RVLM,raphe obscurus nucleus(ROb),lateral paragigantocellular nucleus(LPGi), hypoglossal nucleus,inferior olive,medial nucleus(IOM).In the RVLM,the nNOS-like IR were mainly located in the rostral and medial parts with clear positive cells and fibers,while the distribution of iNOS-like IR was scattered.AMI rats' nNOS-like IR was significantly increased compared with the sham control group, whereas inducible nitric oxide synthase(iNOS)-like IR was decreased in the RVLM. Acupuncture treatment could reduce the nNOS but increase iNOS-like IR in the RVLM of AMI rats.(7) Both in control and in AMI rats,microinjection of NO donor, L-arginine(50 nmol,100 nmol/100 nl),could reduce HR and MAP,MAP decreased in a dose-depend manner,and this was much more obviously in the AMI rats. Microinjection of L-NAME(15 nmol/100 nl) into the RVLM increased both the HR and MAP in the control and AMI rats,Aminoguanidine(AG,250 pmol/100 nl) had the similar effect on HR and MAP.However,application of 7-NI(5 pmol/100 nl) in the RVLM of rats leaded to a significantly reduced MAP and HR;(8) At the same time of microinjection AG into the RVLM,the level of inhibitory amino acid(IAA):γ-aminobutyric acid(GABA) and taurine(Tau) decreased while the excitatory amino acid(EAA):Aspartic acid(Asp) increased in the dialysate during the first and second 10 min of microinjection.While 7-NI microinjection,mainly decreased the level of EAA Glu,and increased the IAA neurotransmitters,glycine(Gly) and Tau in the RVLM.(9) In the dialysate from the RVLM of AMI rats,EAA,Glu,and Asp increased while IAA,GABA and Tau decreased.Acupuncture could decrease Glu and increase both GABA and Tau in the RVLM.Conclusions:(1) By the method of ligating left anterior descending(LAD) coronary artery could successfully duplicate the rat model of AMI.This model cardiac function parameters were abnormal and accompanied augmented sympathetic activity.In the RVLM of AMI rats,nNOS expression increased while iNOS was decreased;(2) Acupuncture at Neiguan acupoint on AMI rats could improve the cardiac function,and regulate nNOS and iNOS expression in the RVLM. The effect of EA might mediate by NO.(3) Acupuncture could reduce the EAA,Glu, and increase IAA,GABA and Tau,in the RVLM of AMI rats,which might relate to the decreased sympathetic activity induced by acupuncture.(4) NO in the RVLM could decrease the MAP and HR of control and AMI rats,it was more significantly in the AMI rats.(5) NO produced from nNOS in the RVLM could mainly promote Glu releasing to educe the effect of increasing HR and MAP,while NO from iNOS mainly through GABA and Gly releasing to produce the effect of decreasing HR and MAP;(6) One of the central effect of EA on improving AMI rats might was by decreasing nNOS but increased iNOS expression in the RVLM,further,through NO regulated the ratio of excitatory/inhibitory amino acid neurotransmitters,which could reduce the over-excited sympathetic outflow of AMI rats.