针刺内关穴调节延髓NO/NOS系统及其有关神经递质改善大鼠心肌缺血作用机制探讨
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摘要
目的:观察急性心肌缺血(AMI)大鼠模型心功能、心肌梗死面积的变化及针刺内关穴(Pe 6)对急性心肌缺血的改善作用。通过检测急性心肌缺血和针刺组大鼠心血管调节中枢延髓头端腹外侧区(RVLM)一氧化氮/一氧化氮合酶(NO/NOS)含量及分布变化,以及该部位相关氨基酸递质改变,探讨针刺与中枢NO/NOS及其氨基酸递质在RVLM对AMI大鼠血压和心功能的调节作用。进而探究针刺改善AMI的中枢机制和意义。
方法:通过冠状动脉左前降支结扎手术制备AMI大鼠模型;采用针刺大鼠双侧内关穴位观察针刺改善心肌缺血作用,同时监测心率(HR),平均动脉压(MAP),心室内压(IVP);采用氯化四唑(TTC)染色检测梗死心肌面积以及苏木精和伊红(HE)染色观察梗死心肌组织形态;采用放射免疫法(RIA)测定急性心肌缺血大鼠血浆去甲肾上腺素(NE)和脑钠肽(BNP)的含量;采用荧光定量聚合酶链式反应(Realtime-PCR)、免疫组织化学(IHC)等方法,观察AMI大鼠RVLM神经元型一氧化氮合酶(nNOS)和诱导型一氧化氮合酶(iNOS)的表达变化:应用中枢核团微量注射方法观察NO及NOS抑制剂在RVLM对针刺作用的影响以及NO/NOS在对AMI大鼠血压和心功能的调节作用;采用微透析结合高效液相色谱-荧光检测研究参与上述过程的氨基酸递质的改变。
结果:(1)冠状动脉左前降支结扎后,大鼠立即出现心电图S-T段抬高,结扎线远端心肌发白,运动减弱,说明AMI模型制备成功。冠脉结扎术后5天,TTC及HE染色显示AMI后坏死的心肌组织,心梗组大鼠外周血液中脑钠肽(BNP),去甲肾上腺素(NE)水平较假手术组升高;(2)针刺AMI大鼠内关穴20min,连续5天,可以降低AMI大鼠心肌梗死面积及血液中BNP和NE的水平;(3)针刺组大鼠HR,MAP下降,其它各项心功能指标均有明显改善,包括LVSP,-dp/dt,+dp/dt和LVEDP下降;(4)若电针前于RVLM微量注射NOS非选择性抑制剂左旋硝基精氨酸甲基酯(L-NAME15 nmol/100nl),或者iNOS抑制剂氨基胍(AG,250pmol/100 nl)或者nNNOS抑制剂7-硝基吲唑(7-NI,5 pmol/100 nl),除HR和MAP外,电针改善AMI心功能的作用被取消或减弱;(5)Realtime-PCR结果表明:正常Wistar大鼠iNOS与nNOS在RVLM均有表达。与对照组假手术相比,AMI大鼠RVLM区nNOS mRNA表达明显增加而iNOS mRNA的表达减少;(6)IHC结果显示,在延髓,nNOS和iNOS阳性神经元主要分布在RVLM、中缝核、巨细胞旁外侧核背侧部。舌下神经核和下橄榄核等处也有阳性细胞分布。AMI大鼠RVLM的nNOS阳性细胞明显多于对照大鼠,而iNOS阳性细胞数明显降低,针刺治疗可以降低AMI大鼠nNOS阳性细胞数,升高其iNOS阳性细胞数;(7)在正常和AMI大鼠,微量注射NO供体L-精氨酸(L-arginine 50 nmol,100 nmol/100nl)至RVLM能够在上述2组大鼠产生明显的MAP和HR降低,MAP呈现剂量依赖性降低并且在AMI大鼠更明显。RVLM注射NOS非选择性抑制剂L-NAME(15nmol/100 nl)能够升高血压加快心率。RVLM注射AG(250 pmol/100 nl)能够产生与L-NAME类似效应,血压升高,心率明显加快。与此相反,7-NI(5 pmol/100nl)能够降低血压和心率(8)在引起血压心率变化的同时,在RVLM注射AG后第一,二个10 min RVLM透析液中抑制性氨基酸(IAA)γ—氨基丁酸(GABA),牛磺酸(Tau)释放减少而兴奋性氨基酸(EAA)天门冬氨酸(Asp)释放增加;在RVLM注射7-NI后第一,二个10 min可见RVLM透析液中EAA谷氨酸(Glu)释放减少IAA甘氨酸(Gly)和牛磺酸(Tau)释放增多;(9)AMI大鼠RVLM透析液中EAA:Glu,Asp释放增多而IAA:GABA和Tau释放减少。针刺可以减少Glu,而增加GABA和Tau的释放。
结论:(1)采用结扎大鼠左冠状动脉前降支(LAD)的方法,能成功造成AMI大鼠模型,该模型大鼠外周交感活性增高伴随心功能各项指标减弱。(2)针刺AMI组大鼠内关穴,可以降低交感活性,改善大鼠血流动力学指标,改善心功能。NOS抑制剂能够部分取消针刺改善心功能的效应,说明这种效应可能与NO介导有关。(3)针刺内关穴可以调节nNOS和iNOS在RVLM内表达量,同时可以减少AMI大鼠RVLM区EAA递质GLU释放,而增加IAA GABA和Tau释放,这可能与针刺降低AMI大鼠中枢交感活性有关。(4)NO在RVLM能降低正常大鼠及AMI大鼠升高的血压和心率,在AMI大鼠上效果更明显。(5)nNOS来源的NO在RVLM能促进兴奋性GLU释放,而抑制性Gly,Tau释放减少而发挥其升压和加快心率的效应,而iNOS来源的NO在RVLM主要通过增加IAA递质GABA,Tau和减少EAA Asp发挥其降压和减慢心率的作用。(6)针刺改善大鼠AMI作用的中枢机制之一可能是在RVLM通过增加iNOS和减少nNOS的表达而影响NO的生成,从而调节兴奋性/抑制性氨基酸递质的比例,进而减少交感传出,这可能是针刺治疗伴有交感活性升高的AMI大鼠的机制之一。
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.
方法:通过冠状动脉左前降支结扎手术制备AMI大鼠模型;采用针刺大鼠双侧内关穴位观察针刺改善心肌缺血作用,同时监测心率(HR),平均动脉压(MAP),心室内压(IVP);采用氯化四唑(TTC)染色检测梗死心肌面积以及苏木精和伊红(HE)染色观察梗死心肌组织形态;采用放射免疫法(RIA)测定急性心肌缺血大鼠血浆去甲肾上腺素(NE)和脑钠肽(BNP)的含量;采用荧光定量聚合酶链式反应(Realtime-PCR)、免疫组织化学(IHC)等方法,观察AMI大鼠RVLM神经元型一氧化氮合酶(nNOS)和诱导型一氧化氮合酶(iNOS)的表达变化:应用中枢核团微量注射方法观察NO及NOS抑制剂在RVLM对针刺作用的影响以及NO/NOS在对AMI大鼠血压和心功能的调节作用;采用微透析结合高效液相色谱-荧光检测研究参与上述过程的氨基酸递质的改变。
结果:(1)冠状动脉左前降支结扎后,大鼠立即出现心电图S-T段抬高,结扎线远端心肌发白,运动减弱,说明AMI模型制备成功。冠脉结扎术后5天,TTC及HE染色显示AMI后坏死的心肌组织,心梗组大鼠外周血液中脑钠肽(BNP),去甲肾上腺素(NE)水平较假手术组升高;(2)针刺AMI大鼠内关穴20min,连续5天,可以降低AMI大鼠心肌梗死面积及血液中BNP和NE的水平;(3)针刺组大鼠HR,MAP下降,其它各项心功能指标均有明显改善,包括LVSP,-dp/dt,+dp/dt和LVEDP下降;(4)若电针前于RVLM微量注射NOS非选择性抑制剂左旋硝基精氨酸甲基酯(L-NAME15 nmol/100nl),或者iNOS抑制剂氨基胍(AG,250pmol/100 nl)或者nNNOS抑制剂7-硝基吲唑(7-NI,5 pmol/100 nl),除HR和MAP外,电针改善AMI心功能的作用被取消或减弱;(5)Realtime-PCR结果表明:正常Wistar大鼠iNOS与nNOS在RVLM均有表达。与对照组假手术相比,AMI大鼠RVLM区nNOS mRNA表达明显增加而iNOS mRNA的表达减少;(6)IHC结果显示,在延髓,nNOS和iNOS阳性神经元主要分布在RVLM、中缝核、巨细胞旁外侧核背侧部。舌下神经核和下橄榄核等处也有阳性细胞分布。AMI大鼠RVLM的nNOS阳性细胞明显多于对照大鼠,而iNOS阳性细胞数明显降低,针刺治疗可以降低AMI大鼠nNOS阳性细胞数,升高其iNOS阳性细胞数;(7)在正常和AMI大鼠,微量注射NO供体L-精氨酸(L-arginine 50 nmol,100 nmol/100nl)至RVLM能够在上述2组大鼠产生明显的MAP和HR降低,MAP呈现剂量依赖性降低并且在AMI大鼠更明显。RVLM注射NOS非选择性抑制剂L-NAME(15nmol/100 nl)能够升高血压加快心率。RVLM注射AG(250 pmol/100 nl)能够产生与L-NAME类似效应,血压升高,心率明显加快。与此相反,7-NI(5 pmol/100nl)能够降低血压和心率(8)在引起血压心率变化的同时,在RVLM注射AG后第一,二个10 min RVLM透析液中抑制性氨基酸(IAA)γ—氨基丁酸(GABA),牛磺酸(Tau)释放减少而兴奋性氨基酸(EAA)天门冬氨酸(Asp)释放增加;在RVLM注射7-NI后第一,二个10 min可见RVLM透析液中EAA谷氨酸(Glu)释放减少IAA甘氨酸(Gly)和牛磺酸(Tau)释放增多;(9)AMI大鼠RVLM透析液中EAA:Glu,Asp释放增多而IAA:GABA和Tau释放减少。针刺可以减少Glu,而增加GABA和Tau的释放。
结论:(1)采用结扎大鼠左冠状动脉前降支(LAD)的方法,能成功造成AMI大鼠模型,该模型大鼠外周交感活性增高伴随心功能各项指标减弱。(2)针刺AMI组大鼠内关穴,可以降低交感活性,改善大鼠血流动力学指标,改善心功能。NOS抑制剂能够部分取消针刺改善心功能的效应,说明这种效应可能与NO介导有关。(3)针刺内关穴可以调节nNOS和iNOS在RVLM内表达量,同时可以减少AMI大鼠RVLM区EAA递质GLU释放,而增加IAA GABA和Tau释放,这可能与针刺降低AMI大鼠中枢交感活性有关。(4)NO在RVLM能降低正常大鼠及AMI大鼠升高的血压和心率,在AMI大鼠上效果更明显。(5)nNOS来源的NO在RVLM能促进兴奋性GLU释放,而抑制性Gly,Tau释放减少而发挥其升压和加快心率的效应,而iNOS来源的NO在RVLM主要通过增加IAA递质GABA,Tau和减少EAA Asp发挥其降压和减慢心率的作用。(6)针刺改善大鼠AMI作用的中枢机制之一可能是在RVLM通过增加iNOS和减少nNOS的表达而影响NO的生成,从而调节兴奋性/抑制性氨基酸递质的比例,进而减少交感传出,这可能是针刺治疗伴有交感活性升高的AMI大鼠的机制之一。
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.
引文
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