| BACKGROUNDIschemic heart disease has been the leading cause of disability and death worldwide.Ischemia-reperfusion injury is one of the main factors leading to the increase of morbidity and mortality of the disease.One of the typical manifestations of ischemia-reperfusion injury is arrhythmia.Therefore,it is of great value to find a way to prevent and improve the arrhythmia caused by ischemia-reperfusion.Acupuncture plays a positive role in the prevention and treatment of diseases.Clinical studies have confirmed that acupuncture can improve the clinical symptoms of patients with myocardial ischemia;at the same time,acupuncture can significantly inhibit the occurrence of arrhythmia.Basic research has also shown that acupuncture pretreatment can reduce arrhythmia scores.The previous work of this laboratory has shown that electroacupuncture can inhibit myocardial ischemic arrhythmia,and the mechanism of electroacupuncture against ischemia-reperfusion arrhythmia has rarely been involved so far.Modern medical research suggests that the κ-opioid receptor system plays an important role in regulating the physiological and pathological activities of the cardiovascular system;on the other hand,acupuncture analgesia studies have shown that acupuncture can regulate the expression of opioid receptors and activate the opioid receptor system However,it is unclear whether the opioid receptor system has a specific mediating effect in the mechanism of acupuncture in improving ischemia-reperfusion arrhythmia.The previous research in this laboratory showed that electroacupuncture can improve myocardial ischemic injury,and κ-opioid receptor specific blockers can partially block the protective effect of electroacupuncture to improve myocardial ischemic injury,suggesting that electroacupuncture is very likely Improve the ischemia-reperfusion arrhythmia by modulating the κ-opioid receptor and its signal transduction pathway after the receptor,and introduce the discussion of the opioid receptor and its signal transduction pathway into the mechanism of acupuncture to improve arrhythmia Related research is still blank.OBJECTIVEThe aim of the present study was to observe the protective effect of electroacupuncture on ischemia-reperfusion arrhythmia,and at the same time determine whether κ-opioid receptor is involved in the protective effect of intervening acupuncture;and on this basis,explore the signal pathway after κ-opioid receptor to conduct acupuncture The mechanism of anti-ischemia-reperfusion arrhythmia.The completion of the research is not only of great significance for the application of acupuncture in the prevention and treatment of ischemia-reperfusion arrhythmia,but also will find a new idea or a new entry point for systematically exploring the law and mechanism of the cardiovascular effects of acupuncture.METHODSThis experiment is divided into two parts.In the first part of the experiment,rats were randomly divided into four groups:normal control group(Normal Control,NC group),ischemia-reperfusion model group(Model,M group),electroacupuncture group(Electroacupuncture,EA group),electroacupuncture+resistance Discontinuation group(Electroacupuncture+NOR-BNI,NOR-BNI group).We will use the arrhythmia score to observe the protective effect of electroacupuncture on ischemia-reperfusion arrhythmia,and at the same time determine whether the κ-opioid receptor is involved in the protective effect of the intervention of the conductive acupuncture;the second part,the experiment is clear On the basis that the receptor participates in the protective effect of the conductive needle to improve the ischemia-reperfusion arrhythmia,the rats are divided into three groups:normal control group(Normal Control,NC group),ischemia-reperfusion model group(Model,M group)),Electroacupuncture group(Electroacupuncture,EA group).Detection ofκ-opioid receptors and their post-receptor signaling pathway sites-Gq protein,Inositol 1,4,5-triphosphate(IP3),Diacylglycerol(DAG),protein kinase c(protein Kinase C,PKC),adenylate cyclase(Adenylatecyclase,AC),cyclic adenosine monophosphate(Cyclic Adenosine monophosphate,cAMP),protein kinase A(Protein Kinase A,PKA),L-type calcium channel To explore which signal molecules on the signal pathway after κ-OR are involved in mediating the protective effect of electroacupuncture against ischemia-reperfusion arrhythmia.RESULTS1 Determination of the efficacy of electroacupuncture in improving ischemia-reperfusion arrhythmias.Compared with the NC group,the arrhythmia scores of the M group during reperfusion and the EA group were significantly higher(P<0.01),and after electroacupuncture intervention,the EA group was compared with the M group during the reperfusion of the arrhythmia scores.It was significantly lower(P<0.01),and theκ-OR specific antagonist—Nor-BNI was given before electroacupuncture,and the arrhythmia score group during reperfusion had arrhythmia compared with the NC group and the EA group The aberration score increased significantly(P<0.05 or P<0.01).2 The role of κ-opioid receptor and its post-receptor signal pathway in mediating electroacupuncture against ischemia-reperfusion arrhythmias.2.1 The role of κ-opioid receptor and its post-receptor direct signal pathway in mediating electroacupuncture against ischemia-reperfusion arrhythmias.2.1.1 Effect of electroacupuncture on κ-opioid receptor protein in rats with myocardial ischemia-reperfusion.The content of κ-opioid receptor protein in M group was higher than that in NC group,which was considered as a stress protective response of myocardium to ischemia-reperfusion injury,while the content of κ-opioid receptor protein in EA group was significantly higher than that in NC group and M group(all P<0 01).2.1.2 Effect of electroacupuncture on Gq protein in myocardial tissue of rats with myocardial ischemia-reperfusion.Compared with the normal group,the relative content of Gq protein in the ischemia-reperfusion model group increased slightly(P<0.05).The relative content of Gq protein in the electroacupuncture group was further increased compared with the normal group and the ischemia-reperfusion group(P<0.01).2.1.3 Effect of electroacupuncture on IP3 in myocardial tissue of rats with myocardial ischemia-reperfusion.The relative expression of IP3 in the ischemia-reperfusion model group increased compared with that in the normal group(P<0.01).The relative expression of IP3 in the electroacupuncture group was significantly increased compared with the normal group and the ischemia-reperfusion model group(P<0.01).2.1.4 Effect of electroacupuncture on DAG in myocardial tissue of rats with myocardial ischemia-reperfusion.Compared with the normal group,the expression of DAG in the ischemia-reperfusion model group had an increasing trend,but there was no statistical difference;while the expression of DAG in the electroacupuncture group was significantly increased compared with the normal group and the ischemia-reperfusion group(P<0.01).2.1.5 effect of electroacupuncture on PKC reactivity of cardiomyocytes in simulated global ischemia-reperfusion rats.2.1.5.1 Calcium transient of single cardiomyocyte under the action of PKC agonist-PMA effect of electroacupuncture on calcium transient of isolated single cardiomyocyte isolated from simulated global myocardial ischemia-reperfusion rat under the action of PKC agonist-PMA.The amplitudes of calcium transient waveforms in each group increased in varying degrees under the action of PKC agonist-PMA.Compared with NC group,the amplitude of calcium transient of cardiomyocytes in M group increased significantly under the action of PKC agonist-PMA,and decreased in EA group after electroacupuncture(P<0.01).2.1.5.2 Changes in contractile amplitude of isolated single cardiomyocytes isolated from simulated global ischemia-reperfusion rats under the action of PKC agonist-PMA.Compared with the Control of this group,the contractile amplitude of cardiomyocytes in NC group increased by 40.00%under the stimulation of PKC agonist-PMA,while that in M group increased by 83.73%compared with that of Control in this group(P<0.01).The contractile amplitude of cardiomyocytes excited by PKC agonist-PMA increased by 83.73%.Compared with Control in this group,the contractile amplitude of cardiomyocytes stimulated by PKC agonist-PMA in EA group increased by 31.79%,which was significantly lower than that in M group.2.2 The role of κ-opioid receptor and its post-indirect signal pathway in mediating electroacupuncture against ischemia-reperfusion arrhythmias.2.2.1 Effect of electroacupuncture on AC reactivity of cardiomyocytes in simulated global ischemia-reperfusion rats.2.2.1.1 Electroacupuncture at calcium transients of isolated single cardiomyocytes isolated from simulated global ischemia-reperfusion rats under the action of AC agonist-Forskolin.The amplitudes of calcium transient waveforms in each group increased in varying degrees under the action of AC agonist-Forskolin.Compared with NC group,the calcium transient amplitude of cardiomyocytes in M group increased significantly under the action of AC agonist-Forskolin(P<0.01).Compared with M group,the increase of EA group decreased after electroacupuncture(P<0.01).2.2.1.2 The effect of electroacupuncture on the contractile amplitude of isolated single cardiomyocytes isolated from simulated global ischemia-reperfusion rats under the action of AC agonist-Forskolin.Compared with the Control of this group,the normal group was stimulated by the PKA agonist-8-Br-cAMP,the contraction amplitude of a single cardiomyocyte increased by 58.72%.Compared with the control in the ischemia-reperfusion model group,the contraction amplitude of cardiomyocytes increased by 90.87%under the excitement of the PKA agonist-8-Br-cAMP.Compared with the control in the electroacupuncture group,the contraction amplitude of cardiomyocytes increased by 20.63%under the excitement of the PKA agonist-8-Br-cAMP,which was significantly lower than that of the ischemia-reperfusion model group.2.2.2 Effect of electroacupuncture on cAMP protein in Myocardial tissue of Rats with Myocardial Ischemia-reperfusion.There was no significant difference in cAMP between M group and NC group,but cAMP in EA group was significantly lower than that in M group(P<0.01).2.2.3 Effect of electroacupuncture on PKA reactivity of cardiomyocytes in simulated global ischemia-reperfusion rats.2.2.3.1 The effect of electroacupuncture on calcium transient of isolated single cardiomyocytes isolated from simulated global ischemia-reperfusion rats under the action of 8-Br-cAMP,an agonist of PKA.The amplitude of calcium transient waveforms in each group increased to different degrees under the action of PKA’s agonist-8-Br-cAMP.As far as the normal group is concerned,the myocardial cells of the ischemia-reperfusion model group have a significant increase in calcium transient amplitude under the action of 8-Br-cAMP(P<0.01).Regarding the ischemia-reperfusion model,the increase in the electroacupuncture group decreased after electroacupuncture(P<0.01).2.2.3.2 The effect of electroacupuncture on the contractile amplitude of isolated single cardiomyocytes isolated from simulated global ischemia-reperfusion rats under the action of 8-Br-cAMP,an agonist of PKA.Compared with the Control of this group,the normal group was stimulated by the PKA agonist-8-Br-cAMP,the contraction amplitude of a single cardiomyocyte increased by 58.72%.In the ischemia-reperfusion model group,compared with this group of Control,the contraction amplitude of cardiomyocytes increased by 90.87%under the excitement of the PKA agonist--8-Br-cAMP.Compared with the Control in the electroacupuncture group,the contraction amplitude of cardiomyocytes increased by 20.63%under the excitement of the PKA agonist-8-Br-cAMP,which was significantly lower than that in the M group.2.2.4 Effect of electrocardiogram on L-type calcium channel reactivity in cardiomyocytes of rats with simulated global ischemia-reperfusion.2.2.4.1 The effect of electroacupuncture on calcium transients in isolated single cardiomyocytes isolated from simulated global ischemia-reperfusion rats under the action of L-type calcium channel agonist Bay-K8644.Under the action of Bay-K8644,an L-type calcium channel agonist,the amplitude of calcium transients in each group increased to varying degrees.Compared with the normal group,under the action of the L-type calcium channel agonist Bay-K8644,the changes in the calcium transient amplitude of myocardial cells in the ischemia-reperfusion model group were significantly increased(P<0.01).Compared with the ischemia-reperfusion model group,after electroacupuncture,the increase of the electroacupuncture group was reduced(P<0.01).2.2.4.2 The effect of electroacupuncture on the contractile amplitude of isolated single cardiomyocytes isolated from simulated global ischemia-reperfusion rats under the action of L-type calcium channel agonist Bay-K8644.Compared with Control,the contractile amplitude of cardiomyocytes in NC group increased by 40.01%under the stimulation of L-type calcium channel agonist-Bay-K8644,while that in M group increased by 86.01%under the stimulation of L-type calcium channel agonist-Bay-K8644 compared with Control in this group.Compared with Control in this group,the contractile amplitude of cardiomyocytes stimulated by L-type calcium channel agonist-Bay-K8644 in EA group increased by 25.11%,which was significantly lower than that in M group.CONCLUSION1 Electroacupuncture can reduce the occurrence of arrhythmia in rats with ischemia-reperfusion,and the κ-OR specific blocker nor-BNI can partially block the protective effect of electroacupuncture in reducing the occurrence of arrhythmia in rats with ischemia-reperfusion.Tip:κ-OR may be involved in mediating the protective effect of electroacupuncture in improving arrhythmia caused by ischemia-reperfusion.2 On the one hand,the specific mechanism of electroacupuncture intervention may be through increasing the content of κ-OR,and then acting on the relevant sites of the signal transduction system directly after κ-OR,that is,enhancing the expression of Gq,IP3 and DAG,and inhibiting the excessive activation of PKC.On the other hand,by inhibiting the related sites of indirectly acting signal transduction pathways,that is,down-regulating the content of cAMP,inhibiting the excessive activation of AC,PKA,and L-Ca2+channels,and playing the role of anti-ischemia-reperfusion arrhythmia. |
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