| Myocardial ischemia is a major cause leading to death and disability worldwide.In the treatment of the coronary heart disease,ischemia reperfusion injury has always been an important factor affecting the clinic efficacy.It is of great value to find effective prevention and treatment methods.Myocardial ischemia preconditioning is known to exert myocardial protection by activating the endogenous protection mechanism.However,it is greatly limited for the clinical application of ischemia preconditioning to treat the disease due to its inconvenience in the clinical practice.More and more attention has been paid to the acupuncture pretreatment based on the theory of "treating pre-disease" in traditional Chinese medicine.With almost no side-effect acupuncture pretreatment is known to be relatively economic and easy to operate,making it to be high value for clinical application.Previous clinical trials have also demonstrated that acupuncture pretreatment can improve ischemia reperfusion injury,reduce the incidence of ischemic arrhythmia and mortality.Our previous experimental studies also showed that acupuncture pretreatment can mimic ischemia preconditioning and protect the ischemic myocardium from injury.The specific mechanism underlying the cardioprotection of acupuncture pretreatment is still less known so far.A large amount of experimental evidences indicated that Adenosine receptor 2b may play a key role in the protective effect of myocardial ischemia reperfusion.Acupuncture pretreatment has been proved to effectively regulate the expression of adenosine and its corresponding receptors.Thus,the acupuncture pretreatment-induced cardioprotective effects is most likely related to the regulation of A2b receptor.Myocardial calcium overload is one of the important causes related to the ischemia-reperfusion injury.Related studies have shown that A2b receptor is involved in the regulation of intracellular calcium.With focus on A2b receptor and the key factors of calcium signal regulation in cardiac myocytes,the present study was aimed to investigate the mechanism of acupuncture pretreatment in the reduction of ischemic myocardial injury.so as to provide scientific evidence for acupuncture pretreatment to prevent and treat myocardial ischemic diseases.The outcomes of the present study will be also helpful for highlighting the importance of the preventive thought of traditional Chinese medicine in solving the worldwide medical problems as well.Objective:Based on the confirmation of the cardioprotective effect of electroacupuncture pretreatment on myocardial injury induced by ischemia and reperfusion,the present study was aimed to verify whether A2b receptor and its related intracellular calcium regulators are involved in the mediation of the cardioprotective effects produced by electroacupuncture pretreatmentMethods:The present study includes two series experiments.The first one was focused on confirming the protective effect of acupuncture pretreatment on the myocardial injury induced by ischemia and reperfusion and determining whether A2b receptor is involved in the protective effect.Forty-eight male rats were randomly divided into normal control(NC)group,model(M)group,electroacupuncture pretreatment(EA)group and electro-acupuncture plus A2b antagonist pretreatment(EAG)group.In the NC group,the rats were performed without ligation and electroacupuncture treatment,and the other groups were treated with ischemia/reperfusion.The rats in EA group were treated with electroacupuncture for three consecutive days before modeling,and in EAG group the rats were treated with GS6201,a specific A2b antagonist,by intraperitoneal injection 30min before each time of electroacupuncture treatment.The hemodynamic indexes including LVEDP,EF,dp/dt max,ESPVR and EDPVR were measured.Evans blue-TTC staining was used to measure the myocardial infarction area.After the confirmation of the curative effect,specific antagonist of A2b receptor(GS6201)was pretreated.The relative expression of A2b protein in ischemic myocardial tissue was detected by fluorescent Western Blot in order to determine whether the A2b receptor is involved in the mediation of the cardioprotective effect of acupuncture pretreatment.In the second series of the experiments,the content of the proteins including ryanodine receptor 2,NCX1,SERCA 2a.PLB,p-PLB in ischemic myocardial tissues was detected by multi-fluorescence Western Blot to explore the roles played by the aforementioned key regulators of intracellular calcium in the A2b receptor-mediated cardioprotection of electroacupuncture pretreatment.Results:1.A2b receptor was involved in the cardioprotective effect of electroacupuncture pretreatment on the myocardial injury induced by ischemia and reperfusion.(1)ECG:As compared with the corresponding time points in NC group,the ST segment in M group was significantly elevated after ligation(all P<0.01).Although the ST segment in EA group was significantly elevated(P<0.01)immediately after ligation in compared with NC group,the level of elevated ST segment was then returned to the baseline gradually as time passed.In particular,at the time points of 15min after ligation and 15min after reperfusion the ST segment in EA group was decreased significantly in comparison with M group(P<0.05,P<0.01).After ligation for 30min and reperfusion for 15min,the ST segment in EAG group was significantly elevated in comparison with EA group(P<0.01).(2)Arrhythmia scores:As compared with NC group,arrhythmia scores in the M group was significantly increased(all P<0.01),while the score in the EA group was significantly decreased in comparison with M group(P<0.01).In EAG group the score was significantly increased as compare with EA group(P<0.05).(3)Evans blue-TTC staining:In comparison with NC group the infarct size in M group was significantly enhanced.The infarct size in EA group was significantly smaller than that in M group.The ratio of infarct size/risk zone in EAG group was significantly higher than that in EA group.(4)Hemodynamic and cardiac functional parameters in different groups:?LVEDP:There was no significant difference in the level of LVEDP among NC,M and EA group at all time points(P>0.05).The LVEDP level in EAG group was higher than that of the NC and EA groups after ligation for 15min and 30 min(P<0.05).?EF:After ligation for 15min,there was no significant difference between the NC and EAG group(P>0.05),while EF values of both M and EA group were decreased as compared with NC group(P<0.05).EF values of M.EA and EAG groups were significantly lower than those of NC group(P<0.01)after ligation for 30min and reperfusion for 15min,and showing a continuous decreasing trend.After reperfusion for 15min,EF value of M group was lower than that of EA group (P<0.05),but significantly higher than that of EAG group(P<0.01).?dp/dt max:As compared with the NC group,dp/dt max in EA group showed no remarkable change,while M and EAG groups were significantly reduced after ligation for 15min(P<0.01).And dp/dt max in M,EA and EAG groups were significantly reduced after ligation for 30min and reperfusion for 15min(P<0.01),as compared with that in NC group.The dp/dt max in EAG group was significantly lower than that in EA group after reperfusion for 15min(P<0.01).?ESPVR and EDPVR:After reperfusion for 15 min End-systolic elastance(Ees) in the NC group showed no significant change as compared with that before ligation(P>0.05),and the Ees in M,EA and EAG group were all decreased significantly in comparision with NC group(all P<0.01),the values of Ees in different groups were in an order as NCEes>EAEes>MEes>EAGEes.Before ligation,there was no significant difference in EDPVR slope among NC,M and EA groups (P>0.05),but the Eed of EAG group was increased as compared with other groups.At the time point 15min after reperfusion,there was no significant change in Eed in NC group compared with that before ligation(P>0.05),but the Eed in other 3 groups was increased significantly(P<0.01).Among all the 4 groups the order of the Eed values after ligation was NCEed<EAEed<MEed<EAGEed.(5)A2b content:The relative expression of A2b in M was significantly higher than that in NC group(P<0.01).As compared with M group,A2b content in EA group was much higher(P<0.01).In EAG group A2b content was significantly lower in comparison with EA group,even lower than that in NC group(P<0.01).2.The key regulators of intracellular calcium were involved in the A2b receptor-mediated cardioprotection of acupuncture pretreatment(1)RyR2 content:RyR2 expression was significantly increased after the completion of modeling in group M as compared with that before ligation(P<0.01).The relative expression of RyR2 protein in M group was much higher than that in NC group.However,as compared with M group the content of RyR2 in EA group was significantly lower(P<0.01).In EAG group the content of RyR2 protein was significantly higher than EA group(P<0.01)(2)PLB,P-PLB and P-PLB/PLB ratio:The relative expression of PLB in both EA group and EAG group was significantly lower than that in NC and M group(P<0.01).The relative expression level of P-PLB protein in M,EA and EAG group was significantly lower than that in NC group(P<0.01).As compared with M group,the relative expression of P-PLB in EA group was higher,while in EAG group it was decreased markedly in comparison with EA group(P<0.01).As compared with NC group,the P-PLB/PLB ratio of M and EAG group was lower markedly(P<0.01),while in EA group it was higher significantly(P<0.01).In comparison with EA group the ratio in EAG group was decreased markedly(P<0.01).(3)NCX1 content:As compared with NC group,the relative expression of NCX1 in M,EA and EAG group did not change significantly(P>0.05).The relative expression level of NCX1 in EAG group was lower than that in M group(P<0.05).(4)SERCA 2a content:SERCA 2a in M group was significantly lower than that in NC group(P<0.01),while as compared with M group the protein content in EA group was significantly higher(P<0.05).In EAG group it was reduced a little bit but not significantly in comparison with EA group.Conclusion:(1)Electroacupuncture pretreatment can effectively reduce both myocardial infarct size and ischemic arrhythmias and improve the cardiac function in the rats subjected to myocardial ischemia and reperfusion.(2)Adenosine receptor 2b antagonist could block the cardioprotective effects of acupuncture pretreatment,which indicates that A2b receptor is involved in the cardioprotective effect of electroacupuncture pretreatment on myocardial injury induced by ischemia and reperfusion.(3)Electroacupuncture pretreatment could protect the heart from the impairment induced by ischemia and reperfusion via increasing the A2b content and regulating the key Ca2+ signaling components,namely,inhibiting RyR2 and PLB contents,enhancing P-PLB(S 16)and SERCA2a proteins,so as to diminish possibly the intracellular Ca2+ overload and consequently lessen the myocardial injury. |
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