基于标本理论研究针刺后处理触发阿片/腺苷受体间相互应答对心肌缺血再灌注损伤的保护作用及机制

Both ischemia preconditioning(IPC) and ischemia postconditioning(IPO) play an important role in pretection of myocardial ischemia reperfusion injury(MIRI), while the IPO is more suitable for clinical practice.Some researches indicate that acupuncture preconditioning is effective for MIRI protection, but the acupuncture postconditioning study is relatively less. Our preliminary researches show acupuncture at Neiguan(PC6) point exert its curative effect for myocardial ischemia via activating multiple adenosine receptor(ADRs) on the heart. Meanwhile, lots of studies have found that crosstalked G-protein-coupled receptors(GPCRs)could trigger the reperfusion jury salvage kinase(RISK) pathway to relieve the MIRI. Thus, we raise the hypothesis that acupuncture postconditioning could multiply trigger the crosstalk between ADRs and opioid receptors(OPRs),then acitvate RISK pathway to pretect MIRI. Based on Branch-Root(Biao Ben) theory, we choose acupoints on the pericardium and heart meridians, estabilish MIRI models rat, by means of qPCR, Western blot, Luminex xMAP,to do research firstly that if acupuncture postconditioning at the different time duration before or after reperfusion have the curative effect on MIRI. Secondly, we assess the regulative role of acupuncture postconditioning at the best curative time duration in ADRs and OPRs expression and RISK pathway activation. Last but not the least, after using ADR/OPR antagonist, the regulation effect of acupuncture postconditioning at another receptor will be estimated to verify the the significant status of crosstalk between ADRs and OPRs in acupuncture postconditioning for MIRI. This research will provide scientific evidence and study target for MIRI prevent and treatment via acupuncture.

缺血预处理和后处理均具有保护心肌缺血再灌注损伤(MIRI)的作用，但后处理更具临床适用性。研究表明针刺预处理对MIRI有较确切保护作用，然针灸后处理报道甚少。前期研究表明针刺内关可激活心脏多种腺苷受体发挥心肌缺血保护效应；且有研究证实GPCRs交互应答可激活RISK通路达到MIRI保护作用。故我们提出“针灸后处理能触发阿片/腺苷受体间的交互应答，激活RISK通路而保护MIRI”的工作假说。本项目基于标本理论选心包经、心经本穴，建立大鼠MIRI模型，运用qPCR、蛋白质印迹法、液相芯片等技术，研究：⑴再灌注前后不同时间点针刺后处理对MIRI的保护效应；⑵在干预效应最佳时间点，针刺对心脏腺苷、阿片受体表达及RISK 通路的调控作用；⑶腺苷/阿片受体分别拮抗后，针刺对另一受体的调控作用，论证阿片/腺苷受体间相互应答在针刺保护MIRI的关键地位。将为针刺防治MIRI临床运用提供新的科学依据和靶点。

本项目以经脉学说理论、组合受体网络模型概念为立项基础，以人类重大疾病“冠心病”的病理改变“心肌缺血再灌注损伤”为研究载体，考虑到腺苷受体与其他多种 G 蛋白偶联受体的交互应答作用，本项目以心脏腺苷受体、阿片受体为研究靶点，以两类受体间的cross-talk为切入点，采用TUNEL、Western blot、qPCR等技术，观察针刺后处理改善心肌缺血再灌注损伤的最佳干预时间窗并阐释其机制。研究结果表明针刺后处理可显著抑制ST段的抬高，降低心肌缺血面积，减少心肌组织中心肌细胞凋亡率，上调心肌组织耗氧量，调控A1 ADR、δ-OPR的表达水平，激活RISK通路；再灌注前后均针刺的治疗效果、调控作用、激活作用均优于再灌注前针刺及再灌注后针刺；A1ADR和δ-OPR抑制剂无论单独使用或联合使用均能抑制针刺后处理的治疗效果及其对RISK通路的调控作用，但并未下调A1ADR和δ-OPR表达。本研究验证了“阿片/腺苷受体交互应答激活RISK信号通路是针刺后处理改善心肌缺血再灌注损伤的作用机制”的假说，并为针刺治疗冠心病心肌缺血再灌注损伤提供了最佳干预时间及科学依据。