颈部交感神经节P2X2、P2Y12受体与心肌缺血引发神经元与卫星胶质细胞的病理性相互作用研究

Acute myocardial ischemia leading to acute coronary syndrome seriously harms to human health. The postganglionic fibers of cervical sympathetic ganglia [superior cervical ganglia (SCG) and stellate ganglion (SG)] are involved in the regulation of cardiovascular function. Peripheral nervous injury causes abnormal neuron- satellite glial cells (SGC) interactions, such as SGC hypertrophy, the increase of cytokine content in SGCs and the enhancement of calcium waves. Blocking abnormal neuron-SGC interactions reduced sensory neuron abnormal firing and pain sensitivity in pain animal models. Our preliminary studies showed that the P2X2 expression of neurons and the P2Y12 expression of SGC (P2Y12 co-expressed with SGC marker GFAP) in SCG and SG of myocardial ischemic rats were upregulated. The project assumption is P2X2, P2Y12 receptors-mediated abnormal interactions of neurons-SGCs in SCG and SG caused by myocardial ischemia, leading to pathological damage of cardiac sympathetic function. In our project, myocardial ischemic rat model and "hypoxic-ischemic" cell model will be used to study the abnormal interactions between neurons and SGCs caused by myocardial ischemia and the relationship between the neurons-SGC abnormal interactions and the cardiac sympathetic pathological changes. By inhibiting the upregulated expressions of P2X2, P2Y12 receptors to understand the effects of P2X2, P2Y12 receptors on the abnormal changes, it will help to explore new targets for prevention and treatment of cardiac sympathetic damage caused by myocardial ischemia and coronary heart disease.

急性心肌缺血导致急性冠脉综合征严重危害人类健康。颈上神经节(SCG)和星状神经节(SG)等颈部交感神经节的节后纤维影响心血管功能。外周神经损伤激活卫星胶质细胞(SGC)导致SGC异常增厚、细胞因子含量增加、细胞间钙波增强等神经元-SGC间形态与功能的异常变化，阻抑此变化可减少痛模型动物感觉神经元的异常放电、降低痛敏。预实验显示心肌缺血上调大鼠模型SCG和SG神经元中P2X2、SGC中P2Y12 (与SGC标志物GFAP共表达)水平。项目假设P2X2、P2Y12受体介导心肌缺血引发SCG和SG神经元-SGC形态与功能的异常变化，参与心肌缺血引发的病理过程。本项目应用大鼠心肌缺血模型、"缺氧缺血"细胞模型研究心肌缺血引起神经元-SGC间病理变化与心交感功能异常的关系，通过阻抑P2X2、P2Y12受体上调表达观察对以上异常变化的影响，为探寻心肌缺血引发心交感损伤及冠心病防治的新靶点提供实验基础。

按计划完成研究工作。本项目首先采用结扎大鼠冠状动脉左前降支建立心肌缺血大鼠模型，观察P2Y12受体、P2X4受体是否参与心肌缺血损伤引起的病理变化，及研究P2Y12shRNA、黄芩苷、uc.48+ shRNA及P2X4shRNA对心肌缺血大鼠病理变化的影响及机制。结果显示心肌缺血模型大鼠心电图出现病理性Q波及ST段抬高，血压升高，心率加快，心交感神经活性增强，P2Y12shRNA、黄芩苷、uc.48+ shRNA及P2X4shRNA可改善心肌缺血大鼠心电图、血压、心率及交感神经活性的异常改变。心肌缺血大鼠颈交感神经节与颈部背根神经节（DRG）中P2Y12及P2X4受体mRNA及蛋白表达显著升高。P2Y12shRNA、黄芩苷、uc.48+ shRNA可降低P2Y12受体的表达；P2X4shRNA后可降低心肌缺血大鼠P2X4受体的表达。心肌缺血大鼠SCG和DRG卫星胶质细胞（SGC）激活，胶质纤维酸性蛋白GFAP及细胞炎性因子TNF-α与IL-1β的表达增加，P38MAPK与Akt的磷酸化水平增强。P2Y12shRNA、黄芩苷、uc.48+ shRNA及P2X4shRNA可降低GFAP表达，TNF-α与IL-1β表达及P38MAPK与Akt的磷酸化水平。黄芩苷可以降低表达P2Y12受体的卫星胶质细胞及转染了P2Y12的HEK293细胞内的钙离子浓度。功能获得和功能丧失实验证实uc.48+与P2Y12受体具有相互作用。总之，上述结果表明颈部交感神经节及背根神经节P2Y12、P2X4受体参与了心肌缺血损伤诱发的交感兴奋反射增强过程并加重心肌缺血，P2Y12shRNA、黄芩苷、uc.48+ shRNA通过调节P2Y12受体表达及功能，而P2X4shRNA通过降低P2X4受体的表达来降低心肌缺血诱发的异常心交感兴奋反射，从而改善心功能保护心肌。本项目研究为探寻心肌缺血引发心交感损伤及冠心病防治的新靶点提供实验基础。