颈部交感神经节P2受体参与糖尿病心交感神经病理损伤的机理研究

Cervical sympathetic ganglion postganglionic fibers including superior cervical ganglia (SCG) and stellate ganglia (SG) were involved in the regulation of cardiac function. Diabetic cardiac sympathetic neuropathy (DCSN) is a common complication. The project will observe the changes of cardiac dysfunction in type 2 diabetic rat model in combined with the expression changes of P2X3, P2Y12 receptors in SCG and SG to understand the relevance between P2X3, P2Y12 receptors and cardiac dysfunction caused by diabetic injury. Peripheral nervous injury causes abnormal neuron- satellite glial cells (SGC) interactions. Blocking abnormal neuron-SGC interactions reduced sensory neuron abnormal firing in nervous injury models. The project assumption is that P2 receptor-mediated abnormal interactions between neurons-SGC induced by diabetic injury, leading to pathological changes of cervical sympathetic ganglia. Pre-experiments showed the upregulated expression of neuron P2X3 receptor, SGC P2Y12 receptor (P2Y12 co-expressed with SGC marker GFAP) in SCG and SG of type 2 diabetic rat model. In our project, abnormal interactions between neurons and SGCs caused by diabetic injury will be observed by application of diabetic rat model and cell model treated with high sugar and high fat, and whether the pathological changes are involved in the mechanism of cardiac dysfunction. By inhibiting P2X3, P2Y12 receptors, we will observe how to affect these abnormal changes and its mechanism, which will be benefit to explore a new target for prevention and treatment of diabetic cardiac sympathetic neuropathy.

自主神经病变是糖尿病最常见的并发症之一。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参与心脏功能调节。本项目将2型糖尿病大鼠模型心功能异常变化与SCG及SG中P2X3、P2Y12受体表达研究相结合，了解其与P2受体的相关性。外周神经损伤激活卫星胶质细胞(SGC)导致神经元-SGC间形态与功能异常变化，阻抑此变化可减少神经损伤模型动物初级感觉神经元的异常放电。项目假设P2受体介导糖尿病引发神经元-SGC间异常作用，导致病理性损害。预实验显示糖尿病模型大鼠SCG和SG神经元中P2X3、SGC中P2Y12受体（与SGC标志物GFAP共表达）水平上调。研究用2型糖尿病大鼠模型、高糖高脂细胞损伤模型观察糖尿病引起神经元-SGC间的病理变化是否涉及心功能异常的病理机制，通过阻抑P2X3、P2Y12受体了解对以上病理变化的影响及机理，为探寻糖尿病引起交感神经损伤的防治提供实验基础。

采用2型糖尿病(DM)大鼠模型，建立糖尿病心脏自主神经病变动物模型。结果显示糖尿病大鼠的血压升高、心率加快、心率变异性将低、血清肾上腺素浓度增加，P2X3 shRNA、P2Y12 shRNA、黄芩苷和蛇床子素处理后升高的血压心率和血清肾上腺素浓度降低，心率变异性得到改善。DM大鼠星状神经节(SG)中P2Y12受体mRNA和蛋白表达水平升高，P2X3 shRNA、P2Y12 shRNA或黄芩苷处理后明显下降，P2Y12受体与卫星胶质细胞标志物谷氨酰氨合成酶共表达。DM大鼠颈部交感神经放电明显增加，经P2Y12 shRNA处理后颈部交感神经放电显著降低，而正常大鼠过表达P2Y12受体可使部交感神经放电明显增加。以上结果表明SG卫星胶质细胞中的P2Y12受体参与糖尿病心脏自主神经病变。DM大鼠SG中P2X3受体表达增加，P2X3 shRNA、P2Y12 shRNA或蛇床子素处理后大鼠SG中P2X3受体表达均降低。DM大鼠SG中IL-1β、TNF-α、缝隙连接蛋白Cx43的表达和p38磷酸化水平均升高，P2X3 shRNA、P2Y12 shRNA、黄芩苷和蛇床子素处理后均显著下降。电镜结果显示，与Ctrl组相比，DM组SGCs间缝隙连接增多，细胞间耦联增强。分子对接结果显示黄芩苷与P2Y12、蛇床子素与P2X3的结合能高，均存在相互作用位点。糖尿病心脏自主神经损伤后，神经元释放ATP增加，SGCs被激活，P2Y12受体表达增加。上调的P2Y12、P2X3受体增加了糖尿病大鼠SG中IL-1β、TNF-α的表达并且SGCs之间及SGCs和神经元之间的缝隙连接增加，表明SGCs-神经元之间的交流信息增强。敲低P2Y12、P2X3受体可以降低炎症反应、缝隙连接，并改善糖尿病心脏自主神经损伤后的交感神经和副交感神经失衡。在2型糖尿病大鼠中，p38MAPK磷酸化水平升高，表明p38MAPK信号通路激活涉及星状神经节糖尿病心脏自主神经病变的病理变化过程。黄芩苷通过P2Y12受体、蛇床子素通过P2X3受体从而缓解糖尿病自主神经病变。