ATP-P2X4信号调控NLRP3炎性体活化在PD发病机制中的作用

Parkinson’s disease (PD) is a neurodegenerative disease, but its precise mechanisms remain unclear. Oxidative stress and inflammation are closely related with its progression, and ATP-P2X signals are involved in the neuroinflammatory response. Recent evidence revealed that ATP binding PX24R can promote the subunits rearrangement and iron channels open; whereas the iron ions interact with α-synuclein accumulation, and its deposition is the crucial point in terms of the dopaminergic neurodegeneration. In addition, ATP-P2X4 signal mediated NLRP3 inflammasome activation, while dopamine could inhibit NLRP3 activation. Our preliminary results indicate that P2X7 1513 A>C polymorphism is a risk factor for PD, and the following study showed that P2X4R was closely correlated to PD. Therefore, we hypothesized that in the pathophysiology of PD, ATP -P2X4 signal may through mediating NLRP3 inflammasome activation to regulate microglial cell activation, DA neurodegeneration , iron metabolism and maintain the steady of α-synuclein and dopamine. In order to verify our hypothesis, we use PD rat, brain slices and primary cultured cell lines as PD models. Laser confocal, molecular biology, siRNA and overexpression technology were applied to examine the expression and distribution of P2X4R and its affect in PD models. As to identify the mechanism of ATP-R2X4R signal axis,we regulated P2X4R expression. This study will present a new perspective in elucidating the pathogenesis of PD and provides a potential therapeutic target for PD.

帕金森病(PD)机制目前认为与氧化应激及炎症反应相关。研究证实ATP-P2X4信号与小胶质细胞活化及神经炎症密切相关，且ATP结合P2X4受体能促进其亚单位重排及铁离子通道的开放，而铁离子与α-synuclein相互作用及沉积聚集是多巴胺能神经元退变的核心环节。此外，ATP-P2X4信号能够介导NLRP3炎性体激活，而多巴胺可以抑制NLRP3活化。前期研究表明P2X7是PD的危险因素，后续实验初步表明P2X4R与PD机制相关。据此我们推测在PD发生发展中ATP -P2X信号可能通过介导NLRP3活化而调节小胶质细胞活化参与多巴胺能神经元的退变行为、调控铁代谢、α- synuclein及多巴胺稳态环境。为证实假说，课题以PD模型鼠和PD脑片及原代培养细胞系为模型，采用激光共聚焦、分子生物学、RNA干扰等技术，观察P2X4R在PD模型中表达；通过减少或过表达P2X4研究其在PD中的重要作用机制。

帕金森病（Parkinson’s disease, PD）发病的分子生物学机制至今尚未明确，包括氧化应激和炎症反应可能参与了疾病的发展过程。既往研究发现，ATP-P2X4信号通路可能通过介导NLRP3炎性小体活化而参与了炎症反应机制。我们的研究旨在探讨ATP-P2X4R信号通路在PD发病中的作用，运用LPS建立NLRP3体外活化模型，进而探索调控ATP-P2X4R信号轴对于NLRP3炎性体活化、炎性因子分泌、α-synuclein表达的影响；通过激光共聚焦显微镜、分子生物学检测方法、小干扰RNA和过表达技术对6-OHDA诱导的大鼠PD模型的脑黑质组织进行评估，以确定P2X4R在6-OHDA诱导的大鼠PD发病过程中的作用。我们发现，三磷酸腺苷（ATP）能够上调小胶质细胞P2X4 mRNA的表达水平，诱导小胶质细胞中NLRP3mRNA、caspase-1蛋白水平表达上调、促进炎性因子的释放，而P2X4的特异性受体阻断剂5-BDBD能抑制上清液中炎性因子水平的上调；在6-OHDA诱导的PD大鼠模型的脑内，P2X4R、NLRP3、caspase-1以及炎性因子表达均显著增加；通过P2X4R受体抑制剂5-BDBD以及立体定位注射携带P2X4R干扰RNA的慢病毒下调P2X4R表达，NLRP3炎性体的活化减少，导致下游炎性因子的释放减少，从而减缓DA能神经元的变性过程；通过立体定位注射携带P2X4R目的基因的慢病毒上调P2X4R表达，使得NLRP3炎性体的活化增加，从而使得下游炎性因子释放增加，促进了DA能神经元的变性。综上，我们的结果提示ATP-P2X4R信号通路可能通过激活下游NLRP3炎性体活化进而促使胶质细胞活化，炎性因子释放，促进DA能神经元的变性而参与了PD的发病过程，这可能为PD的靶向治疗提供一个新的靶点。