P2X4受体靶向抑制对新生鼠缺氧后脑内谷氨酸及血清Tau蛋白表达的调控机制研究

Hypoxic and ischemic encephalopathy (HIE) is gaining interest by many because of its high mortality and disability rate. Indeed, the modulatory mechanism and serum biomarkers related to HIE have been the focus of many studies. The defining feature of neuropathology in HIE is the injury susceptibility of the periventricular white matter (PWM). It has been reported that inflammation and glutamate metabolic disorder are involved in the PWM damage after hypoxia in neonatal brain. The activation of amoeboid microglial cells (AMC) have been linked to the inflammatory response in PWM after hypoxia in neonatal. On the other hand, the underlying mechanism leading to AMC activation has remained uncertain. In our previous study,we showed that AMC expressed ATP receptors, namely, P2X4 notably the first-named receptor in hypoxia in neonatal brain. It was suggested that the P2X4 may be linked to regulation of AMC activation for production of proinflammatory cytokines in hypoxic exposure. Recently it has been reported that there is a crosstalk between the purinic signal and glutamate metabolic disorder. However, the putative roles of P2X4 in regulating glutamate metabolism in PWM after hypoxia in neonatal brain has remained to be further explored. Tau is an microtubules assosiated protein whose expression is localized in the axons of neurons. Some studies have reported that Tau can be released into the prepherial blood after cerebral injury. A recent study has reported that Tau expression is increased as a result of reduced glutamate trasporter expression after brain ischemia. We have reported previously that glutamate transporter 1 expression was decreased after hypoxia in neonatal rats. In the present study, we first aim to invetigate the underlying mechanism of P2X4 in its regulation of glutamate release in the PWM. Along with this, the potential relationship between the glutamate metabolism and serum Tau protein after hypoxia in rat pups will be investigated. P2X4 will be blocked by pyridoxal phosphate-6-azo (benzene-2, 4-disulfonic acid) tetrasodium salt hydrate (a slective blockade for P2X1-3,5-7) and 2', 3'-0-(2, 4, 6-Trinitrophenyl) adenosine 5'-triphosphate (a slective blokcade for P2X1-4) by intraperitoneal injection wihtin 4 hours after hypoxic exposure in neonatal rats. To confirm the blockade efficiency of P2X4, the differences between the hypoxic and blokade groups will be compared. An ultra-high performance liquid chromatography-tandem mass-spectrometry method will be adopted to meaurse the concentration of glutamate in PWM. Glutamate concentration and cytokine expression levels will be used to evaluate the blockade effciency. Tau and phosphate Tau will also be tested in the seurm and brain at the same time points after hypoxia and P2X4 blockade. It is hoped the information obtained would provide to basis for designing of a new therapeutic target and potential serum biomarker for hypoxia induced injuries in the postnatal brain.

缺血缺氧性脑病（HIE）是新生儿致死致残的主要原因，探索其发生机制，寻找其血清学监测指标，具有重要的研究意义。目前的研究发现，HIE发生时脑室周围白质(PWM)呈选择性易损，其中炎症反应、谷氨酸代谢紊乱可能起关键作用，但机制不清。前期研究首次报道阿米巴样小胶质细胞（AMC）表达ATP受体P2X4，缺氧后其表达上调，并伴随谷氨酸转运体1（EAAT1）表达水平降低。但二者间的关系，及P2X4对其可能的调控机制迄今未见报道。脑损伤时脑内Tau蛋白异常表达并释放入血，且与EAAT1的表达间可能存在密切联系，但其在HIE中的意义尚不清楚。本项目拟对P2X4进行受体靶向抑制，研究对新生鼠缺氧后PWM区域谷氨酸浓度、EAAT1、炎症介质表达的作用；重点以Tau蛋白作为血清学指标，揭示其对PWM损伤可能的监测作用。从而为HIE的发生机制提供新证据，为临床寻找监测HIE后脑损伤程度的血清学指标提供新依据。

项目背景：发育期的缺血缺氧性脑损伤(HBD)，常导致远期行为异常，给家庭和社会带来极大的负担。 本研究聚焦发育期HBD的病理生理学机制，重点揭示炎症反应及谷氨酸代谢紊乱两大核心机理在其间可能的作用，落脚于Tau的病理性磷酸化及其调控，为该病远期行为异常的机理提供证据。.研究内容：研究首先对脑内炎症反应、谷氨酸代谢紊乱、Tau病理性磷酸化改变进行研究，进而以P2X4受体和CD36为靶点，深入研究脑内炎症反应的细胞水平调控机制；其次，从发育阶段的特殊性出发，了解发育期脑内谷氨酸转运体的发育特点、缺血缺氧性脑损伤后脑内谷氨酸浓度的改变，并以P2X4受体为切入点，揭示炎症反应与谷氨酸代谢间可能存在的调控；最后，在清楚脑内Tau蛋白病理性磷酸化后，瞄准外周血，深入了解病理情况下外周血Tau的改变及P2X4受体的作用。.重要结果及意义：1.HIE后Tau蛋白病理性磷酸化集中于脑室周围区域，为HIE后该区域的选择性易损现象提供新的依据。 2. P2X4活化参与了脑室周围区域（PWM）Tau蛋白病理性磷酸化过程，但与海马Tau蛋白病理性表达及磷酸化未见显著相关。3.但脑内Tau不同，缺血缺氧性脑损伤后血清Tau浓度升高但没有统计学意义。P2X4阻断及给予EPO后，脑损伤修复，脑内Tau蛋白水平出现升高。 4.P2X4活化对HBD后PWM和海马区域谷氨酸浓度的升高贡献较大，这与其对谷氨酸转运体EAAT2的调控更加密切。5. P2X4阻断在早期对脑室周围区域炎症介质能够发挥较好的作用，但是在海马该受体对炎症反应的抑制作用在损伤后第7天才发挥。6.进一步研究小胶质细胞功能的调控,发现急性新生儿脑损伤中，缺乏CD36改变了小胶质细胞的功能：促进趋化因子MCP-1的积聚，CD11b+/CD45+细胞增多，使其共受体TLR2表达升高，但不影响小胶质细胞内超氧化物产物的积聚以及损伤区细胞因子TNF-α和IL-1β的表达。.成果产出：项目资助下共完成6篇文章，其中SCI收录2篇，PUBMED检索1篇，北大核心3篇。授权专利3项。培养研究生4人。