肺炎链球菌脑膜炎中proBDNF-P75NTR调控TrkB受体拮抗BDNF的神经修复及再生功能

Neurological sequelae occur in 30%-50% of survivors from pneumococcal meningitis (PM). Our previous studies have shown that BDNF declines in PM after the treatment of antibiotics. We found that exogenous BDNF could promote the proliferation of neural stem cells (NSCs) in PM, however few NSCs differentiation into mature neurons. The underlying mechanism is still unclear. In the prelimary experiments, we found that proBDNF increased obviously 7 days after PM. We hypothesize that proBDNF-P75NTR signal pathway probably changes the construction of TrkB receptor, then impairs neurogenesis and neuroregeneration functions of BDNF in PM. In this project, we want to activate proBDNF-P75NTR pathway or block TrkB T1 receptor both in vitro and in vivo experiments to explore the expression of proBDNF-p75NTR, identify the types of TrkB receptor, and the effects on neurogenesis and neurogeneration, and to explore whether inhibition of TrkB T1 in vivo could eliminate the impairment of proBDNF-P75NTR signal pathway on neurogenesis and neurogeneration in PM. Our study would provide new control strategy and theoretical basis for prevention and treatment of neurological sequelae after PM.

肺炎链球菌脑膜炎存活者中仍有30%-50%患者留有神经后遗症。申请人前期研究发现，在肺炎链球菌脑膜炎使用抗生素后内源性BDNF减少而给予外源性BDNF辅佐治疗后，尽管能促进肺炎链球菌脑膜炎内源性神经干细胞增殖，但向神经元分化成熟较少，其机制尚不清楚。预实验发现脑膜炎7天后proBDNF明显增多，申请人提出假说在肺炎链球菌脑膜炎中proBDNF-P75NTR可能通过影响TrkB受体蛋白结构从而抑制了BDNF的神经修复及再生作用。本研究分别激活proBDNF-P75NTR通路或抑制TrkB T1受体，观察肺炎链球菌脑膜炎中proBDNF-P75NTR及其TrkB受体各类型的表达变化，及对神经元凋亡、神经干细胞增殖分化的影响，并验证抑制TrkB T1受体是否可以消除proBDNF-P75NTR对BDNF的拮抗作用。本研究为防治肺炎链球菌脑膜炎神经系统后遗症提供新的防治策略和科学的理论依据。

目的：尽管随着疫苗和抗生素的不断应用，肺炎链球菌脑膜炎的病死率及神经系统后遗症的发生率仍居高不下。剧烈的炎症反应及内源性神经修复的不足是肺炎链球菌脑膜炎造成不良预后的重要原因。课题组前期研究探究了调控BDNF-TrkB信号通路在肺炎链球菌脑膜炎神经炎症和神经再生修复中的作用。而与BDNF-TrkB具有相反生物学作用的proBDNF-p75NTR信号通路，在肺炎链球菌脑膜炎中的作用仍待探索。本研究主要探讨proBDNF-p75NTR信号通路在肺炎链球菌脑膜炎病程中的动态变化及调控p75NTR后对肺炎链球菌脑膜炎神经炎症及神经生发的影响。.方法：1、采用3周龄SD大鼠，构建肺炎链球菌脑膜炎模型，分别于造模后第24小时、7天、14天及28天取材，探究proBDNF-p75NTR信号通路的表达变化；2、采用小分子配体LM11A-31调控p75NTR及基因敲减p75NTR在肺炎链球菌脑膜炎急性期大鼠大脑中的表达，探究p75NTR是否参与了肺炎链球菌脑膜炎的炎性损伤过程；3、构建肺炎链球菌脑膜炎模型24小时后，采用LM11A-31或赋形剂长时程辅助治疗7天，观察肺炎链球菌脑膜炎不同时间点海马DG区中神经生发情况。.结果：第一部分 造模后第24小时、7天、14天及28天，脑膜炎组大鼠脑组织中p75NTR的表达水平较生理盐水对照组显著增高，proBDNF的表达在感染后第7天开始显著升高。同时，p75NTR主要表达于神经元及星形胶质细胞中，而在小胶质细胞中无表达。.第二部分 LM11A-31的预处理可显著减轻肺炎链球菌脑膜炎急性期大鼠的临床症状、病理严重程度，减少小胶质细胞和星形胶质细胞的激活以及神经元的凋亡和坏死，同时减少促炎转录因子及促炎因子的表达。侧脑室注射腺病毒敲减p75NTR的表达，也可显著减轻肺炎链球菌脑膜炎大鼠病理严重程度及促炎介质的生成。 .第三部分 LM11A-31的长时程干预，加速了肺炎链球菌脑膜病程中神经炎症的消退。同时长时程LM11A-31干预组大鼠海马DG区中EdU&DCX、EdU&NeuN阳性细胞数较赋形剂干预组显著增多，而EdU&DCX/EdU、EdU&NeuN/EdU比例在各组大鼠中的无显著差异。