脑源性神经营养因子（BDNF）/TrkB和模式识别受体TLRs-Myd88及Nod2通路在肺炎链球菌脑膜炎的交互作用

Neurological sequelae still occur in 30 to 52% of survivor from Streptococcus pneumoniae meningitis. Our previous animal studies have shown that BDNF and its receptor, TrkB, was strongly expressed in the inflammatory cells of the CSF in Streptococcus pneumoniae meningitis animal model, and was also up-regulated in cortical and hippocampal. But after antibiotics treatment, their expressions were significantly down-regualted. We also found exogenous BDNF adjuvant therapy could rescue the cortical and hippocampal neurons from inflammatory brain injury and showed a significant neuroprotective effects. However, its mechanism is still unclear. We hypothesize that it is associated with host inflammatory response. Thus, in this study, we are constructing two kinds of Streptococcus pneumoniae meningitis animal models, respectively in Myd88 and Nod2 knock-out mice, to explore whether TLRs-Myd88 and Nod2 pathway regulate the expression of BDNF in host immune cells, inflammatory cells and nerve cells. We are also constructing another two kinds of models in BDNF knock-out mice as well as in rats with exogenous BDNF treatment, to explore the effects of BDNF / TrkB signal transduction on TLRs-Myd88 and Nod2 pathways. Our results will provide a new theoretical basis for brain injury pathophysiology and clinical intervention therapy of Streptococcus pneumoniae.

肺炎链球菌脑膜炎存活者中仍有30-52%患者留有神经后遗症。我们前期实验研究发现，肺炎链球菌脑膜炎时在脑脊液的炎症细胞中BDNF及其受体TrkB高表达，在皮层和海马中表达上调。而抗生素治疗后, 在皮层和海马的表达明显下调，存活神经元减少。BDNF补充治疗后，呈显著的神经保护作用，但其机制尚不清楚。我们假设这些变化与宿主炎症反应有关。因此，本研究分别用Myd88 、Nod2和BDNF基因敲除及外源性BDNF处理的肺炎链球菌脑膜炎模型，探讨宿主免疫细胞、炎症细胞和神经细胞TLRs-Myd88 、Nod2通路对BDNF表达的调节，研究BDNF/TrkB信号传导对TLRs-Myd88和Nod2通路的影响，阐明BDNF/TrkB与上述模式识别受体通路的交互作用，为肺炎链球菌脑膜炎后遗症发生的病理机制和临床干预提供新的理论依据，也为研究BDNF在免疫系统的作用和模式识别受体在神经系统的作用奠定基础。

课题组前期研究发现，在肺炎链球菌脑膜炎急性期，皮层和海马区神经元广泛受损，而脑源性神经营养因子(Brain derived neurotrophic factor, BDNF)及其受体TrkB的表达明显升高，但应用抗生素治疗后，其表达水平明显低于正常。给予外源性BDNF补充治疗后，可以明显提高皮层及海马区神经元的存活率，保护肺炎链球菌脑膜炎引起的听力损伤。以上结果提示，在肺炎链球菌脑膜炎时，不仅存在TNF-α、IL-1β等炎症因子的破坏作用，还存在神经营养因子的保护作用，这两个系统共同推进肺炎链球菌脑膜炎的病理生理过程。本项目首先采用myeloid differentiation factor 88 (MyD88) 基因敲除小鼠（myd88-/-），建立肺炎链球菌脑膜炎模型，观察TLRs/MyD88信号通路对BDNF表达的影响。随后，提前补充外源性BDNF或高效TrkB受体阻断剂，激活或阻断BDNF/TrkB通路后，再次建立肺炎链球菌脑膜炎模型，观察BDNF/TrkB通路对TLRs/TLR信号通路的影响。第一部分结果：肺炎链球菌感染24h后，myd88-/-脑膜炎组脑膜炎症反应明显弱于WT脑膜炎组，而神经元损伤及凋亡数目明显多于WT脑膜炎组；ELISA结果提示myd88-/-脑膜炎组中枢及外周炎症因子TNF-α、IL-1β、IL-6、IL-10均明显低于WT脑膜炎组。同时，myd88-/-脑膜炎组皮层及海马内BDNF mRNA及蛋白升高均低于WT脑膜炎组。ChIP-PCR结果提示在小胶质细胞内BDNF启动子区域存在Nf-κB有效结合位点。第二部分结果：提前补充外源性BDNF后，可以减轻脑膜炎症反应，皮层及海马存活神经元增加，凋亡神经元数目减少。皮层及海马中MyD88的表达明显减低，趋炎因子TNF-α、IL-1β和IL-6表达明显降低，抗炎因子IL-10表达升高。同时，皮层及海马区TrkB表达增高，PI3K-AKT活性增强。以上结果提示：1、在肺炎链球菌脑膜炎急性期，炎症信号通路MyD88/Nf-κB激活，释放趋炎因子、细胞因子，产生炎症级联反应，同时该通路也有助于产生具有神经保护作用的BDNF。2、提前给予外源性BDNF补充后，可能通过激活PI3K/AKT通路调节自身免疫反应，提高神经元应激能力，明显改善肺炎链球菌脑膜炎神经损伤。