NOD2在幼鼠肺炎链球菌脑膜炎中的作用和分子机制

The gram-positive bacterium, Streptococcus pneumoniae, is the leading cause of bacterial meningitis in adults Such central nervous system (CNS) infections are associated with an intense inflammatory host response and a high degree of mortality Furthermore, patients that survive S. pneumoniae-associated meningitis often exhibit serious neurological sequelae. As such, delineating the pathogenesis of these infections is essential if we are to improve disease outcome. While the brain has traditionally been viewed as a ''victim organ'' of infiltrating leukocytes, it has become increasingly apparent that resident glial cells such as microglia and astrocytes play an important role in the initiation and/or progression of immune responses following pathogen invasion We have previously demonstrated that astrocytes and microglia constitutively express nucleotide-binding oligomerization domain-2 (NOD2), a member of the novel nucleotide-binding domain leucine-rich repeat region-containing family of proteins (NLR) that functions as an intracellular receptor for a minimal motif present in all bacterial peptidoglycans. Importantly, we have recently confirmed that NOD2 is an important component in inflammatory CNS responses to the gram- positive bacterial pathogens, Streptococcus pneumoniae. NOD2 have been identified as cytosolic pattern recognition receptors that play a role in the initiation of inflammatory host immune responses to bacterial challenge. However, the manner of NOD2 in astrocyte and microglial recognize S. pneumoniae have not been reported. In the present study, we will study the mechanism that NOD2 in astrocyte and microglial are involved in the intracellular recognition of S. pneumoniae and streptococcal cell walls. In addition, we will show NOD2 and its signalling pathways in the pathophysiology of bacterial meningitis. These data will contribute to disclose the mechanism of bacterial meningitis and provide the scientific basis for the treatment of bacterial meningitis.

肺炎链球菌脑膜炎是儿科常见病，有较高的致残率和死亡率，其确切发病机制尚未阐明， 多认为与细菌诱导脑组织免疫反应有关。近年研究发现NOD2参与细胞内细菌成分的识别并诱导炎症反应。我们前期研究发现，神经胶质细胞存在NOD2受体，NOD2敲除后，肺炎链球菌脑膜炎小鼠脑组织中细胞因子含量显著下降。但是，NOD2作为胞内识别受体，肺炎链球菌及其菌体成分MDP怎样由胞外转移到神经胶质细胞内供NOD2识别？NOD2及其信号通路在肺炎链球菌脑膜炎脑损伤发病机制中的作用如何？这些问题迄今国内外尚无报道。本项目在既往工作的基础上，利用基因敲除小鼠和突变重组质粒，采用分子生物学、免疫荧光等技术，从细胞、整体两个层次，探讨肺炎链球菌及其菌体成分由胶质细胞外转移到胞内供NOD2识别的途径以及NOD2信号通路在脑膜炎发病机制中的作用，以期深入理解肺炎链球菌脑膜炎的发病机制，为其防治提供新思路。

肺炎链球 脑膜炎是儿科常见病，有较高的死亡率和致残率，其确切发病机制尚未阐明，多认为与细菌诱导脑组织免疫反应有关。近年研究发现NOD2参与细胞内细菌成份的识别并诱导炎症反应。本项目采用分子生物学、免疫荧光等技术，从细胞、整体两个层次，探讨肺炎链球菌及其菌体成份由胶质细胞外转移到胞内供NOD2识别的途径以及NOD2信号通路在脑膜炎发病机制中的作用。我们的细胞实验发现间接免疫荧光双染法显示肺炎链球菌细胞壁的胞壁酰二肽（MDP）和网格蛋白共定位存在。MDP可以刺激小胶质细胞上调NOD2和RIP2的mRNA和蛋白表达。氯丙嗪可以抑制MDP引起的小胶质细胞上调NOD2和RIP2的mRNA和蛋白表达的作用。MDP 可引起IL-1β表达增加，但是这种作用，可被氯丙嗪抑制。但是MDP和氯丙嗪对小胶质细胞分泌IL-6.没有作用。MDP可通过网格蛋白进入小胶质细胞，MDP对于NOD2和RIP2的影响，可以被氯丙嗪抑制。我们的整体实验发现感染组NOD2表达高于对照组和治疗组，头孢曲松+地塞米松组和NOD2-siRNA组NOD2表达较头孢曲松组低。数据表明地塞米松和基因敲除技术可能下调肺炎球菌性脑膜炎大鼠脑组织NOD2表达,肺炎链球菌感染后，RIP2的表达抑制可明显降低炎性转录激活因子NF-kB以及相关炎性细胞因子的生成。我们的研究结果显示感染组动物RIP2蛋白的表达水平明显高于生理盐水组。维生素B6辅助治疗肺炎球菌性脑膜炎可以改善临床症状，并通过对犬尿氨酸（KYN）通路的影响，导致细胞烟酰胺腺嘌呤二核苷酸（NAD） +和ATP稳态的维持及显著降低脑组织中的细胞因子。此外，它还可以改善认知行为及保护大脑皮层的神经元免受炎症性损伤。