Piperlongumine下调NLRC5抑制大肠杆菌脑膜炎形成的分子机制研究

Escherichia coli meningitis is a serious infectious disease in the central nervous system of the newborn, with high morbidity and mortality. The bacteria passing through the blood brain barrier (BBB) is the key to the occurrence of meningitis, but its mechanism is still unclear.The inhibition of bacteria invasion cells and killing intracellular bacteria have been the difficulties in the study of bacterial meningitis treatment. Our previous studies have demonstrated that NLRC5 can induce Escherichia coli E44 to invade human brain microvascular endothelial cells (HBMEC), piperlongumine (PL) can inhibit E.Coli E44 invasion of brain microvascular endothelial cells (BMECs), and can prevent the happening of the meningitis in mice caused by E44. we have also found PL can regulate the gene expression of related signaling pathways of bacterial adhesion cell , and can dramatically inhibited the gene expression of NLRC5. Knockout of NLRC5 significantly inhibits bacteria invasion HBMEC. Basing on the results above, we put forward the hypothesis that PL can suppress E44 invasion of cell by down-regulation of NLRC5 and inhibit the formation of bacterial meningitis, but some molecular mechanisms should be further elucidated. Trough this project, we intend to use in vitro model of BBB and the knock-out animal models, chromatin Immunoprecipitation (ChIP), and Next-generation sequencing technology to study the mechanism of PL reduced expression of NLRC5, to reveal the target genes of NLRC5 regulated (as a receptor when bacterial invasioncell), and to determine the mediated ligand of the bacteria.This project will help further elucidate the interaction and recognition between the bacteria and cells, and it will be potential for new targets and new approaches for treatment of bacterial meningitis .

大肠杆菌脑膜炎是新生儿中枢神经系统严重的感染性疾病，发病率和死亡率均高。细菌穿越血脑屏障（BBB）是脑膜炎发生的关键，其机制仍不清楚；抑制细菌侵袭细胞、杀灭胞内细菌是细菌性脑膜炎防治的难点。我们前期发现NLRC5能介导大肠杆菌E44侵袭人脑微血管内皮细胞（HBMEC）；Piperlongumine(PL)具有抑制E44侵袭HBMEC、降低NLRC5基因的表达、预防E44致小鼠脑膜炎发生的作用。据此提出科学假说：PL下调NLRC5抑制E44对BMEC的侵袭从而抑制细菌性脑膜炎的形成。本项目拟从体外BBB模型和基因敲除动物模型两个层面揭示NLRC5所调控的靶基因（作为细菌侵袭细胞的受体）在E44侵袭细胞中的作用，确定介导细菌侵袭细胞配体，探讨PL下调NLRC5抑制大肠杆菌脑膜炎形成的具体分子机制。本项目的实施进一步丰富了对细菌、细胞之间互作及识别的认识，为治疗细菌性脑膜炎提供新的靶点和思路。

大肠杆菌脑膜炎是新生儿时期严重的感染性疾病，发病率和死亡率均高。细菌穿越血脑屏障（BBB）是脑膜炎发生的关键，其机制仍不清楚，也是脑膜炎防治的难点。课题组前期发现NLRC5能介导大肠杆菌E44侵袭人脑微血管内皮细胞（HBMEC）；Piperlongumine（PL）具有抑制E44侵袭HBMEC、降低NLRC5基因的表达的作用。本研究构建过表达和敲除NLRC5的HBMEC细胞系、体外血脑屏障模型和新生小鼠脑膜炎模型，引进NLRC5基因敲除小鼠，运用二代测序、Cas-9技术、免疫共沉淀、流式细胞术等实验技术，从体内外探究PL下调NLRC5抑制E44穿越血脑屏障诱发脑膜炎的机制。结果发现：①NLRC5可促进细菌对HBMEC的侵袭，抑制细胞的迁移和内吞，加剧神经元的丢失、脑内及HBMEC的炎症反应，在大肠杆菌脑膜炎形成过程中发挥重要作用；②PL相似的自由基刺激剂—佛波酯同样能抑制E44侵袭HBMEC、降低NLRC5基因的表达，并提高细胞内ROS的水平，而氮乙酰半胱氨酸（NAC，活性氧拮抗剂）能逆转PL及佛波酯下调NLRC5的表达、降低E44对HBMEC的侵袭作用，证实ROS是PL调控NLRC5表达、防止E44侵袭HBMEC的关键因子。③PL可以抑制E44的侵袭及诱发的新生小鼠脑膜炎，对脑损伤有保护作用，其防治机制可能与PL促进ROS的产生，从而降低STAT1及转录因子Sox5与NLRC5启动子的结合、NLRC5与PPARγ的相互作用，进而下调NLRC5及其调控的NF-κB炎症信号通路，抑制E44穿越血脑屏障，防止大肠杆菌脑膜炎的发生有关。. 该研究结果明确了NLRC5在E44侵袭BMEC、诱发脑膜炎发生中的重要作用；阐明了PL促进ROS的产生，下调NLRC5及其调控的炎症信号通路，防止大肠杆菌脑膜炎发生的机制。为大肠杆菌脑膜炎的防治提供了新的靶点和思路，具有重要的临床意义和社会应用价值。