A2AR-mGluR5相互作用促进中性粒细胞穿越血脑屏障的机制及其意义研究

Neutrophil is a key factor to induce over-inflammation and secondary injury of brain after brain injury. The first step of neutrophil exert this effect is to go through the blood-brain barrier. However, the molecular mechanism of the neutrophil invasion is not clear. In our previous study, we found glutamate could induce the interaction of adenosine A2A receptor (A2AR) and metabotropic glutamate receptor 5 (mGluR5) on neutrophil, cerebral microvascular endothelial cell or between the two types of cells, which significantly enhanced the ability of neutrophil going through the blood-brain barrier in vitro. And during this course, neutrophil chemokine CXCL1 in endothelial cell and its receptor CXCR1 in neutrophil were both upregulated. Online analysis indicated that the promoter region of CXCL1 has NF-κB binding site while the promoter region of CXCR1 has CREB binding site. Both NF-κB and CREB are the downstream molecule of A2AR/mGluR5 signaling. Accordingly, combination with protein molecule interaction analysis and above results, we speculate that the glutamate-induced and -activated A2AR-mGluR5 interaction on one hand promotes neutrophil adhere via the extracellular region contact of the two receptors, on the other hand, the A2AR-mGluR5 heteromeric complex activation stimulates NF-κB to upregulate CXCL1 while CREB to upregulate CXCR1 to enhance neutrophil chemotaxis. These mechanisms promote neutrophil breaks through blood-brain barrier to infiltrate into brain. In the present study, we plan to elucidated the way and key amino residues for A2AR-mGluR5 interaction, and verify the hypothesis described above via gene expression regulation research, in vitro blood-brain barrier model, traumatic brain model, Live Cell Imaging System and animal MRI. This study will provide insight of neutrophil function in brain immune regulation under abnormal immune conditions.

中性粒细胞密切参与颅脑损伤等免疫异常条件下脑组织过度炎症反应，其如何突破血脑屏障浸润入脑是本领域关注的重要科学问题。基于前期研究发现谷氨酸所诱导的中性粒细胞、内皮细胞及两者之间A2AR与mGluR5的相互作用可促进中性粒细胞穿越血脑屏障的能力，并伴随趋化因子及受体CXCL1/CXCR1表达上调，结合蛋白质分子对接模拟及生物信息学分析，我们推测：A2AR-mGluR5相互作用通过胞外区分子对接直接促进中性粒细胞黏附，并通过激活其下游分子NF-κB、CREB从转录水平上调CXCL1/CXCR1，增强中性粒细胞趋化，从而促进中心粒细胞穿越血脑屏障。因此，本研究拟对A2AR-mGluR5互作的形式及关键位点进行解析，并借助基因表达调控研究、体外血脑屏障模型、颅脑创伤模型、活细胞工作站、MRI等证实假设。旨在阐明中性粒细胞在穿越血脑屏障的分子机制及其在颅脑损伤等免疫异常条件下脑组织免疫调节中的意义。

正常情况，血脑屏障将脑组织与外周血白细胞隔绝而使脑组织具有免疫豁免器功能。但在颅脑创伤、脑缺血性损伤等病理情况下，外周血白细胞却能穿越血脑屏障浸润入脑，密切参与促发脑组织的炎症损伤，且直接关系着损伤与疾病发生发展及预后和转归。因此外周炎症系统与中枢的炎症密切相关。. 本项目的主要研究内容为：探索颅脑创伤后，外周血中性粒细胞穿越血脑屏障参与脑内炎症调控及损伤的新机制，为颅脑创伤等中枢神经系统损伤的防治新策略的提出奠定理论基础。通过研究，我们获得的重要研究结果和意义为：（1）探索了颅脑创伤中中枢与外周mGluR5在炎症调控中的作用差异与机理，证实脑微血管内皮细胞及中性粒细胞上的mGluR5缺失可以一方面可抑制血脑屏障通透性增高及血脑屏障重要组成细胞之一的脑微血管内皮细胞上中性粒细胞相关的趋化因子表达；另一方面，可降低中性粒细胞上趋化因子受体的表达。通过这两方面的作用，共同减少了颅脑创伤后中性粒细胞的浸润及促炎作用，从而实现抑炎保护效果。这为颅脑创伤的治疗策略提供了新依据。（2）阐明了全身系统性炎症反应中A2AR对中性粒细胞自身命运的调控机制及意义，即发现A2AR可以通过ROS-JNK及βγ-AKT途径抑制全身系统性炎症条件诱导的中性粒细胞的自噬进而抑制该自噬，进而通过抑制caspase3/8及PARP途径而抑制该自噬依赖的中性粒细胞凋亡。这是对A2AR抑炎新机理的发现，为全身系统性炎症反应的发生发展及控制提供了新的观点。（3）证实了颅脑创伤中中性粒细胞对神经修复的新功能，即发现浸润入脑的中性粒细胞可与神经元间发生异种细胞间的A2AR-mGluR5 互作，进而解析了其互作位点并证实其降低神经元NPR及PPP1R12，影响神经元修复。这提出了GPCR互作的新模式并为中枢神经系统损伤后基于中性粒细胞的临床干预的新理念奠定了基础。.