proBDNF信号介导的小胶质/巨噬细胞极化在脊髓缺血再灌注损伤中的作用研究

Spinal cord ischemia-reperfusion injury (SCIRI) and the following paralysis is a central issue of perioperative organ protection for the clinicians. Despite the development of various strategies, it remains a great challenge for the research field of the perioperative organ protection and requires novel therapeutic approaches. Microglia/macrophage activation has been reported to contribute to the progress of SCIRI. Microglia/macrophage can be polarized to two functionally different types of macrophage: M1 macrophages which mediate profinlammatory response and M2 macrophages which mediate anti-inflammatory response. However, it is elusive the polarization of microglia/macrophage and their roles during the SCIRI progress. Shedding new light on microglia/macrophage polarization and their role may offer a novel approaches to alleviate the progress of SCIRI. . Our previous studies have shown that brain derived neurotrophic factor precursor (proBDNF) could recruit inflammatory cells infiltration and upregulate the proinflammatory cytokine expression, suggesting that proBDNF may regulate the macrophage polarization. The preliminary experiments in the bone marrow derived macrophage (BMDM) showed that proBDNF could promote the M1 type macrophage polarization. The present project thus hypothesizes that SCIRI could induce the upregulation and release of proBDNF, which subsequently promote M1 microglia/macrophage polarization. The M1 microglia/macrophages would exacerbate the disease progress and promote the development of paralysis. . In attempt to test this hypothesis, the present project would establish the microglia/macrophage specific over-expression of proBDNF(mut) knock-in mice, the LysMCre/proBDNF(mut)f/f-KI mice. In the SCIRI mice, the microglia/macrophage polarization will be firstly examined by the flow cytometry assay, Western-blot and quantitative PCR. The expression of proBDNF and its receptors p75NTR and sortilin are examined by Western-blot. In the microglia or bone marrow derived macrophage culture, the role of proBDNF in the microglia/macrophage polariztion will be tested by up-regulation or down-regulation of proBDNF. In SCIRI model, the role of proBDNF on microglia/macrophage polarization will be also tested in the LysMCre/proBDNF(mut)f/f-KI mice or Ab-proBDNF treatment. In addition, the present study will also investigate whether the SCIRI results in more severe disease progress in the over-expression of proBDNF-KI mice. The effect of Ab-proBDNF on SCIRI disease progress will be also examined by neurologic functions, histopathologic and biochemical examinations. . Conducting the present project would shed light on the role of macrophage polarization and its modulation in the SCIRI progress in various relating surgeries. It may also suggest that anti-proBDNF is a potential drug candidate to attenuate the SCIRI progress.

脊髓缺血再灌注损伤（SCIRI）是围术期脏器保护领域的难题和热点。小胶质/巨噬细胞(MΦ)作为SCIRI进程的主要参与细胞，其M1（促炎）和M2（抗炎）型极化状态在SCIRI进程中的调控和作用尚不清楚。申请者前期研究及预实验提示抑制M1型细胞可减轻SCIRI诱发的神经功能损伤；脑源性神经生长因子前体（proBDNF）可促进MΦ向M1型极化，故推测proBDNF调控MΦ极化可能是促进SCIRI进程的重要机制。为此，本课题拟采用MΦ特异性过表达proBDNF敲入小鼠，分离、培养原代MΦ从细胞水平探讨proBDNF调控MΦ极化的机制；构建SCIRI模型从整体水平干预proBDNF表达，明确SCIRI进程中MΦ极化状况及proBDNF对其的调控作用。本研究将从新视角揭示proBDNF调控小胶质/巨噬细胞极化的机制，并为以proBDNF为靶点进行SCIRI药物研发提供坚实基础。

本课题主要探讨主动脉夹层单核/小胶质细胞极化及proBDNF信号在其中发挥的作用。研究内容包括：建立小鼠脊髓缺血再灌注模型探究脊髓小胶质细胞极化及其和疾病发展进程关系；分离主动脉夹层（AAD）患者外周血单个核细胞，研究proBDF信号在主动脉夹层单核巨噬细胞的调控作用；以及探索proBDNF信号介导的中枢神经系统损伤作用。重要发现包括：1）在AAD患者proBDNF在CD14+CD163+CX3CR1+M2类单核细胞上proBDNF表达增加，上调的proBDNF可能是促进主动脉夹层患者循环系统性炎症反应综合征的重要因子（FASEB Journal，JCR分区1区，IF：5.39）；2）发现CD4+T细胞自发性凋亡导致的淋巴细胞耗竭和围术期不良事件具有显著相关性 （Frontiers in Cardiovascular Medicine, 2021, JCR 1区，IF：6.05）；3）发现ProBDNF通过抑制脑膜CD4+T细胞的免疫活性促进脓毒症相关脑病的发生 （J Neuroinflammation， 2020，JCR 1区，IF：8.32）；4）首次发现免疫细胞来源的proBDNF信号在多发性硬化症患者脑部标本高表达，促进EAE小鼠病情进程，给予自主研发的proBDNF单克隆抗体可改善EAE小鼠症状（Theranostics 2021, JCR 1区，IF：11.556）；5）揭示了proBDNF对B细胞亚群抗体生成细胞的调控作用，发现上调的proBDNF+ASCs是促进系统性红斑狼疮疾病进程的重要机制 （Science Advances，2022， JCR1区，IF：14.14）。在本课题及先前国家自然科学基金资助下，本课题组在proBDNF信号的免疫调控功能开展了系列和开拓性的研究，自主研发的proBDNF单抗有可能成为治疗免疫炎性疾病潜在新药物。