脑卒中后HMGB1释放对T细胞免疫功能抑制的效应及机制研究

Stroke-induced immunodepression (SIID)，characterized by lymphopenia, results in infection, which is considered to be the major complication leading to delayed mortality in stroke patients, but its mechanism is still far from clear. Our previous data showed that T cells were the most crucial lymphocytes for SIID. Hign-mobility group box 1 protein (HMGB1), a novel cytokine-like protein, is released by necrotic neurons, which circulates in the peripheral blood after stroke through damaged blood brain barrier. In vitro, low concentration of HMGB1 promotes T-cell mediated immunity. However, T cell function is inhibited by high concentration of HMGB1. Previous studies on thermal injury reported HMGB1 might be associated with immunodepression in splenic T lymphocytes in rats. In our pilot experiments, we found the level of HMGB1 was significantly increased in the plasma after cerebral ischemia/reperfusion injury (CIRI) in rat, however, numbers of total peripheral blood mononuclear cells (PBMCs) or splenocytes and lymphocyte subsets, including T cells, CD4+ or CD8+ T cells, B cells were decreased. Glycyrrhizin, as a HMGB1 inhibitor, protecting against postischemic brain by preventing HMGB1 phosphorylation and secretion, inhibted HMGB1 release in the plasma and attenuated the lymphopenia. Therefore, we speculated that HMGB1 might induce SIID through regulating T cell-mediated immunity. In this study, we will investigate the effect of HMGB1 release on T-cell mediated immunity, as well as its receptor and signaling pathway after stroke by using different immunological and molecular biological techniques in rat ischemic stroke model. Our study will offer new insight into the mechanism of SIID and provide new target for precaution and treatment of stroke patients.

脑卒中诱导的免疫抑制(SIID)，以淋巴细胞数量减少、功能改变为特征，是患者继发感染，愈后较差的重要原因，目前机制不完善，缺乏特异性治疗手段。我们前期工作发现，T细胞是SIID的关键因素；而体外研究报道，细胞因子样蛋白-高迁移率族蛋白B1(HMGB1) 小剂量促进而大剂量抑制T细胞介导的免疫应答，提示其可调控T细胞功能。脑卒中后HMGB1大量释放入血已被证实；预实验中，我们发现，脑缺血再灌注损伤（CIRI）大鼠，血浆HMGB1水平升高而脾脏和血液T细胞数量减少，抑制HMGB1释放，T细胞数量增加。因此，我们推测，脑卒中后HMGB1释放可能通过介导T细胞数量和功能改变诱导免疫抑制。本项目拟利用大鼠CIRI模型，采用多种免疫学及分子生物学实验技术，探讨脑卒中后HMGB1对T细胞免疫功能改变的效应及受体、信号转导机制。本项目将从新的视角揭示SIID的发生机制，为脑卒中及继发感染的防治提供新靶标。

脑缺血再灌注损伤（CIRI）诱导脑组织炎症反应和外周免疫抑制（SIID）双向效应，目前机制不明。CIRI致坏死神经元释放高迁移率蛋白B1(HMGB1)，其致炎效应与浸润入脑的T细胞有无关联，尚未可知。CIRI后HMGB1经破损血脑屏障释放入血，外周高水平HMGB1是否介导SIID，尚无报导。本项目采用局灶性脑缺血再灌注损伤模型及HMGB1抑制剂Glycyrrhizin，观察CIRI后，HMGB1在缺血脑组织的表达、定位，脑组织炎症反应，外周器官T细胞数量和功能以及HMGB1对T细胞作用的受体信号，同时利用T细胞缺陷动物，离、在体观察T细胞对HMGB1效应的影响及二者的相关性。结果：①CIRI 12 h，HMGB1从神经元胞核转至胞浆，48 h释放至胞外伴神经元数量减少；同时，CIRI 24 h脑脊液和血浆检测到高水平HMGB1表达；②CIRI 后缺血区小胶质细胞/巨噬细胞、中性粒细胞以及淋巴细胞及其亚型数量增多，浸润的小胶质细胞/巨噬细胞表达HMGB1；③Glycyrrhizin致大鼠脑梗塞体积减小，神经元死亡减少，HMGB1转位和胞外释放减轻，缺血区小胶质细胞/巨噬细胞、中性粒细胞和淋巴细胞浸润减轻；④Glycyrrhizin减小野生型（WT）MCAO、dMCAO大鼠及MCAO小鼠脑梗塞体积，但在裸大鼠及T、B联合缺陷SCID小鼠中，脑保护作用丧失；SCID小鼠接受WT脾细胞过继回输，脑梗塞体积增大，此增大被Glycyrrhizin抑制；Glycyrrhizin改善离体WT而非SCID脾细胞与神经元共培养体系中脾细胞诱导的神经元死亡；⑤Glycyrrhizin显著抑制MCAO 诱导的大鼠脑脊液和血清HMGB1水平增高；⑥与HMGB1升高相反，MCAO后外周血及脾脏单个核细胞总数，尤其是淋巴细胞及其亚型数量减少，Glycyrrhizin抑制血清高水平HMGB1释放的同时，显著改善淋巴细胞数量的减少；⑦MCAO诱导Th1型细胞因子IL-2和IFN-γ 基因表达下降，Glycyrrhizin改善CIRI诱导的淋巴细胞功能抑制，促进辅助性T细胞向Th1漂移；⑧抗RAGE抗体逆转HNGB1介导的T细胞免疫功能抑制，促进Th1极化。本项目的意义在于将HMGB1作为靶标，既改善中枢炎症反应脑损伤，又调控其对T细胞功能的抑制，改善SIID，为脑卒中及继发感染治疗提供理论和实验依据