肥大细胞外泌体介导miR-27a靶向内质网应激IRE1调节脑型疟神经元损伤的作用机制

Long-term neurological impairments are frequently detected in surviving cerebral malaria (CM) patients. The pathophysiology and therapy of long lasting cognitive deficits in CM patients after treatment of the parasitic disease is a critical area of investigation, which is called for exploring the mechanism of neuronal damage in CM. It was reported that mast cells modulated function and behavior of brain. Our preliminary data was showed that, both the numbers and degranulated percentage of mass cells were significantly increased in the brain tissue of experimental CM; activation of mast cells by Compound 48/80 intracerebroventricular injection resulted in enhanced neuronal apoptosis, p-IRE1 (that is related to endoplasmic reticulum stress-induced apoptosis), and exosome in the brain tissue of experimental CM; the elevated expression of miR-27a was also observed on the exosomes secreted form activation of mast cells in vitro. It was reported that cross-talk was among brain mast cells and neurons through exosomal microRNAs. Our central hypothesis is that mast cells degranulation can aggravate the pathogenesis of neuronal damage in CM through the secretion of exosomes. This project will use murine CM model, mast cells and neuron in vitro experiments, test that, 1) exosomal miR-27a secreted form mast cells can participate in cross-talk among brain mast cells and neurons, 2) exosomal miR-27a secreted form mast cells can aggravate the pathogenesis of neuronal damage in CM through targeting endoplasmic reticulum stress-IRE1 signaling pathway. Our findings will provide new treatment ideas and new adjuvant drugs for the therapies of CM disease.

脑型疟幸存者并发的持久神经功能障碍后遗症是亟待解决的临床问题，而探索神经元损伤机制对后遗症治疗具有重要意义。既往研究表明脑部肥大细胞可调控脑功能。预实验显示：脑型疟小鼠脑部肥大细胞数目和脱颗粒程度显著升高；侧脑室注射肥大细胞激活剂后，脑型疟小鼠脑部神经元凋亡程度、内质网应激介导凋亡的p-IRE1蛋白以及释放的外泌体升高；激活的肥大细胞上调外泌体-miR-27a的表达。以往研究表明脑部肥大细胞释放的外泌体-microRNAs参与肥大细胞和神经元间的信息交流，故我们提出假说：脑部肥大细胞通过其释放的外泌体加剧内质网应激介导的脑型疟神经元损伤。本课题拟采用体内外实验，证实脑部肥大细胞外泌体介导miR-27a在肥大细胞和神经元间转运，靶向内质网应激的IRE1通路，从而调控脑型疟神经元损伤。研究结果将为以肥大细胞的外泌体通路作为靶点的脑型疟辅助药物治疗提供科学依据。

疟疾是全球重要传染病之一，严重威胁人口健康和生命。脑型疟是重症疟疾的常见类型，高发于5岁以下儿童和疟疾无免疫力人群，病情发展迅速、死亡率高，且近三分之一的幸存者长期呈神经功能后遗症。尽管机械阻塞和免疫学说作为目前研究脑型疟发生的主流学说，但其发病机制尚未完全明确。脑部肥大细胞可调控脑功能，但其在疟疾感染以及脑型疟致病机制中的作用不详。本项目采用肥大细胞激活剂(C48/80)和稳定剂(DSCG)分别处理感染伯氏疟原虫昆明小鼠、且采用尾静脉注射肥大细胞外泌体至脑型疟模型小鼠，探讨：（1）肥大细胞活化对感染小鼠的原虫繁殖和组织病理损伤影响；（2）采用尾静脉注射肥大细胞释放的外泌体对脑型疟小鼠血脑屏障破坏的影响。研究结果显示：（1）感染小鼠淋巴结和皮肤、脑型疟小鼠脑部的肥大细胞数目和脱颗粒程度升高；（2）C48/80干预组小鼠生存时间缩短、原虫感染率增加和肝、淋巴结组织的病理损害加重，而DSCG干预组小鼠生存时间延长、原虫感染率下降和肝、淋巴结组织的病理损害减轻；（3）激活的肥大细胞促进疟原虫在小鼠体内的繁殖和加重组织病理损害，其原因可能通过分泌介质（如趋化因子和MMPs等）、增加白细胞浸润以及血管通透性；（4）尾静脉注射肥大细胞外泌体后，小鼠的脑型疟发生率、肝和脑组织病理损伤、血清Th1细胞因子表达水平（IFN-γ、TNF-α、IL-1α以及IL-1β）脑微血管内皮细胞活化程度（即ICAM-1和VCAM-1蛋白表达升高）和血脑屏障通透性（即脑组织伊文思蓝含量升高、而ZO-1和 Claudin-5蛋白表达下降）均显著升高。本项目的研究结果提示，激活的肥大细胞可促进疟原虫在小鼠体内繁殖和组织病理损害、及肥大细胞释放的外泌体可通过诱导血脑屏障破坏促进脑型疟的发生和发展，为以抑制肥大细胞功能作为疟疾治疗的靶点提供科学依据。