在脑膜炎脑水肿中NF-κB激活对AQP4内化的作用及机制研究

Aquaporin-4(AQP4) plays an important role in the development of brain edema induced by meningitis. In the previous study, we have found that AQP4 was internalized from the astrocytes membrane to the intracellular endosome in the brain with meningitis and that the internalization of AQP4 was involved in the development of brain edema. Therefore, elucidating the mechanism of AQP4 internalization and taking corresponding measures to regulate the process of AQP4 internalization are expected to open up new ways for the treatment of brain edema. The results of some researches demonstrated that inflammation stimulation can make the activation of NF-κB, which serves as a pivotal transcription factor upregulating the expression of matrix metalloproteinases (MMPs). Other literatures showed that MMPs can cleave dystroglycan and laminin, which are important factors for AQP4 anchored at proper membrane domain. Based on above research foundings, we propose that the degrading of AQP4 anchoring factors caused by activation of NF-κB may be involved in the internalization of AQP4. In this study, we will study the effect and mechanism of NF-κB activation on AQP4 internalization in the brain with meningitis following the axial line of ‘inflammatory stimulations—NF-κB activation—upregulation of MMPs—degrading of AQP4 anchoring factors—AQP4 internalization’. The results of this study can supply an experimental basis for clarifying the mechanism of AQP4 internalization and developing therapeutic drugs for the treatment of brain edema.

具有水转运功能的水通道蛋白-4（AQP4）对脑膜炎脑水肿的发生具有重要作用。我们前期在幼鼠脑膜炎脑内发现：AQP4从星形胶质细胞膜进入细胞内的内化过程参与了脑膜炎脑水肿的发生发展。因此探讨AQP4内化的发生机制并干预其过程，有望为脑水肿的治疗开辟新途径。研究表明，炎症激活的NF-κB可上调基质金属蛋白酶(MMPs)表达；另有研究发现MMPs可促进dystroglycan（DG）和laminin蛋白的降解；而DG和laminin等是使AQP4锚定在细胞膜上的重要分子。因此，在脑膜炎脑水肿中，AQP4内化的发生可能与NF-κB活化导致的AQP4锚定因素破坏相关。本课题将以“炎症刺激—NF-κB激活—MMPs表达上调—AQP4锚定因素破坏—AQP4内化”为轴线，研究在脑膜炎脑水肿中NF-κB激活对AQP4内化的影响及机制。本课题有望阐明AQP4内化的分子机制，为开发治疗脑水肿新药提供实验依据。

背景：水通道蛋白4（AQP4）对脑水肿的发生具有重要作用。前期发现：AQP4从星形胶质细胞膜进入细胞内的内化过程与了脑膜炎脑水肿的发生。探讨AQP4内化的发生机制并干预其过程，有望为脑水肿的治疗开辟新途径。研究内容：以“炎症刺激—Poldip2表达上调—NF-κB激活—MMPs表达上调—AQP4锚定因素破坏—AQP4内化”为轴线，研究在脑膜炎脑水肿中NF-κB激活对AQP4内化的影响及机制。结果及关键数据：在体实验中，与对照小鼠比较，小鼠脑膜炎模型中脑含水量增加，行为学评分降低。模型鼠脑内Poldip2, p-NF-κB, MMPs表达增高, β-dystroglycan（β-DG）和血脑屏障相关蛋白表达降低；AQP4与EEA1共表达，发生内化。上调和下调Poldip2可分别激活和抑制p-NF-κB表达。通过抑制Poldip2对模型鼠脑内激活的NF-κB进行抑制后，可提高行为学评分，减轻脑水肿程度，逆转MMPs、β-DG和血脑屏障相关蛋白的表达变化，同时，抑制Poldip2 还可逆转AQP4内化发生；上调Poldip2与上述作用相反。抑制MMPs的活性后，可提高行为学评分、减轻脑水肿程度、上调β-DG及血脑屏障相关蛋白的表达水平，同时逆转AQP4内化的发生。离体实验中，用Poldip2激活星形胶质细胞中NF-κB后，MMPs和β-DG表达下调，AQP4发生内化。MMPs抑制剂可逆转β-DG的表达变化及AQP4内化。结论及科学意义：建立了小鼠脑膜炎脑水肿模型，明确在该模型中发生AQP4的极性分布缺失，发生内化；明确了在脑膜炎脑水肿模型中Poldip2 表达增高、NF-κB通路被激活；明确了脑膜炎脑水肿模型中NF-κB可通过促进MMPs表达上调，使AQP4锚定相关蛋白降解，从而促进AQP4内化发生；完成了原代星形胶质细胞的取材，培养及传代等工作，并在体外细胞模型中进一步证实了NF—κB通路激活可通过“MMPs表达上调-AQP4锚定相关蛋白降解”这一通路促进AQP4内化发生； 明确了在新生小鼠GBS感染的脑膜炎脑水肿模型中，Poldip2可激活NF-κB通路；阐明了小鼠脑膜炎脑水肿发生的分子机制，并为开发脑水肿治疗新药奠定了实验基础。