卡拉胶寡糖基于AMPK/ULK1通路诱导小胶质细胞自噬作用机理的研究

Some resent research works showed that autophagy of microglia can mediate the degradation of extracellular Aβ and induction of autophagy has been regarded as a new therapeutic target for Alzheimer's disease (AD), but the target point of inducting the autophagy of microglia under the progress of AD is still unrevealed..Our previous work showed that carrageenan oligosaccharides (KOS), which were prepared in our lab, can induce autophagy of microglia and inhibit neuroinflammation. But there still are some unsolved issues which are barriers for us to understand the mechanism of autophagy induced by KOS, such as the regulation pathway of autophagy induced by KOS, the interaction between the autophagy induction and inhibition of neuroinflammation and the neuroprotection through the autophagy induced by KOS. In order to solve above issues, our project will use KOS as probe compound and focus on the AMPK/ULK1 pathway of the microglia autophagy to explore the regulation signal pathway of autophagy and the neuroprotection induced by KOS with methods of western blotting, siRNA and co-culture cell et al. At the end, the mechanism of autophagy induced by KOS, the interaction between autophagy induction and neuroinflammation inhibition and the neuroprotection by increasing autophagy will be revealed in our project. The key factor and target point for the regulation of microglia autophagy through the AMPK/ULK1 pathway will be identified in this project. All results of this project will provide scientific basis for the AD treatment and drug screening with the target of the induction of microglia autophagy.

自噬可以介导小胶质细胞对细胞外Aβ的清除作用，诱导小胶质细胞自噬对阿尔茨海默病防治具有重要意义，但是在阿尔茨海默病(AD)发病条件下诱导小胶质细胞自噬的作用靶点不明确。.我们在制备卡拉胶寡糖（KOS）的基础上，发现KOS具有诱导小胶质细胞自噬和降低神经炎症的双重功能，因此本项目拟以KOS作为探针化合物，以小胶质细胞和AD小鼠作为研究对象，以AMPK/ULK1自噬调控途径为切入点，通过蛋白印迹、siRNA干扰、细胞混合培养等技术，研究KOS诱导小胶质细胞自噬的的作用途径和位点，探讨KOS诱导自噬与抑制炎症之间的相互作用规律，揭示KOS通过诱导小胶质细胞自噬对神经损伤的保护机制。项目最终不仅将阐明KOS基于AMPK/ULK1通路诱导小胶质细胞自噬的作用机理，而且能够明确AMPK/ULK1通路中调控小胶质细胞自噬的关键因子和作用靶点，为以诱导小胶质细胞自噬为靶标的AD治疗和药物筛选提供科学依据。

首先，研究了卡拉胶寡糖（KOS）对小胶质细胞过度活化的抑制作用。研究结果表明，经过KOS预保护后，LPS诱导的小胶质细胞增殖力相对减弱，炎症因子释放量均有所抑制，而细胞内精氨酸酶的活性相对减弱，并且这种抑制作用呈剂量相关。与KOS相比，其脱硫酸基衍生物的抑制作用相对较弱。经激光共聚焦技术和流式细胞术检测，KOS可以透过细胞膜进入细胞核，并且与LPS有一定竞争关系。Western Blot结果表明KOS能够显著抑制TLR4等蛋白的表达和JNK、p38 蛋白的磷酸化，表明KOS能通过抑制TLR4/NF-κB、JNK/p38 MAPKs信号通路的激活、抑制氮应激和氧应激反应降低小胶质细胞的炎症反应。.其次，研究了KOS诱导小胶质细胞自噬的作用机理及其与炎症之间的关系。结果表明KOS对小胶质细胞自噬通路标志蛋白LC3-I/II、自噬通路各阶段关键蛋白Beclin1等具有上调作用，通过对自噬上游调节通路蛋白表达的检测，发现KOS作用后上调了AMPK及磷酸化水平、ULK1及磷酸化水平，抑制mTOR表达水平；LC3-Ⅰ/Ⅱ表达升高并且出现了自噬小体聚集，自噬水平显著提高，炎症因子和炎性小体NLRP3表达显著下降；自噬抑制剂3-甲基腺嘌呤可以抑制KOS诱导小胶质细胞的自噬水平，结果发现细胞形态发生“阿米巴”样变化，细胞处于激活状态，小胶质细胞TNF-α、IL-6、IL-1β及炎性小体NLRP3表达水平升高。.最后研究了KOS通过抑制小胶质细胞炎症反应对神经的保护作用。结果表明在共培养体系下发现KOS能够通过抑制小胶质细胞过度激活维持神经细胞骨架蛋白表达的稳定，维持细胞膜电位、上调Bcl-2的表达、下调Bax、Cytochrome c和Caspase-3的表达，从而抑制炎症引起的神经细胞凋亡。在体内实验中，KOS能够抑制小胶质细胞过度激活，降低阿尔兹海默症（AD）模型小鼠脑内炎症，并减少APP和Aβ1-42的沉积、上调CSP-α的表达、下调iNOS、NF-L、Tau、ApoE等与AD病相关蛋白的表达，提高了模型小鼠空间学习和记忆能力。.总之，通过本项目的完成，阐明了KOS基于AMPK/ULK1通路诱导小胶质细胞自噬的作用机理，明确了AMPK/ULK1通路中调控小胶质细胞自噬的关键因子和作用靶点，为以诱导小胶质细胞自噬为靶标的AD治疗和药物筛选提供科学依据。