TLR2信号通路在间歇性低氧导致的认知功能障碍中的作用及分子机制

Cognitive impairment is one of the most common non-respiratory complications of obstructive sleep apnea syndrome (OSAS) and severely impairs the quality of life of patients. Immunological stress induced by chronic intermittent is a major cause of cognitive impairment in OSAS patients. However, the related mechanisms are still unknown. It has been demonstrated that TLR2 plays a crucial role in intermittent hypoxia (IH) induced cognitive impairment. Our previous studies have found the abilities of learning and memory in wild type C57BL/6 have significantly declined and the increased expressions of TLR2 have been detected in hippocampus and frontal cortex after chronic intermittent hypoxia. Compared with wild type mice, TLR2-/- mice showed obvious cognitive impairments. However, no significant changes of the ability in learning and memory have been found in TLR2-/- mice after chronic intermittent hypoxia. Whether TLR2 play an important part in the cognitive impairment caused by OSAS and what are the detailed mechanisms, still need to be clarified. In this study, we intend to detect the expression level of TLR2, functional molecules and cytokines after chronic intermittent hypoxia in vivo and in vitro. It will be help to explore the mechanisms of OSAS induced cognitive impairment by analyzing the relationship between the damage or death of neurocyte and the changing expression of functional molecules and cytokines regulated by TLR2 signalling pathway. We aim to demonstrate the effects and mechanisms of TLR2 in the impairment of learning and memory caused by chronic intermittent hypoxia and search the molecular target to prevent and treat OSAS-related cognitive deficit. Until now, there has been no relative report.

间歇性低氧能够引发免疫应激，造成神经细胞损伤、死亡，与OSAS导致的认知障碍密切相关，但其相关机制尚不清楚。我们前期工作发现TLR2在其中发挥了重要作用。本研究拟从TLR2入手：在个体水平上，利用C57BL/6与TLR2敲除小鼠构建动物间歇性低氧模型，研究间歇性低氧后，小鼠海马、皮层中TLR2信号通路激活引起的细胞因子IL-1β、IL-6、IL-10表达水平的变化，并找到这些关键细胞因子介导的免疫炎症、免疫毒性反应与间歇性低氧造成的神经损伤间的相关性。同时，构建细胞间歇性低氧模型，探索间歇性低氧暴露导致神经元、胶质细胞、小胶质细胞损伤及应激的过程，结合RNAi手段，研究TLR2途径在间歇性低氧导致的神经细胞损伤中的作用机制。综合in vivo与in vitro实验结果，揭示TLR2在间歇性低氧导致的认知障碍中的作用及分子机制，从而为找到治疗OSAS神经系统并发症的有效分子靶点奠定理论基础。

睡眠呼吸暂停综合征（OSAS）是在呼吸睡眠障碍这一领域最常见的病症之一。间歇性低氧（CIH）是OSAS的典型特征，能够引发免疫应激，造成神经细胞损伤、死亡，与OSAS导致的认知障碍密切相关，但其相关机制尚不清楚。在前期工作中，我们发现Toll样受体（TLRs），尤其是TLR2在其中发挥了重要作用。课题组构建了C57BL/6与TLR2敲除小鼠间歇性低氧模型，通过三箱实验及水迷宫实验表明CIH能导致野生型及TLR2缺失型小鼠认知功能障碍，而TLR2基因缺失减轻了CIH引起的小鼠社交记忆、空间学习记忆等认知功能障碍，明确了TLR2在间歇性低氧导致的认知障碍中的作用。为了找到其中潜在的病理机制，分别研究了海马中神经元、胶质细胞、小胶质细胞损伤及应激的过程，HE结果，我们发现CIH不仅可以引起海马神经元排列紊乱、细胞形态异常，甚至导致神经元脱失，而敲除TLR2减少了海马内神经元的相关损伤；免疫荧光结果证明CIH条件下，野生型及TLR2 KO型实验鼠海马神经元NeuN的表达量减少，而小胶质细胞标志物Iba-1以及星型胶质细胞的特异性标记物GFAP的荧光强度却较正常对照组显著增加，值得注意的是，与TLR2基因缺失的小鼠相比，间歇性低氧条件下野生型的海马神经元NeuN减少更为明显，相反Iba-1和GFAP相对增加更为显著，表明TLR2参与了CIH诱发的神经元损伤以及胶质细胞异常。此外，通过检测TLR2信号通路以及相关细胞因子的表达水平，发现CIH可以激活TLR2-MyD88-NF-κB信号通路，增加TNF-α，IL-6，IL-1β等炎症因子的产生、释放；但当TLR2介导的信号通路被阻断后，CIH诱导的炎症因子表达量显著减少。由此可以看出，CIH能够导致海马神经细胞的损伤，进而影响小鼠社交记忆、空间学习记忆等认知功能，TLR2-MyD88-NF-κB途径在这个过程中扮演了重要的角色，抑制TLR2信号通路的活化可以改善间歇性低氧引起的认知功能的障碍，这为揭示CIH导致认知功能障碍的分子机制提供了新的思路。