运动训练抑制NLRP3介导的小胶质细胞焦亡改善阿尔茨海默病小鼠焦虑样行为和认知障碍的作用与机制研究

关键词：阿尔茨海默病  认知康复  运动训练  小胶质细胞  焦亡  NLRP3

Keywords:Alzheimer's disease  cognitive rehabilitation  physical exercise  microglia  pyroptosis  NLRP3

摘要：

      摘要 目的：探讨运动训练改善阿尔茨海默病（Alzheimer's disease，AD）情绪和认知功能的作用，及NLRP3[NACHT-，LRR，and pyrin（PYD）-domain-containing protein 3]炎症小体介导的小胶质细胞焦亡的参与机制。 方法：选取10只5月龄的雄性5xFAD小鼠为AD模型，分为对照组和运动训练组，选取年龄匹配的C57/BL6小鼠为野生型（wild type，WT）组。WT组和对照组小鼠在普通饲养笼里饲养，运动训练组小鼠在装配有跑轮的笼子里饲养，小鼠可以自由跑步训练。采用旷场试验检测小鼠的情绪功能，Morris水迷宫试验检测小鼠的空间认知功能，免疫荧光染色检测神经元存活、淀粉样斑块沉积和小胶质细胞激活，以及小胶质细胞溶酶体的聚集和焦亡相关蛋白（GSDMD）的表达情况。通过Western Blot实验检测小鼠促炎因子、抑炎因子、GSDMD蛋白的表达。 结果：在旷场试验中，与WT组小鼠相比，5xFAD对照组小鼠在中心区域停留的时间显著缩短，而5xFAD运动训练组小鼠在中心区域停留的时间较对照组显著延长。在Morris水迷宫训练期间，WT组和5xFAD运动训练组小鼠到达平台的潜伏期逐渐缩短，而5xFAD对照组小鼠到达平台的时间无明显变化。在空间探索实验中，5xFAD对照组小鼠穿越平台的次数比WT组显著减少，而5xFAD运动训练组小鼠较对照组显著增加。5xFAD运动训练组小鼠皮质和海马的神经元数量较对照组显著增加，而Aβ1-42斑块所占面积显著减小。5xFAD对照组小鼠Aβ淀粉斑块周围可见明显的溶酶体相关膜蛋白1抗体（Lysosomal associated membrane protein 1，Lamp1）聚集，而运动训练小鼠的Lamp1荧光强度较对照组显著减少。Western Blot结果显示，5xFAD对照组小鼠皮质和海马的Lamp1和（Cathepsin B，CtsB）的表达比WT组小鼠显著增加，而运动训练可降低5xFAD小鼠皮质和海马的Lamp1和CtsB的表达。5xFAD对照组小鼠皮质和海马的促炎因子的表达较WT组小鼠显著增加，运动训练可减少5xFAD小鼠皮质和海马促炎因子的表达、增加抑炎因子的表达。与WT小鼠比较，5xFAD对照组小鼠皮质和海马的GSDMD蛋白的表达显著增加，而运动训练可降低5xFAD小鼠GSDMD的表达，且GSDMD蛋白与Iba-1阳性小胶质细胞共定位。 结论：运动训练通过抑制NLRP3介导的小胶质细胞焦亡改善阿尔茨海默病情绪和认知功能。

Abstract:

      Abstract Objective： To investigate the protective effect of exercise training on emotional and cognitive dysfunction in Alzheimer's disease （AD）， as well as the involvement of NLRP3 mediated microglial pyroptosis. Method： Male 5xFAD mice at age of 5 months were randomly divided into control and physical exercise， （PE） groups. Age-matched C57/BL6 mice were used as wild type （WT） group. The mice in the WT and the control groups were fed in a common cage， while the mice in the PE group were fed in a cage equipped with a running wheel， in which mice were freely to running. Open field test was used to detect the emotional function， the Morris water maze test was used to detect the spatial cognitive function， and immunofluorescence staining was used to detect the neuronal survival， Amyloid beta （Aβ） plaque deposition， the microglial activation， the aggregation of microglial lysosomes and the expression of GSDMD， which is related to pyroptosis. The expression of inflammatory cytokines、proinflammatory cytokines and GSDMD protein were detected by Western Blots. Result： In open field test， when comparing with WT group， the time spent in the central area was significantly shortened in the control-5xFAD group， while which was significantly extended in the PE-5xFAD group. During the Morris water maze training period， the latencies of mice in the WT and PE-5xFAD groups to the platform were gradually decreased， while there was no significant differences in the control-5xFAD group. During the probe trial test， the times crossing the platform in the control-5xFAD group was significantly reduced compared with that in the WT group， while which was significantly increased in the PE-5xFAD mice. The number of neurons in the PE-5xFAD group was significantly increased in cortex and hippocampus compared with those in the control-5xFAD group， while Aβ1-42 plaques were significantly decreased in the PE-5xFAD group. Compared with the WT group. In addition, the lysosomal associated membrane protein-1 （Lamp1） was obviously detected around the Aβ plaques in the control-5xFAD group. However, physical exercise significantly reduced the expressions of Lamp1 and CtsB in the cortex and hippocampus of 5xFAD mice. In the control-5xFAD group，the proinflammatory cytokines were significantly increased in cortex and hippocampus when compared with the WT mice, while PE decreased the expression of proinflammatory cytokines and increased the expression of anti-inflammatory cytokine. When compared with the WT mice, the 5xFAD mice exhibited a significantly increased expression of GSDMD protein in cortex and hippocampus, while PE decreased the expression of GSDMD protein. Moreover，GSDMD protein was co-localized with Iba-1-positive microglia. Conclusion： Physical exercise improves cognitive function in Alzheimer's disease by inhibiting NLRP3-mediated microglial pyroptosis.