规律性有氧运动对大脑中动脉栓塞大鼠NLRP3/caspase-1/GSDMD轴及学习记忆功能的影响

关键词：有氧运动  学习记忆  小胶质细胞焦亡  缺血性脑损伤  神经炎症

Keywords:aerobic exercise  learning and memory  microglia pyroptosis  ischemic cerebral injury  neuroinflammation

摘要：

      摘要 目的：研究规律性有氧运动对MCAO/R大鼠学习记忆功能的影响及作用机制。 方法：将36只SD大鼠随机分为3组，假手术组（IS组）、有氧运动组（AE组）及模型组（IC组）。IC组及AE组大鼠制备MCAO/R模型，IS组进行相同动脉分离即可。成模后AE组大鼠接受14d跑步机训练，IS组、IC组大鼠仅需在相同的持续时间于跑台自由活动。对各组大鼠进行神经功能评分；应用小动物磁共振T2WI扫描观察干预前后各组大鼠脑梗死灶；应用新物体识别实验检测各组大鼠干预前后的识别记忆能力；采用HE染色对缺血侧海马CA1区神经元损害情况进行研究；应用免疫组化检测缺血侧海马CA1区小胶质细胞活化水平；应用Western Blot检测缺血侧海马NOD样受体蛋白3 （NOD-like receptor protein 3，NLRP3）、半胱氨酸蛋白酶-1（cysteinyl aspartate specific proteinase 1，caspase-1）、消皮素D （gasdermin D，GSDMD）蛋白表达情况；应用Elisa法检测缺血侧海马白细胞介素1β（IL-1β）、白细胞介素18（IL-18）水平；应用免疫荧光共标记实验检测缺血侧海马IBA1与NLRP3、caspase-1、GSDMD的荧光表达及共定位情况。 结果：干预前，与IS组相比，IC组神经功能评分显著升高（P<0.05），并可见明显高信号梗死灶（P<0.05），新物体辨别率显著下降（P<0.05），而AE组与IC组神经功能评分、脑梗死体积百分比及新物体辨别率无显著差异（P>0.05）；干预14d后，与IC组相比，AE组大鼠神经功能评分显著降低（P<0.05）、脑梗死体积显著减小（P<0.05）、物体辨别能力显著提高（P<0.05）、缺血侧海马CA1区病理情况改善、小胶质细胞活化水平降低（P<0.05）、IL-1β、IL-18含量下降（P<0.05），NLRP3、caspase-1、GSDMD 蛋白表达显著减少（P<0.05）。除此之外，与IC组相比，AE组大鼠缺血侧海马CA1区NLRP3、GSDMD、CA3区caspase-1累计光密度显著性下降，IBA1、NLRP3与IBA1、GSDMD双阳性细胞数量明显减少。 结论：规律有氧运动可提高MCAO/R大鼠的识别记忆功能，其作用机制可能是通过抑制小胶质细胞焦亡和活化来实现的。

Abstract:

      Abstract Objective：To explore the possible mechanism of regular aerobic exercise in improving the learning and memory function in rats with middle cerebral artery occlusion reperfusion （MCAO/R）. Method： Total of 36 SD rats were randomly divided into 3 groups： sham operation group （IS group）， aerobic exercise group （AE group）， and model group （IC group）. MCAO/R models were established in the IC and AE groups, while the IS group underwent the same arterial dissection without occlusion. After model establishment， the rats in the AE group were trained on a treadmill for 14 days， while the rats in the IS group and IC group were only placed on the treadmill for the same duration without running. The neurological function of the rats in each group was scored， the cerebral infarction focus of the rats was observed by magnetic resonance imaging T2WI scanning before and after the intervention， and the recognition and memory ability of the rats was detected by the new object recognition experiment before and after the intervention. The Western Blot was used to detect the expression of NLRP3， caspase-1， and GSDMD in the hippocampus， and immunohistochemistry was used to detect the activation of microglia in the CA1 region of the ischemic hippocampus. The levels of IL-1β and IL-18 were detected by ELISA. The expression and co-localization of IBA1 with NLRP3， caspase-1， and GSDMD were detected by immunofluorescence co-labeling assay. Result： Before intervention， the neurological function score and the signal intensity of the left cerebral cortex hippocampus of the IC Group were significantly higher than that of the IS group （P<0.05）， and the resolution of new objects decreased significantly （P<0.05）. There were no significant differences between the AE and IC groups in the proportion of neurological deficit， cerebral infarction volume and novel object recognition rate（P>0.05）. After 14 days of treadmill training intervention， compared with IC group， the AE group showed significantly lower neurological function scores（P<0.05）， reduced the percentage of cerebral infarction volume（P<0.05）， improved object recognition ability（P<0.05）， improved pathology in hippocampal CA1 area， reduced microglia activation level （P<0.05）， decreased levels of IL-1β and IL-18（P<0.05）， and significantly reduced expression of NLRP3， caspase-1 and GSDMD proteins（P<0.05）. In addition， compared with the IC group， the accumulative optical density of NLRP3， GSDMD in CA1 and caspase-1 in CA3 regions of the ischemic hippocampus in the AE group was significantly decreased， and the number of IBA1 and NLRP3， IBA1 and GSDMD double-positive cells was significantly decreased（P<0.05）. Conclusion： Regular aerobic exercise can improve the ability of recognition and memory in MCAO/R rats， which may be related to the inhibition of hippocampal microglia pyroptosis and activation.