带针下有氧运动通过NLRP 3/Caspase-1/GSDMD调节小胶质细胞焦亡改善MCAO/R大鼠认知功能的机制研究

关键词：学习记忆功能障碍  带针下有氧运动  小胶质细胞焦亡  神经炎症  小胶质细胞活化

Keywords:learning and memory dysfunction  aerobic exercise with needles  microglia pyroptosis  neuroinflammation  microglia activation

摘要：

      摘要 目的：观察带针下有氧运动对大脑中动脉闭塞再灌注（MCAO/R）大鼠学习记忆能力的影响并探索相关作用机制。 方法：SD雄性大鼠随机等分为假手术组、模型组、带针下有氧运动组和抑制剂组，每组12只。假手术组大鼠仅暴露左侧颈总动脉、颈内动脉和颈外动脉，其余三组大鼠制备左侧大脑MCAO/R模型。抑制剂组造模前60min需腹腔注射MCC 950以复制NLRP 3低表达MCAO/R大鼠。于造模第1天采用Zea-Longa评分法及小动物磁共振T2 WI筛选合格MCAO/R大鼠。于造模第2天对带针下有氧运动组、抑制剂组大鼠进行为期14d干预，穴取“神庭”“百会”，疏密波形，频率2/10Hz，电流强度1—3mA，同时将大鼠置于电动跑道，干预后1—3d跑台速度4m/min，4—7d跑台速度6m/min，8—14d跑台速度8m/min，每天30min。干预后再次评定神经功能；新物体识别实验检测学习记忆能力；HE染色观察缺血侧海马病理形态改变；Western Blot法、免疫荧光法检测缺血侧海马NLRP 3、GSDMD、Caspase-1蛋白表达；Elisa法检测促炎因子IL-1β、IL-18含量；免疫荧光双标法检测IBA 1及NLRP 3表达及定位情况。 结果：模型组大鼠缺血侧海马细胞排列疏松紊乱；与假手术组相比，模型组大鼠神经功能及新物体识别能力显著下降（P<0.01），缺血侧海马NLRP 3、GSDMD、Caspase-1蛋白表达显著增加（P<0.01），IL-1β、IL-18含量显著增加（P<0.01），另外IBA 1、NLRP 3荧光表达显著增强（P<0.01），镜下可见大量IBA 1+/NLRP 3+双阳性细胞（P<0.01）；与模型组相比，带针下有氧运动组神经功能评分及新物体识别指数显著增加（P<0.05，P<0.01）；海马病理损伤减轻；NLRP 3、GSDMD及Caspase-1蛋白表达下降（P<0.01）；IL-1β及IL-18含量降低（P<0.05）；小胶质细胞活化程度及IBA 1+/NLRP 3+双阳性细胞数量减少（P<0.01）；抑制剂组大鼠的神经功能及新物体识别能力较带针下有氧运动组进一步恢复（P<0.05，P<0.01），NLRP 3、GSDMD、Caspase-1蛋白表达（P<0.01）及IL-1β、IL-18因子水平显著下降（P<0.01，P<0.05），小胶质细胞活化水平、神经细胞数量、排列及形态均趋于假手术组，镜下几乎未观察到有NLRP 3表达的小胶质细胞（P<0.01）。 结论：“神庭”“百会”带针下有氧运动可能通过抑制小胶质细胞焦亡和活化，进而下调神经炎症水平，改善MACO/R大鼠大学习记忆功能。

Abstract:

      Abstract Objective：To investigate the effect of aerobic exercise with acupuncture needles on the learning and memory ability in rats subjected to middle cerebral artery occlusion/reperfusion（MCAO/R） and to explore the underlying mechanisms. Method： Male SD rats were randomly divided into four groups: sham，model，aerobic exercise with acupuncture needles，and inhibitor groups(n=12 per group). In the sham group, only the left common carotid artery，internal carotid ar tery，and external carotid artery were exposed without inducing ischemia，while the other three groups underwent MCAO/R modeling. To establish an MCAO/R model with reduced NLRP3 expression, rats in the inhibitor group received an intraperitoneal injection of MCC950 60 minutes before modeling. On the first day of post-modeling， the Zea-Longa score and T2 WI were used to screen the MCAO/R rats. From the second day， rats in the aerobic exercise with acupuncture needles and the inhibitor groups were underwent a 14-day intervention. The acupoints were selected for “Shenting”（GV 24）and “Baihui”（GV 20），with sparse waveform at a frequency 2/10Hz and a current intensity of 1—3mA. Simultaneously，the rats performed treadmill exercise, with speeds of 4m/min (day 1—3)，6m/min (day 4—7)，and 8m/min (day 8—14) for 30min/day. After the intervention， neurological function was reassessed， and the new object recognition test was used to measure the learning and memory ability，and the pathological changes of the hippocampus were observed by HE staining. The protein expressions of NLRP 3，GSDMD，and Caspase-1 were detected by Western Blot and immunofluorescence，ELISA detected the contents of IL-1β and IL-18，the expression and localization of IBA 1 and NLRP 3 were detected by double immunofluorescent staining. Result：The hippocampal neurons in the ischemic side of the rats were loosely and disorderly arranged in the model group. Compared to the sham group, the model group showed significantly impaired neurological function and novel object recognition ability（P<0.01），with significant increased expression of NLRP 3，GSDMD，and Caspase-1（P<0.01），and elevated IL-1β and IL-18 contents（P<0.01）. In addition，IBA 1 and NLRP 3 fluorescence expression were enhanced（P<0.01），and many IBA 1+/NLRP 3+ double-positive cells were observed under the microscope（P<0.01）. Compared with the model group，the aerobic exercise with acupuncture needles group showed significant improvements in neurological function and the new object recognition index（P<0.05, P<0.01），reduced hippocampal pathological injury，decreased expression of NLRP 3，GSDMD，and Caspase-1 d（P<0.01） and lower IL-1β and IL-18 levels （P<0.05）; the activation of microglia and the expression of IBA 1，NLRP 3 and the number of IBA 1+/NLRP 3+double-positive cells decreased（P<0.01）. The inhibitor group demonstrated even greater improvements in neural function and new object recognition performance than the aerobic exercise with acupuncture needles group （P<0.05，P<0.01），with further reductions in NLRP 3，GSDMD，Caspase-1 protein expression（P<0.01） and lower level of IL-1β，IL-18（P<0.01，P<0.05）; the microglia activation level，the number，arrangement and morphology of nerve cells were closely resembled those in the sham group，and there was minimal NLRP 3 microglia observed under the microscope（P<0.01）. Conclusion： Aerobic exercise with acupuncture needles at “Shenting” （GV 24） and “Baihui” （GV 20） points could improve the learning and memory function in MACO/R rats by inhibiting microglia pyroptosis and activation， thereby reducing neuroinflammation.