带针下有氧运动调控AMPK-Drp1-线粒体分裂平衡对大鼠脑缺血再灌注损伤的保护效应

关键词：带针下有氧运动  神庭  百会  线粒体分裂  腺苷酸活化蛋白激酶  动力相关蛋白1

Keywords:aerobic exercise with needles  Shenting  Baihui  mitochondrial fission  AMPK  Drp1

摘要：

      摘要 目的：探讨神庭、百会带针下有氧运动对大脑中动脉闭塞再灌注（MCAO/R）模型大鼠认知功能的改善效果，及其对海马腺苷酸活化蛋白激酶（AMPK）-动力相关蛋白1（Drp1）-线粒体分裂的调控作用。 方法：将48只清洁级健康雄性SD大鼠随机分为假手术组（Sham）（12只）及造模预筛组（36只）。造模预筛组大鼠采用改良Longa线栓法制备MCAO/R模型， 经“三步法”筛选的PSCI大鼠再次随机分为模型组（Model）、带针下有氧运动组（EA+AE）和抑制剂组（AMPK Inh.），每组12只。带针下有氧运动组、抑制剂组大鼠予“神庭” “百会”带针下跑台训练，每次30min，1次/天，持续14d。抑制剂组选用AMPK抑制剂（Compound C）进行腹腔注射（2mg/kg），每日1次，连续14d。干预后，采集大鼠脑的T2WI图像并计算脑梗死体积百分比；采用Zea Longa评分评估大鼠的神经功能；新物体识别实验评估大鼠的空间学习记忆能力；HE染色观察大鼠缺血侧海马形态学；免疫组化检测海马AQP4表达以评估脑水肿情况；透射电镜观察海马线粒体形态及线粒体分裂情况；Western Blot法检测海马AMPK、Drp1、Drp1S616及Drp1S637蛋白的表达；磷钼酸比色法检测海马三磷酸腺苷（ATP）的浓度。 结果：与假手术组相比，模型组大鼠左侧脑室梗死灶明显（P<0.01），新物体识别指数及神经功能评分显著下降（P<0.01），缺血侧海马细胞排列紊乱并出现坏死；AQP4大量阳性表达于星形胶质细胞终足（P<0.01）；镜下海马线粒体结构破坏严重且观察到大量线粒体分裂；另外；AMPK、Drp1S637表达、ATP含量显著下降（P<0.05，P<0.01，P<0.01），而Drp1、Drp1S616表达显著增加（P<0.01，P<0.01）；相较于模型组，带针下有氧运动组大鼠脑梗死体积下降（P<0.01），新物体识别能力及神经功能改善（P<0.01），海马细胞病理损伤、线粒体分裂情况减轻；AQP4表达显著下降（P<0.01）；AMPK、Drp1S637表达、ATP含量显著增加（P<0.05，P<0.01，P<0.01），Drp1、Drp1S616表达显著下降（P<0.05，P<0.01）；抑制剂组较带针下有氧运动组脑梗死体积增加（P<0.01），新物体识别能力及神经功能水平下调（P<0.01），海马细胞病理损伤、线粒体分裂情况趋于模型组；AQP4表达显著增加（P<0.01）； AMPK、Drp1S637表达、ATP含量显著下降（P<0.05，P<0.01，P<0.01），Drp1、Drp1S616表达显著增加（P<0.01，P<0.01）。 结论：带针下有氧运动可提高MCAO/R大鼠的学习记忆能力，其作用机制可能是通过AMPK/Drp1轴重新恢复Drp1S616、Drp1S637磷酸化平衡，进而恢复线粒体分裂、维持线粒体动力学。

Abstract:

      Abstract Objective： To investigate the effects of aerobic exercise combined with acupuncture at Shenting and Baihui on MCAO/R rats， and its regulatory role on hippocampal AMPK-Drp1-mediated mitochondrial fission. Method： A total of 48 male Sprague-Dawley rats were randomly divided into the sham group （n=12） and the modeling pre-screening group （n=36）. MCAO/R models were established in the pre-screening group using the modified Longa suture method. PSCI rats successfully screened via a "three-step method" were then randomly re-divided into the Model group， the aerobic exercise with needles group （EA+AE）， and the inhibitor group （AMPK Inh.）， with 12 rats per group. Rats in the EA+AE and AMPK Inh. groups received treadmill training while retaining needles at "Shenting" and "Baihui" for 30 minutes daily， over 14 days. The inhibitor group additionally received intraperitoneal injections of the AMPK inhibitor Compound C （2 mg/kg） once daily for 14 days. Post-intervention， T2-weighted MRI was performed to calculate the percentage of cerebral infarct volume； neurological function was assessed using the Zea Longa score； spatial learning and memory were evaluated via the novel object recognition test； hippocampal morphology on the ischemic side was observed using HE staining； AQP4 expression in the hippocampus was detected by immunohistochemistry to assess cerebral edema； mitochondrial morphology and fission in the hippocampus were observed under transmission electron microscopy； expression levels of AMPK， total Drp1， Drp1S616，Drp1S637 in the hippocampus were measured by Western blot； and ATP concentration in the hippocampus was quantified using the phosphomolybdate colorimetric method. Result： Compared with the Sham group， the Model group exhibited significant left cerebral infarction （P<0.01）， a significantly decreased novel object recognition index and neurological function score （both P<0.01）， disordered cell arrangement and necrosis in the ischemic hippocampus， extensive AQP4 positive expression in astrocyte end-feet （P<0.01）， severe ultrastructural damage and prominent mitochondrial fission observed in hippocampal mitochondria， as well as significantly decreased AMPK and p-Drp1S637protein expression and ATP content （P<0.05，P<0.01，P<0.01）， while total Drp1 and p-Drp1S616expression were significantly increased （both P<0.01）. Compared with the Model group， the EA+AE group showed reduced cerebral infarct volume （P<0.01）， improved novel object recognition ability and neurological function （both P<0.01）， alleviated pathological hippocampal damage and mitochondrial fission， significantly decreased AQP4 expression （P<0.01）， significantly increased AMPK and p-Drp1S637protein expression and ATP content （P<0.05，P<0.01，P<0.01）， and significantly decreased total Drp1 and Drp1S616expression （P<0.05，P<0.01）. Compared with the EA+AE group， the AMPK Inh. group demonstrated increased cerebral infarct volume （P<0.01）， worsened novel object recognition ability and neurological function （both P<0.01）， pathological hippocampal damage and mitochondrial fission returning to levels similar to the Model group， significantly increased AQP4 expression （P<0.01）， significantly decreased AMPK and Drp1S637protein expression and ATP content （P<0.05，P<0.01，P<0.01）， and significantly increased total Drp1 and Drp1S616expression （both P<0.01）. Conclusion： Aerobic exercise combined with acupuncture at Shenting and Baihui can improve learning and memory abilities in MCAO/R rats. The underlying mechanism may involve the restoration of the phosphorylation balance between Drp1S616 and Drp1S637 through the AMPK/Drp1 axis， thereby restoring mitochondrial fission and maintaining mitochondrial dynamics.