Objectives: To observe the effect of "Shugan Tiaoshen"(soothing the liver and regulating the mental activities) needling on the expressions of AMP-activated protein kinase (AMPK) and autophagy-related proteins in the prefrontal cortex of rats with depression after ischemic stroke (post-stroke depression), in order to explore its mechanisms underlying improvement of post-stroke depression behaviors.
Methods: A total of 60 male SD rats were randomly divided into sham operation (control), stroke, post-stroke depression, inhibitor, acupuncture, and acupuncture + inhibitor groups. The post-stroke depression model was established by occlusion of the middle cerebral artery and reperfusion (MCAO/R), combined with chronic unpredictable mild stimulation (CUMS). Rats in the inhibitor group received bilateral lateral ventricular injection of AMPK inhibitor (0.1 μmol, 10 μL). Manual acupuncture stimulation was applied to "Baihui" (GV20), "Yintang" (GV24+), "Hegu" (LI4), and "Taichong" (LR3) for the acupuncture group, once daily for 14 d. Rats in the acupuncture + inhibitor group received acupuncture treatment after 3 d of AMPK inhibitor injection. Neurologic impairment was assessed by Longa score. Behavioral assessment of depression was conducted by sucrose preference test and open field test. H.E. staining was used to observe the morphological changes of prefrontal cortex. Western blot was used to detect the protein expression levels of AMPK, p-AMPK, Beclin1, P62 and LC3 in the prefrontal cortex tissue. Transmission electron microscopy (TEM) was used to observe the ultrastructural changes of prefrontal neurons.
Results: Compared with the control group, only the total locomotor distance, the percentage of central area dwell time within 5 min and the p-AMPK/AMPK were significantly decreased (P<0.01) in the stroke group while Longa score was increased (P<0.01). Compared with the control group, the sucrose preference, total locomotor distance and the percentage of central area dwell time within 5 min, levels of p-AMPK/AMPK, Beclin1 and LC3Ⅱ/Ⅰ were significantly decreased (P<0.01, P<0.05), while Longa score and P62 expression was significantly up-regulated (P<0.01) in the post-stroke depression group. Compared with the post-stroke depression group, the sucrose preference, total locomotor distance and the percentage of central area dwell time within 5 min, and the levels of p-AMPK/AMPK, Beclin1, and LC3Ⅱ/Ⅰ were considerably increased (P<0.01), while P62 expression was notably decreased (P<0.05) in the acupuncture group. Comparison between the inhibitor group and the acupuncture+inhibitor group showed that the sucrose preference, total locomotor distance and the percentage of central area dwell time within 5 min, and the expression level of p-AMPK/AMPK were even lower in the former group than those in the latter group (P<0.05, P<0.01), and the expression level of P62 was higher in the former group than in the latter group (P<0.01). H.E. staining showed loose arrangement of prefrontal cortex neurons, reduced number, pyknotic and hyperchromatic nuclei, even with degeneration and necrosis of some neurons in the stroke, post-stroke depression and the inhibitor groups, while increase in neuronal number, with relatively complete structure and uniform stain observed in the acupuncture group. Results of TEM showed disrupted neuronal nuclear membrane, reduction in the number of mitochondria with moderate to severe swelling, and decrease or disappearance of the cristae, and transparent matrix with vacuolation in the post-stroke depression group, which was relatively milder (mild swelling, nearly normal structure and more autophagosome forming) in the acupuncture group and acupuncture+inhibitor group.
Conclusions: "Shugan Tiaoshen" needling can improve the depressive symptoms in post-stroke depression rats, which may be related to its function in activating AMPK-dependent autophagy process.
目的: 观察“疏肝调神”针刺对缺血性脑卒中后抑郁模型大鼠前额叶皮质中腺苷酸激活蛋白激酶（AMPK）及自噬相关蛋白表达的影响，探讨“疏肝调神”针刺改善缺血性脑卒中后抑郁的作用机制。方法: 随机选取9只SD大鼠作为假手术组，另45只大鼠采用线栓法建立大脑中动脉阻塞/再灌注（MCAO/R）模型，MCAO/R模型构建成功后，随机分为脑卒中组、脑卒中后抑郁组、抑制剂组、针刺组、针刺+抑制剂组，每组9只。后4组大鼠接受慢性不可预知性温和刺激（CUMS）构建缺血性PSD模型。缺血性脑卒中后抑郁模型构建成功后，抑制剂组予双侧侧脑室注射AMPK抑制剂Compound C （0.1 μmol，10 μL）；针刺组针刺“印堂”“百会”“合谷”“太冲”，每次30 min，每15 min捻转一次，每日1次，连续干预14 d；针刺+抑制剂组在双侧侧脑室注射AMPK抑制剂3 d后，再接受14 d的针刺治疗。Longa评分法评价神经功能缺损情况，糖水偏好实验和旷场实验评估大鼠行为学，HE染色观察前额叶皮质病理形态变化，Western blot法检测前额叶皮质AMPK、磷酸化（p）-AMPK、Beclin1、P62、微管相关蛋白轻链3（LC3）Ⅱ/Ⅰ蛋白的表达，透射电镜观察前额叶神经元超微结构。结果: 与假手术组相比，脑卒中组5 min内总运动距离及中心区域停留时间百分比显著下降（P<0.01），前额叶皮质p-AMPK/AMPK下降（P<0.01）。与假手术组相比，脑卒中后抑郁组Longa评分升高（P<0.01），糖水偏嗜度、5 min内总运动距离及中心区域停留时间百分比显著下降（P<0.01）；缺血侧前额叶皮质神经元排列松散，核固缩深染，可见变性坏死神经元；前额叶皮质p-AMPK/AMPK、Beclin1表达、LC3Ⅱ/Ⅰ下降（P<0.01，P<0.05），P62表达升高（P<0.01）。与脑卒中后抑郁组相比，针刺组糖水偏嗜度、5 min内总运动距离及中心区域停留时间百分比显著升高（P<0.01）；缺血侧前额叶皮质神经元排列较为整齐，形态大小较为均一；前额叶皮质p-AMPK/AMPK、Beclin1表达、LC3Ⅱ/Ⅰ升高（P<0.01），P62表达下降（P<0.05）。与抑制剂组相比，针刺+抑制剂组糖水偏嗜度、5 min内总运动距离及中心区域停留时间百分比显著升高（P<0.01）；缺血侧前额叶皮质神经元呈轻度肿胀，坏死神经元减少；前额叶皮质p-AMPK/AMPK、Beclin1表达、LC3Ⅱ/Ⅰ升高（P<0.05，P<0.01），P62表达下降（P<0.01）。透射电镜结果显示，假手术组前额叶皮质神经元结构完整，脑卒中组、脑卒中后抑郁组和抑制剂组神经元有明显病理变化，针刺组和针刺+抑制剂组均可见典型自噬小体的形成，神经元整体结构尚可。结论: “疏肝调神”针刺治疗可以改善缺血性脑卒中后抑郁大鼠的抑郁症状，其机制可能与激活AMPK依赖性自噬有关。.
Keywords: AMP-activated protein kinase; Acupuncture; Autophagy; Post-stroke depression.