Objective: To observe the effect of electroacupuncture (EA) pretreatment on inflammatory reaction, apoptosis and expression of Yes-associated protein (YAP) of ischemic penumbra of cerebral cortex in cerebral ischemia reperfusion injury rats, and to explore the possible mechanism of its neuroprotection effect.
Methods: A total of 84 SD rats were randomized into a sham operation group (12 rats), a model group (18 rats), an EA group (18 rats), an EA+YAP virus transfection group (18 rats) and an EA+virus control group (18 rats). Except for the sham operation group, thread embolization method was adopted to establish the middle cerebral artery occlusion (MCAO) model in rats of the other groups. EA was applied at "Baihui" (GV 20) and "Dazhui" (GV 14) for 30 min in the 3 EA intervention groups 2 h before model establishment, disperse-dense wave, 2 Hz/15 Hz in frequency and 1 mA in intensity. Adenovirus transfection technique was used to induce gene silencing of YAP in the EA+YAP virus transfection group, and adenovirus vectors was injected as negative control in the EA+virus control group 4 d before model establishment. Twenty-four hours after model establishment, neurological function score was evaluated, the relative cerebral infarction area was observed by TTC staining, the apoptosis in the ischemic penumbra of cerebral cortex was detected by TUNEL staining, the levels of inflammatory factors IL-1β, IL-6 and TNF-α in the ischemic penumbra of cerebral cortex was detected by ELISA method, the expression of YAP was detected by Western blot and immunofluorescence.
Results: Compared with the sham operation group, the expression of YAP was increased in the model group (P<0.05); compared with the model group, the expression of YAP in the ischemic penumbra of cerebral cortex was increased in the EA group (P<0.05). Compared with the sham operation group, the neurological function score, the percentage of TUNEL positive cells and the levels of IL-1β, IL-6 and TNF-α in the ischemic penumbra of cerebral cortex were increased in the model group (P<0.001, P<0.01); compared with the model group, the neurological function score, the relative cerebral infarction area, the percentage of TUNEL positive cells and the levels of IL-1β, IL-6 and TNF-α in the ischemic penumbra of cerebral cortex were decreased in the EA group (P<0.05, P<0.01); compared with the EA group, the neurological function score, the relative cerebral infarction area, the percentage of TUNEL positive cells and the levels of IL-1β, IL-6 and TNF-α in the ischemic penumbra of cerebral cortex were increased in the EA+YAP virus transfection group (P<0.01, P<0.05); compared with the EA+YAP virus transfection group, the neurological function score, the relative cerebral infarction area, the percentage of TUNEL positive cells and the levels of IL-1β, IL-6 and TNF-α in the ischemic penumbra of cerebral cortex were decreased in the EA+virus control group (P<0.01, P<0.05).
Conclusion: Electroacupuncture pretreatment can effectively improve the ischemia reperfusion injury, its mechanism may be related to up-regulating the expression of YAP in the ischemic penumbra of cerebral cortex and relieving the apoptosis and inflammatory reaction.
目的：观察电针预处理对脑缺血再灌注损伤大鼠大脑皮层缺血半暗带炎性反应、细胞凋亡和Yes相关蛋白（YAP）表达的影响，探讨其神经保护作用的可能机制。方法：将84只SD大鼠随机分为假手术组（12只）、模型组（18只）、电针组（18只）、电针+YAP病毒转染组（18只）和电针+病毒对照组（18只）。除假手术组外，其他各组大鼠均采用线栓法制备大脑中动脉阻塞（MCAO）模型。3个电针干预组大鼠于造模前2 h电针“百会”和“大椎”穴30 min，疏密波，频率2 Hz/15 Hz，电流强度1 mA。造模前4 d，电针+YAP病毒转染组大鼠采用腺病毒转染技术沉默大脑皮层YAP基因，电针+病毒对照组大鼠注射阴性对照的腺病毒载体。造模后24 h，记录各组大鼠神经行为学评分，TTC染色观察各组大鼠相对脑梗死面积，TUNEL染色检测大脑皮层缺血半暗带细胞凋亡，ELISA法检测大脑皮层缺血半暗带炎性因子白细胞介素（IL）-1β、IL-6和肿瘤坏死因子（TNF）-α水平，Western blot和免疫荧光染色法检测大脑皮层缺血半暗带YAP表达。结果：与假手术组比较，模型组大鼠大脑皮层缺血半暗带YAP表达增加（P<0.05）；与模型组比较，电针组大鼠大脑皮层缺血半暗带YAP表达增加（P<0.05）。与假手术组比较，模型组大鼠神经行为学评分、大脑皮层缺血半暗带TUNEL阳性细胞百分比及IL-1β、IL-6和TNF-α水平均升高（P<0.001，P<0.01）；与模型组比较，电针组大鼠神经行为学评分、相对脑梗死面积、大脑皮层缺血半暗带TUNEL阳性细胞百分比及IL-1β、IL-6和TNF-α水平均降低（P<0.05，P<0.01）；与电针组比较，电针+YAP病毒转染组大鼠神经行为学评分、相对脑梗死面积、大脑皮层缺血半暗带TUNEL阳性细胞百分比及IL-1β、IL-6和TNF-α水平升高（P<0.01，P<0.05）；与电针+YAP病毒转染组比较，电针+病毒对照组大鼠神经行为学评分、相对脑梗死面积、大脑皮层缺血半暗带TUNEL阳性细胞百分比及IL-1β、IL-6和TNF-α水平降低（P<0.01，P<0.05）。结论：电针预处理可有效改善脑缺血再灌注损伤，其机制可能与上调大脑皮层半暗带YAP表达，进而减轻细胞凋亡和神经炎性反应有关。.
Keywords: Yes-associated protein (YAP); apoptosis; electroacupuncture; inflammatory reaction; ischemia reperfusion injury; ischemia stroke; pretreatment; stroke.