Objective: To investigate the mechanism of electroacupuncture in alleviating cerebral ischemia injury in cerebral ischemia-reperfusion rats by regulating melatonin - NOD-like receptor protein 3 (NLRP3) mediated pyroptosis.
Methods: A total of 48 SD rats were randomly divided into sham operation group, model group, electroacupuncture (EA) group and EA +Luz group, with 12 rats in each group. The focal cerebral ischemia-reperfusion injury model was established by middle cerebral artery embolization. Rats of the EA group was treated with EA stimulation (4 Hz/20 Hz, 0.5 mA,20 min) at "Baihui" (GV20) and "Shenting" (GV24) once a day for 7 consecutive days; rats of EA+Luz group were given the same EA treatment and intraperitoneally administered melatonin receptor antagonist (luzindole, 30 mg/kg), once a day for 7 consecutive days. The neurological impairment was evaluated by Zea Longa score. The level of serum melatonin content at 12:00 and 24:00 was detected by ELISA. The percentage of cerebral infarction volume was evaluated by MRI of small animals. The apoptosis rate of nerve cells in cerebral cortex of infarct side was detected by TUNEL staining. The activation of microglia cells was detected by immunofluorescence staining. The expression levels of pyroptosis-related proteins NLRP3, Caspase-1 and interleukin (IL) -1β were detected by Western blot.
Results: Compared with the sham operation group, the neural function score was significantly increased (P<0.01); the melatonin content was significantly decreased at 24:00 (P<0.01); the percentage of cerebral infarction volume, apoptosis rate of nerve cells in cerebral cortex area of infarction side, the expressions of NLRP3, Caspase-1 and IL-1β proteins were significantly increased (P<0.01); and microglia cells were significantly activated in the model group.Compared with the model and EA +Luz groups, the nerve function score was significantly decreased (P<0.05); the percentage of cerebral infarction volume, the nerve cell apoptosis rate, the activation level of microglia cells, the expression levels of NLRP3, Caspase-1 and IL-1β were significantly decreased (P<0.01, P<0.05) in the EA group. Compared with the model and EA+Luz groups, the melatonin content at 24:00 was significantly increased (P<0.01, P<0.05) in the EA group.
Conclusion: EA at GV20 and GV24 can reduce the neurolo-gical injury in cerebral ischemia reperfusion model rats, which may be related to regulating the expression of endogenous melatonin, inhibiting cell scorchification and reducing cerebral ischemia injury.
目的：探讨电针通过调节褪黑素-核苷酸结合寡聚化结构域样受体蛋白3炎性小体(NLRP3)轴介导的细胞焦亡减轻脑缺血再灌注大鼠脑缺血损伤的机制。方法：SD大鼠随机分为假手术组12只及手术组36只，手术组大鼠采用大脑中动脉栓塞法制备局灶性脑缺血-再灌注损伤模型。大鼠造模成功后进一步分为模型组、电针组和电针+Luz组，每组12只。电针组和电针+Luz组电针“百会”“神庭”，每次20 min，电针+Luz组腹腔注射褪黑素受体拮抗剂luzindole(30 mg/kg)，以上干预均1次/d，干预7 d。采用Zea Longa法评估各组大鼠神经功能缺损情况，ELISA法检测各组大鼠12:00及24:00血清褪黑素含量，小动物MRI评估各组大鼠脑梗死体积百分比，TUNEL法检测各组大鼠梗死侧大脑皮质区神经细胞凋亡水平，免疫荧光技术检测各组大鼠大脑皮质小胶质细胞活化情况，Western blot法检测各组大鼠大脑皮质细胞焦亡相关蛋白NLRP3、半胱氨酸天冬氨酸酶（Caspase)-1、白细胞介素(IL)-1β表达水平。结果：与假手术组比较，模型组大鼠神经功能评分显著升高(P<0.01)，24:00褪黑素含量显著降低(P<0.01)，脑梗死体积百分比、神经细胞凋亡率显著升高(P<0.01)，小胶质细胞明显活化，细胞焦亡相关蛋白NLRP3、Caspase-1、IL-1β表达显著升高(P<0.01)。与模型组和电针+Luz组比较，电针组神经功能评分显著降低(P<0.05)，24:00褪黑素含量显著升高(P<0.01，P<0.05)，脑梗死体积百分比、神经细胞凋亡率显著降低(P<0.01，P<0.05)，小胶质细胞活化水平降低，细胞焦亡相关蛋白NLRP3、 Caspase-1、IL-1β表达显著降低(P<0.01)。结论：电针“百会”“神庭”可减轻脑缺血再灌注模型大鼠神经功能损伤，其机制可能与调控内源性褪黑素表达、抑制细胞焦亡减轻脑缺血再灌注大鼠脑缺血损伤有关。.
Keywords: Cerebral ischemia; Electroacupuncture; Melatonin; NOD-like receptor protein 3; Pyroptosis.