目的 探讨葛根素通过腺苷一磷酸活化蛋白激酶（AMPK）-哺乳动物雷帕霉素靶蛋白（mTOR）-Unc-51样激酶1（Ulk1）通路抑制自噬改善大鼠脑缺血再灌注损伤的作用。方法 40只雄性SD大鼠随机分为4组，即假手术组、模型组及葛根素低、高剂量（50、100 mg/kg）组。各组大鼠连续给药7 d，末次给药30 min后，采用线栓法制备大鼠大脑中动脉栓塞（middle cerebral artery occlusion，MCAO）模型，缺血1.5 h再灌注24 h后，进行神经功能评分，TTC染色观察脑梗死体积，电镜观察自噬小体的形成，Western blotting法检测海马组织中微管相关蛋白1轻链3（LC3）、p62、AMPK、p-AMPK、mTOR、p-mTOR、Ulk1、pS757-Ulk1蛋白表达水平。结果 与假手术组比较，模型组大鼠神经功能评分显著增加，脑梗死体积明显增大，自噬小体增多，LC3-Ⅱ/LC3-I显著增加，p62蛋白表达水平显著降低，p-AMPK表达水平显著升高，p-mTOR和pS757-Ulk1蛋白表达水平显著降低。与模型组比较，葛根素各组大鼠的神经功能损伤明显改善，脑梗死体积减小，自噬小体减少，LC3-Ⅱ/LC3-I显著降低，p62蛋白表达水平显著升高，p-AMPK蛋白表达水平显著降低，p-mTOR和pS757-Ulk1蛋白表达水平显著升高。结论 葛根素可能通过调控AMPK-mTOR-Ulk1信号通路抑制自噬的过度发生，从而减轻脑缺血再灌注损伤的发生。

Objective To investigate the effect of puerarin on the regulation of AMPK-mTOR signaling pathway to inhibit autophagy and alleviate focal cerebral ischemia reperfusion injury. Methods Forty male Sprague-Dawley rats were randomly divided into four groups:Sham group, model group, puerarin low-dose (50 mg/kg) group and puerarin high-dose (100 mg/kg) group. Pretreatment with puerarin for 7 d, then the middle cerebral artery occlusion (MCAO) model was established 0.5 h after the last administration according to Longa's method. After 1.5 h of ischemia and 24 h of reperfusion, the neurological deficit scores were assessed, the infarct volume was calculated by TTC staining. The formation of autophagosome was observed by electron microscopy. The expression levels of LC3, p62, AMPK, p-AMPK, mTOR, p-mTOR, Ulk1, and pS757-Ulk1 were detected by Western blotting. Results Compared with the Sham group, the neurological deficit scores and infarct volume in model group were significantly increased, the numbers of autophagosome increased, and the rate of LC3-Ⅱ/LC3-I significantly increased, the expression level of p62 gradually decreased. The expression of p-AMPK was markedly up-regulated, while the expression of p-mTOR and pS757-Ulk1 was significantly down-regulated. Compared with the model group, the neurological deficit scores and infarct volume were significantly reduced, the number of autophagosome and the rate of LC3-Ⅱ/LC3-I decreased, the expression of p62 was significantly up-regulated, the expression of p-AMPK was markedly down-regulated, the levels of p-mTOR and pS757-Ulk1 were significantly up-regulated. Conclusion Puerarin alleviates cerebral ischemia-reperfusion injury may through suppressing autophagy via the AMPK-mTOR-Ulk1 signaling pathway.

湖北省宜昌市科技计划项目（A18-301-25）