褪黑素通过AMPK-mTOR信号通路介导的自噬在神经元缺血缺氧损伤中的作用研究  

Effect of Melatonin on Ischemic Anoxic Neuronal Injury by AMPK-mTOR Signaling Pathway Mediated Autophagy

作　　者：陈继军 王倩梅 赵鹏 陈实 CHEN Ji-jun;WANG Qian-mei;ZHAO Peng;CHEN Shi(Department of Emergency Medicine,Xijing Hospital,Military Medical University of PLA Air Force,Xi'an 710032,China;Department of Emergency Medicine,Honghui Hospital Affiliated to Xi'an Jiaotong University,Xi'an,710054,China)

机构地区：[1]空军军医大学急诊科,西安710032 [2]西安交通大学附属红会医院急诊科,西安710054

出　　处：《解放军医药杂志》2019年第10期10-16,共7页Medical & Pharmaceutical Journal of Chinese People’s Liberation Army

摘　　要：目的观察氧糖剥夺(OGD)对神经元凋亡及自噬的影响,研究褪黑素(MT)对OGD所致神经元损伤的作用,并以AMPK-mTOR信号通路介导的自噬为靶点探讨褪黑素在OGD所致神经元损伤中发挥保护作用的分子机制。方法将培养成熟的原代神经元分为对照组、OGD处理组、氧糖剥夺+褪黑素(OGD+MT)处理组、氧糖剥夺+褪黑素+细胞自噬抑制剂(OGD+MT+3-mA)处理组及氧糖剥夺+褪黑素+化合物C(OGD+MT+Cpd C)处理组。OGD处理组采用免疫荧光染色和免疫印迹法检测凋亡及自噬相关蛋白的表达;OGD+MT处理组用褪黑素对神经元进行预处理前/后,检测神经元凋亡相关指标,观察OGD所致神经元损伤程度,并采用免疫荧光染色和western blot等方法检测神经元自噬;OGD+MT+3-mA处理组使用细胞自噬抑制剂(3-mA)处理神经元后探讨褪黑素对神经元凋亡及自噬的影响;OGD+MT+Cpd C处理组使用AMPK抑制剂化合物C(Cpd C)处理神经元后,检测AMPK-mTOR信号通路关键分子表达水平,探索褪黑素对AMPK-mTOR通路的调控作用。结果OGD可显著增加神经元凋亡及自噬,并阻断自噬流的通畅;褪黑素可以显著减轻神经元凋亡,激动自噬,并维持自噬流的通畅;细胞自噬抑制剂及化合物C可以逆转褪黑素对OGD所致神经元损伤的保护作用。结论褪黑素可以通过AMPK-mTOR信号通路激活自噬,从而减轻OGD所致神经元损伤。Objective To observe effect of oxygen glucose deprivation(OGD)on apoptosis and autophagy of cortical neurons,to investigate effect of melatonin(MT)on OGD-induced neuronal injury,and to study molecule mechanism of melatonin in neuroprotective effect of MT by using AMPK-mTOR signaling pathway mediated autophagy as target point.Methods Mature primary neurons were divided into control group,OGD,OGD+MT,OGD+MT+3-mA and OGD+MT+Cpd C groups.Immunostaining and western blot methods were performed to detect expressions of apoptotic and autophagic related proteins in OGD group.In OGD+MT group,related indexes of neuronal apoptosis were detected before and after MT pretreatment for neurons,and degree of OGD-induced neuronal injury was observed,and neurons autophagy was detected by using immunofluorescence and western blot methods.In OGD+MT+3-mA group,neurons was treated with cell autophagy inhibitor(3-mA),and then effects of MT on neuronal apoptosis and autophagy were investigated.In OGD+MT+Cpd C group,neurons was treated with AMPK inhibitor compound C(Cpd C),expressions of key molecules of AMPK-mTOR signaling pathway were detected,and regulation effect of MT on AMPK-mTOR signaling pathway was studied.Results OGD obviously increased neuronal apoptosis and autophagy,and it blocked smooth of autophagy flow.MT obviously decreased neuronal apoptosis,agitated autophagy and kept smooth of autophagy flow.Cell autophagy inhibitor and AMPK inhibitor compound C could reverse protective effect of MT on OGD-induced neuronal injury.Conclusion Melatonin may activate autophagy by AMPK-mTOR signaling pathway so as to decrease OGD-induced neuronal injury.

关 键 词：褪黑素 神经元 缺血缺氧 脑 自噬 神经保护 小鼠 

分 类 号：R-322[医药卫生] R743