AMPK介导线粒体融合与裂变在抑制P2X7受体抗神经元缺氧/复氧损伤中的作用

目的探讨AMPK介导下线粒体融合与裂变在抑制P2X7受体抗神经元缺氧/复氧（hypoxia/reoxygenation，H/R）损伤中的作用。方法培养原代神经元细胞，并随机分为正常对照（Control）组、缺氧/复氧（H/R）组、H/R+Bright Blue G（BBG）组、H/R+BBG+Dorsomorphin组。采用Calcein-AM/PI双染法检测细胞存活率；采用活性氧（ROS）试剂盒检测ROS的产生；采用Mito Tracker TM Green FM检测线粒体形态；采用Western blotting法检测p-AMPK、p-Drp1、Mfn2蛋白的表达。结果与Control组相比，当神经元细胞发生H/R损伤时，其细胞死亡率、线粒体裂变、ROS的含量都明显增加，BBG预处理后，H/R损伤神经元细胞的死亡率、线粒体裂变、ROS的含量都明显减少，而AMPK抑制剂Dorsomorphin则减弱了BBG对H/R损伤神经元细胞的保护作用（P < 0.01）。Western blotting结果显示，与Control组相比，H/R组神经元细胞p-AMPK与Mfn2的表达显著降低，而p-Drp1的表达显著增高（P < 0.01）；BBG预处理后，H/R损伤神经元细胞p-AMPK与Mfn2的表达显著增高，而p-Drp1的表达则显著降低（P < 0.01）；AMPK抑制剂Dorsomorphin则减弱了BBG增加H/R损伤神经元p-AMPK与Mfn2的表达而降低p-Drp1的表达的作用（P < 0.01）。结论抑制P2X7受体可通过抑制线粒体裂变促进线粒体融合减轻神经元缺氧/复氧损伤，其机制可能与激活AMPK有关。

Role of AMPK-mediated mitochondrial fusion and fission in inhibiting P2X7 receptor anti-hypoxia/reoxygenation injury of neurons

ObjectiveTo investigate the role of mitochondrial fusion and fission regulated by AMPK in the protective effects of inhibiting P2X7 receptor against hypoxia/reoxygenation injury in neurons.MethodsThe primary neurons were cultured, and randomly divided into the Control group, hypoxia/reoxygenation(H/R) group, H/R+Bright Blue G(BBG) group, H/R+BBG+Dorsomorphin group.The viability of neurons was assessed using Calcein-AM/PI kit, the ROS level was detected using ROS kit, the morphology of mitochondrial morphology was detected using Mito Tracker TM Green FM kit, and the expression levels of p-AMPK, p-Drp1 and Mfn2 were detected using Western blotting.ResultsCompared with Control group, the death rate, mitochondrial fission and level of ROS in neurons increased significantly in H/R group.After pretreated with BBG, the death rate, fission of mitochondrial and level of ROS in neurons with H/R injury decreased significantly, while the protective effects of inhibitor of AMPK Dorsomorphin on neurons with H/R injury were attenuated(P < 0.01).Compared with the Control group, the results of Western blotting showed that the expression levles of p-AMPK and Mfn2 decreased significantly, while the expression level of p-Drp1 increased markedly in H/R group(P < 0.01).After pretreated with BBG, the expression levels of p-AMPK and Mfn2 increased significantly, while the expression level of p-Drp1 decreased markedly.The inhibitor of AMPK Dorsomorphin weakened the BBG increasing the expression levels of p-PMAK and Mfn2 in H/R injured neurons and decreas the expression level of p-Drpl(P < 0.01).ConclusionsThe inhibition of P2X7 receptor can reduce the damage of neurons induced by H/R injury through inhibiting mitochondrial fission to promote mitochondrial fusion, which may be involved in the activation of AMPK.