褪黑素通过AMPK通路减轻H_(2)O_(2)诱导的小鼠神经元线粒体损伤

Melatonin attenuates H_(2)O_(2)-induced neuronal mitochondrial damage through AMPK pathway in mice

目的:研究褪黑素对H_(2)O_(2)所致小鼠皮层神经元线粒体稳态失衡的保护作用以及分子机制。方法:将原代小鼠皮层神经元分为对照组(control)、H_(2)O_(2)处理组(H_(2)O_(2))、H_(2)O_(2)+褪黑素处理组(H_(2)O_(2)+Mel)和H_(2)O_(2)+褪黑素+WZ4003处理组(H_(2)O_(2)+Mel+WZ4003)。利用商品化试剂盒检测神经元乳酸脱氢酶(LDH)释放,使用TUNEL染色和Annexin V/PI染色检测神经元凋亡,利用real time RT-PCR检测过氧化物酶体增殖物激活受体-γ共激活因子-1α(PGC-1α)和核呼吸因子1(NRF1)mRNA表达变化,利用Western Blot检测cleaved caspase3、p-AMPK、AMPK、线粒体融合蛋白2(Mfn2)和动力蛋白相关蛋白1(Drp1)等蛋白分子的变化,使用JC-1染色检测线粒体膜电位(MMP)变化。结果:H_(2)O_(2)处理能导致小鼠皮层原代神经元LDH释放增多,线粒体膜稳态失衡,并导致神经元发生凋亡;褪黑素可以显著减轻神经元损伤,恢复线粒体稳态,抑制神经元凋亡;WZ4003可以逆转褪黑素对H_(2)O_(2)导致神经元线粒体损伤的保护作用。结论:褪黑素可以通过激活AMPK相关信号通路,维持神经元线粒体稳态,从而减轻H_(2)O_(2)所致神经元损伤。

Objective:To investigate the protective effect of melatonin on mitochondrial homeostasis imbalance induced by H_(2)O_(2) in mouse cortical neurons and its molecular mechanism.Methods:Cortical neurons of mice were divided into control group(control),H_(2)O_(2) treatment group(H_(2)O_(2)),H_(2)O_(2)+melatonin treatment group(H_(2)O_(2)+Mel),and H_(2)O_(2)+melatonin+WZ4003 treatment group(H_(2)O_(2)+Mel+WZ4003).The release of lactate dehydrogenas(LDH)from neurons was detected by commercial kits.TUNEL staining and Annexin V/PI staining were used to detect cells apoptosis.The mRNA expression of peroxisome-proliferator-activated receptorγcoactivator-1α(PGC-1α)and nuclear respiratory factor 1(NRF1)was detected by real time RT-PCR.The levels of cleaved caspase-3,p-AMPK,AMPK,mitochondrial fusion protein 2(Mfn2),and dynein-associated protein 1(Drp1)were detected by Western Blot.The changes in mitochondrial membrane potential(MMP)were detected by JC-1 staining.Results:H_(2)O_(2) treatment could lead to increased LDH release,disruption of mitochondrial homeostasis and apoptosis of primary neurons in mouse cortex.Melatonin treatment significantly reduced neuronal injury,restored mitochondrial homeostasis and inhibited neuronal apoptosis.WZ4003 reversed the protective effect of melatonin on H_(2)O_(2)-induced mitochondrial damage in neurons.Conclusion:Melatonin can maintain neuronal mitochondrial homeostasis by activating AMPK related signaling pathways,thus alleviating H_(2)O_(2)-induced neuronal injury.