Dai Shu-Hui, Chen Tao, Li Xia, Yue Kang-Yi, Luo Peng, Yang Li-Kun, Zhu Jie, Wang Yu-Hai, Fei Zhou, Jiang Xiao-Fan
Department of Neurosurgery, Xijing Institute of Clinical Neuroscience, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi 710032, China.
Department of Neurosurgery, Xijing Institute of Clinical Neuroscience, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi 710032, China; Department of Neurosurgery, The 101th Hospital of PLA, Rescue Center of Craniocerebral Injuries of PLA, Wuxi, Jiangsu 214044, China; Department of Neurosurgery, The 123th Hospital of PLA, Bengbu, Anhui 233000, China.
Free Radic Biol Med. 2017 Jul;108:345-353. doi: 10.1016/j.freeradbiomed.2017.04.005. Epub 2017 Apr 7.
Sirtuin3 (Sirt3) is a member of the silent information regulator 2 (Sir2) family of proteins located in mitochondria that influences almost every major aspect of mitochondrial biology, including ATP generation and reactive oxygen species (ROS) production. Our previous study showed that Sirt3 exerts protective effects against oxidative stress in neuronal cells. In this study, we investigated the role of Sirt3 in neuronal ischemia using an oxygen and glucose deprivation (OGD) model. Sirt3 was up-regulated by OGD and overexpression of Sirt3 through lentivirus transfection significantly reduced OGD-induced lactate dehydrogenase (LDH) release and neuronal apoptosis. These effects were accompanied by reduced hydrogen dioxide (HO) production, enhanced ATP generation and preserved mitochondrial membrane potential (MMP). The results of immunocytochemistry and electron microscopy showed that Sirt3 increased autophagy in OGD-injured neurons, which was also confirmed by the increased expression of Beclin-1 as well as LC3-I to LC3-II conversion. In addition, the autophagy inhibitor 3-MA and bafilomycin A1 partially prevented the effects of Sirt3 on LDH release and apoptosis after OGD. The results of western blotting showed that overexpression of Sirt3 in cortical neurons markedly increased the phosphorylation of AMPK, whereas the phosphor-mTOR (p-mTOR) levels decreased both in the presence and absence of OGD insult. Furthermore, pre-treatment with the AMPK inhibitor compound C partially reversed the protective effects of Sirt3. Taken together, these findings demonstrate that Sirt3 protects against OGD insult by inducing autophagy through regulation of the AMPK-mTOR pathway and that Sirt3 may have therapeutic value for protecting neurons from cerebral ischemia.
沉默调节蛋白3（Sirt3）是位于线粒体中的沉默信息调节因子2（Sir2）蛋白家族的成员，它几乎影响线粒体生物学的每个主要方面，包括ATP生成和活性氧（ROS）产生。我们之前的研究表明，Sirt3对神经元细胞的氧化应激具有保护作用。在本研究中，我们使用氧糖剥夺（OGD）模型研究了Sirt3在神经元缺血中的作用。OGD可上调Sirt3，通过慢病毒转染过表达Sirt3可显著降低OGD诱导的乳酸脱氢酶（LDH）释放和神经元凋亡。这些作用伴随着过氧化氢（HO）生成减少、ATP生成增强以及线粒体膜电位（MMP）的维持。免疫细胞化学和电子显微镜结果显示，Sirt3可增加OGD损伤神经元中的自噬，这也通过Beclin-1表达增加以及LC3-I向LC3-II的转化得到证实。此外，自噬抑制剂3-MA和巴弗洛霉素A1可部分阻止Sirt3对OGD后LDH释放和凋亡的影响。蛋白质免疫印迹结果显示，皮质神经元中Sirt3的过表达显著增加了AMPK的磷酸化，而在有或没有OGD损伤的情况下，磷酸化mTOR（p-mTOR）水平均降低。此外，用AMPK抑制剂化合物C预处理可部分逆转Sirt3的保护作用。综上所述，这些发现表明，Sirt3通过调节AMPK-mTOR途径诱导自噬来保护细胞免受OGD损伤，并且Sirt3可能对保护神经元免受脑缺血具有治疗价值。
