Stroke is one of the leading causes of death and disability worldwide with limited therapeutic options. Melatonin can attenuate ischemic brain damage with improved functional outcomes. However, the cellular mechanisms of melatonin-driven neuroprotection against post-stroke neuronal death remain unknown. Here, distal middle cerebral artery occlusion (dMCAO) was performed in C57BL/6j mice to develop an ischemic stroke in vivo model. Melatonin was injected intraperitoneally immediately after ischemia, and 24 and 48 hours later. Melatonin treatment, with 5 to 20 mg/kg, elicited a dose-dependent decrease in infarct volume and concomitant increase in sensorimotor function. At the molecular level, phosphorylation of PTEN and Akt were increased, whereas PTEN activity was decreased in melatonin treated animals 72 hours after dMCAO. At the cellular level, oxygenglucose deprivation (OGD) challenge of neuronal cell line Neuro-2a (N2a) and primary neurons supported melatonin’s direct protection against neuronal cell death. Melatonin treatment reduced LDH release and neuronal apoptosis at various time points, markedly increased Akt phosphorylation in neuronal membrane, but significantly suppressed it in the cytoplasm of post-OGD neurons. Mechanistically, melatonin-induced Akt phosphorylation and neuronal survival was blocked by Wortmannin, a potent PIP3 inhibitor, exposing increased PI3K/Akt activation as a central player in melatonin-driven neuroprotection. Finally, PTEN knock-down through siRNA significantly inhibited PI3K/Akt activation and cell survival following melatonin treatment, suggesting that melatonin protection against ischemic brain damage, is at least partially, dependent on modulation of the PTEN/PI3K/Akt signaling axis.

中风是世界范围内导致死亡和残疾的主要原因之一，治疗选择有限。褪黑激素可以减轻缺血性脑损伤并改善功能结果。然而，褪黑激素驱动的针对中风后神经元死亡的神经保护的细胞机制仍然未知。在这里，在 C57BL/6j 小鼠中进行远端大脑中动脉闭塞 (dMCAO)，以在体内发生缺血性中风模型。缺血后立即和 24 和 48 小时后腹腔注射褪黑激素。褪黑激素治疗（5 至 20 mg/kg）引起梗死体积呈剂量依赖性减少，同时感觉运动功能增加。在分子水平上，在 dMCAO 后 72 小时，褪黑激素处理的动物中 PTEN 和 Akt 的磷酸化增加，而 PTEN 活性降低。在细胞水平上，神经元细胞系 Neuro-2a (N2a) 和原代神经元的氧葡萄糖剥夺 (OGD) 挑战支持褪黑激素对神经元细胞死亡的直接保护。褪黑激素处理减少了不同时间点的 LDH 释放和神经元凋亡，显着增加了神经元膜中的 Akt 磷酸化，但在 OGD 后神经元的细胞质中显着抑制了它。机械地，褪黑激素诱导的 Akt 磷酸化和神经元存活被 Wortmannin（一种有效的 PIP3 抑制剂）阻断，暴露出增加的 PI3K/Akt 活化作为褪黑激素驱动的神经保护的核心参与者。最后，通过 siRNA 敲低 PTEN 显着抑制褪黑激素治疗后的 PI3K/Akt 活化和细胞存活，这表明褪黑激素对缺血性脑损伤的保护至少部分依赖于 PTEN/PI3K/Akt 信号轴的调节。