腺苷酸激酶 4 在脑缺血早期神经损伤中的作用研究

Study on the Role of Adenylate Kinase 4 in Nerve Injury in the Early Stage of Cerebral Ischemia

摘要:

〔摘 要〕 目的：脑供血不足导致的神经元损伤是缺血性脑卒中神经功能障碍的直接原因，线粒体功能异常与神经 元损伤密切相关，腺苷酸激酶 4（AK4）是能量代谢关键因子，本研究旨在探讨 AK4 在脑缺血早期神经损伤中的作用。 方法：研究样本为 60 只雄性 C57BL/6J 小鼠，3 ~ 4 周龄，体质量 18 ~ 20 g，构建神经元特异性过表达 AK4 腺相关病 毒（AAV）– 空载（AAV–Ctrl 组）以及 AAV–AK4 过表达（AAV–AK4 组）小鼠。通过建立小鼠大脑中动脉阻塞（MCAO） 模型模拟体内脑缺血，分别在缺血 1 h、2 h 和 3 h 后采用免疫印迹法测定 AK4 的蛋白表达水平；在单纯缺血 3 h 后， 分别采用 2,3,5– 氯化三苯基四氮唑（TTC）染色法检测缺血脑组织损伤程度，末端脱氧核苷酸转移酶介导的 dUTP 缺 口末端标记测定法（TUNEL）法检测神经元凋亡情况。 结果：实验结果表明，MCAO 2 h 和 MCAO 3 h 时梗死侧（I） 相较于非梗死侧（NI）的 AK4 蛋白表达开始下调，其中 MCAO 3 h 最明显，差异均具有统计学意义（P ＜ 0.05）。 与 AAV–Ctrl 组比较，MCAO 3 h 后，AAV–AK4 组小鼠的脑梗死体积明显缩小，且 AAV–AK4 组比 AAV–Ctrl 组小鼠 的神经元凋亡数量明显减少，差异均具有统计学意义（P ＜ 0.05）。 结论：脑缺血早期神经元过表达 AK4 可明显减 少脑梗死体积，抑制神经元凋亡，发挥神经保护作用，AK4 可能是缺血早期神经元损伤干预的新靶点。

Abstract:

〔Abstract〕 Objective Neuronal damage caused by insufficient cerebral blood supply is the direct cause of neurological dysfunction in ischemic stroke. Mitochondrial dysfunction is closely related to neuronal damage. Adenylate kinase 4 (AK4) is a key factor in energy metabolism. The purpose of this study is to explore the role of AK4 in early nerve injury after cerebral ischemia. Methods The study sample consisted of 60 male C57BL/6J mice, 3 to 4 weeks of age and 18 to 20 g body mass, constructed neuron-specific overexpressing AK4 adeno-associated virus (AAV) -unloaded (AAV-Ctrl group) and AAV-AK4 overexpressing mice (AAV-AK4 group). A mouse middle cerebral artery occlusion (MCAO) model was established to simulate cerebral ischemia in vivo, and the protein expression level of AK4 was determined by western blot after 1 h, 2 h and 3 h of ischemia, respectively. After 3 h of pure ischemia, 2,3, 5-triphenyltetrazolium chloride (TTC) staining was used to detect the degree of ischemic brain tissue injury, and TUNEL method was used to detect neuronal apoptosis. Results The experimental results showed that AK4 protein expression on infarct side (I) began to be down-regulated compared with non-infarct side (NI) at MCAO 2 h and MCAO 3 h, and MCAO 3 h was the most significant, with statistical significance (P < 0.05). Compared with AAV-Ctrl group, after 3 h of MCAO, the volume of cerebral infarction in AAV-AK4 group was significantly reduced, and the number of neuronal apoptosis in AAV-AK4 group was significantly reduced compared with AAV-Ctrl group, with statistical significances (P < 0.05). Conclusion Overexpression of AK4 in neurons in the early stage of cerebral ischemia can significantly reduce the volume of cerebral infarction,inhibit neuronal apoptosis and play a neuroprotective effect. AK4 may be a new target for intervention of neuronal injury in the early stage of cerebral ischemia.