[Effect of electroacupuncture on neuronal autophagy in rats with chronic sleep deprivation via the PI3K/AKT/mTOR signaling pathway].

Abstract

<p>OBJECTIVE: To observe the effects of electroacupuncture (EA) at Fengchi (GB20) and Gongxue (Extra) on the phosphatidylinostitol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapanycin (mTOR) signaling pathway and hippocampal neuronal autophagy in rats with chronic sleep deprivation (CSD), and to explore the potential mechanisms of EA in treating CSD-related cognitive impairment. METHODS: A total of 65 Wistar rats with normal cognitive function were screened, and 12 were randomly selected as the control group. The remaining rats were subjected to a modified multiple-platform water environment method to induce the CSD model. Forty-eight successfully modeled rats were randomly divided into a model group, an EA group, an activator group, and an EA+activator group, with 12 rats in each group. The EA group was treated with EA at bilateral Fengchi (GB20) and Gongxue (Extra) for 20 min, once daily. The activator group was treated with intraperitoneal injection of PI3K activator 740Y-P (0.02 mg/kg) once daily. The EA+activator group was treated with EA, followed by intraperitoneal injection of 740Y-P. All interventions lasted for 14 days. Cognitive function was assessed using the Morris water maze. Golgi staining was used to observe dendritic branching and spine density in the hippocampal CA1 region. Western blot was employed to detect protein expression levels of phosphorylated PI3K (p-PI3K), PI3K, p-AKT, AKT, p-mTOR, mTOR, microtubule-associated protein light chain 3 (LC3)Ⅱ, LC3Ⅰ, and Beclin-1 in hippocampal tissues. Immunofluorescence staining was used to detect positive expression of Beclin-1 and LC3 in the hippocampal CA1 region. RESULTS: Compared with the control group, the rats in the model group and activator group exhibited prolonged escape latency and fewer platform crossings (P&lt;0.05), fewer dendritic branches and lower spine density in hippocampal CA1 pyramidal neurons (P&lt;0.05), decreased positive expression of Beclin-1 and LC3 (P&lt;0.05), increased ratios of p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR (P&lt;0.05), and decreased expression levels of LC3Ⅱ/Ⅰ and Beclin-1 (P&lt;0.05). Compared with the model group, the activator group and EA+activator group, the rats in the EA group showed shortened escape latency, increased platform crossings (P&lt;0.05), enriched dendritic branching and higher spine density (P&lt;0.05), increased positive expression of Beclin-1 and LC3 in the CA1 region (P&lt;0.05), decreased ratios of p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR (P&lt;0.05), and elevated protein expression of LC3Ⅱ/Ⅰ and Beclin-1 (P&lt;0.05). CONCLUSION: EA at Fengchi (GB20) and Gongxue (Extra) could promote dendritic spine formation, enhance synaptic plasticity, and improve cognitive function in rats with CSD. These effects may be related to the inhibition of the PI3K/AKT/mTOR signaling pathway and activation of hippocampal neuronal autophagy. 目的：观察电针“风池”和“供血”对慢性睡眠剥夺（CSD）模型大鼠磷脂酰肌醇-3-激酶（PI3K）/蛋白激酶B（AKT）/雷帕霉素靶蛋白（mTOR）信号通路和海马神经元自噬的影响，探讨电针治疗CSD相关认知损伤的可能作用机制。 方法：从筛选出的65只认知功能正常Wistar大鼠中随机取12只为对照组，其余大鼠采用改良的多平台水环境法制备CSD模型，将造模成功的48只大鼠随机分为模型组、电针组、激活剂组和电针+激活剂组，每组12只。电针组予电针双侧“风池”和“供血”，每次20 min，每天1次。激活剂组大鼠腹腔注射PI3K激活剂740Y-P溶液（0.02 mg/kg），每天1次。电针+激活剂组大鼠在电针干预后，进行腹腔注射740Y-P溶液。以上各组连续干预14 d。采用水迷宫试验评价大鼠的认知能力，高尔基染色法观察大鼠海马CA1区树突分支形态和树突棘数量，Western blot法检测大鼠海马组织磷酸化PI3K（p-PI3K）、PI3K、磷酸化AKT（p-AKT）、AKT、磷酸化mTOR（p-mTOR）、mTOR、微管相关蛋白轻链3 （LC3）Ⅱ、LC3Ⅰ、苄氯素-1（Beclin-1）蛋白表达，免疫荧光染色法检测大鼠海马CA1区Beclin-1、LC3阳性表达。 结果：与对照组比较，模型组和激活剂组大鼠逃避潜伏期延长、穿越平台次数减少（P&lt;0.05），海马CA1区锥体神经元主干树突分支较少，树突棘密度降低（P&lt;0.05），海马CA1区Beclin-1和LC3阳性表达降低（P&lt;0.05），海马组织p-PI3K/PI3K、p-AKT/AKT、p-mTOR/mTOR比值升高（P&lt;0.05），LC3Ⅱ/LC3Ⅰ比值和Beclin-1蛋白表达降低（P&lt;0.05）。与模型组、激活剂组和电针+激活剂组比较，电针组大鼠逃避潜伏期缩短、穿越平台次数增加（P&lt;0.05），海马CA1区树突分支丰富，树突棘密度升高（P&lt;0.05），海马CA1区Beclin-1和LC3阳性表达升高（P&lt;0.05），海马组织p-PI3K/PI3K、p-AKT/AKT、p-mTOR/mTOR的比值降低（P&lt;0.05），LC3Ⅱ/LC3Ⅰ比值和Beclin-1蛋白表达升高（P&lt;0.05）。 结论：电针“风池”“供血”能够促进树突棘的形成，提高突触可塑性，改善CSD大鼠的神经认知功能，其机制可能与抑制PI3K/AKT/mTOR通路，激活海马神经元自噬有关。.</p>