基于p-NR2B/PI3K/Akt信号通路逍遥散对抑郁大鼠海马突触可塑性保护机理研究

Depression is one of common psychological illnesses, but its underlying mechanisms remain poorly understood. Accumulating evidence have showed that synaptic plasticity played important roles in the incidence and process of depression. Thus, we proposed a hypothesis that Xiaoyaosan exerts the protective effects against the impairment of hippocampal synaptic plasticity by regulating the p-NR2B/PI3K/Akt pathway in rat model of depression. In current study, both of vivo and vitro experiments are used. In vivo, the procedure of chronic unpredictable mild stress is used to establish the rat model of depression; Xiaoyaosan is used as the treatment drug; fluoxetine is used as the positive drug; the administration of N-Methyl-D-aspartic acid and LY-294002 is separately used as the agonist and antagonist for this pathway, respectively. In vitro, the synaptic plasticity deficit of primary cultures of rat hippocampal neurons is induced by exposure to oxidative stress. The administration of LY-294002 is also used to block the specific signalling pathways. The changes in thep-NR2B/PI3K/Akt pathway and the synaptic plasticity biomarker are determined in both of vivo and vitro experiments. In summary, the purpose of the study is to reveal that the p-NR2B/PI3K/Akt pathway may be one of the key mechanisms underlying Xiaoyaosan against impairment of hippocampal synaptic plasticity in depressive rat. This will help provide scientific evidence for theavailability of Xiaoyaosan in the treatment of depression.

抑郁症是临床常见的精神疾病，越来越多的证据表明其与突触可塑性有着密切关系。针对国内外研究现状结合前期工作基础，本项目提出“逍遥散对抑郁症大鼠海马突触可塑性有保护作用，其机理与p-NR2B/PI3k/Akt信号通路调控有关”的假说。本项目结合体内和体外海马原代细胞培养：采用国际公认的慢性不可预知温和应激建立抑郁症大鼠模型，以中药逍遥散进行干预，西药盐酸氟西汀为阳性对照药，分别采用N-甲基-D-天门冬氨酸与LY-294002为信号通路的激动剂和阻断剂，检测通路上各指标的变化及突触可塑性标志蛋白的表达；体外细胞培养中，氧化应激海马原代细胞，LY-294002为信号通路的阻断剂，检测逍遥散含药血清对该信号通路上各指标的影响及突触可塑性标志蛋白的表达，以阐明逍遥散对抑郁大鼠海马突触可塑性的保护机制，其中p-NR2B/PI3k/Akt信号通路是关键分子事件，将为临床应用逍遥散防治抑郁症提供科学依

背景：抑郁症是临床上常见的、发病机制复杂的精神疾病。已有研究表明逍遥散可降低抑郁大鼠海马谷氨酸水平，谷氨酸代谢和再摄取导致抑郁的病理生理。然而，其根本机制仍不清楚。本研究旨在探讨逍遥散对谷氨酸代谢的影响，以及如何调节由谷氨酸引起的兴奋性损伤。.研究内容：采用慢性不可预测轻度应激(Chronic unpredictable mild stress，CUMS)诱导抑郁模型大鼠。将大鼠随机分为对照组、模型组、模型+逍遥散组、模型+氟西汀组、模型+DMSO组、模型+逍遥散+阻断剂组和模型+阻断剂组。逍遥散口服剂量为2.224 g/kg/d，氟西汀的剂量为2.0 mg/kg，连续3周，并在侧脑室中微量（5 mM）注射Ly294002 (PI3K抑制剂)。采用旷场试验(OFT)、强迫游泳试验(FST)和蔗糖偏好试验(SPT)评价逍遥散的抗抑郁作用。比色法检测谷氨酸水平；Elisa检测血清皮质酮水平；RT-qPCR检测MAP2、NR2B、PI3K的RNA水平；Western blot、免疫化学或免疫荧光检测海马CA1区NR2B、MAP2、PI3K、P-AKT、Akt的表达。体外细胞培养中，谷氨酸诱导海马HT-22细胞，LY-294002为信号通路的阻断剂，检测逍遥散含药血清对PI3K/AKT信号通路上各指标的影响及对海马神经元标志蛋白MAP2表达的影响。.结果：逍遥散和逍遥散+Ly294002对CUMS大鼠的摄食和体重均有增加作用；改善了OFT中央区停留时间和运动总距离；减少SPT的大鼠不动时间，增加蔗糖偏好。降低CUMS大鼠海马CA1区谷氨酸水平和血清皮质酮水平。此外，这些治疗还提高了CUMS大鼠海马CA1区MAP2蛋白的表达，增加了海马CA1区NR2B和PI3K的表达以及P-AKT/AKT比值。体外细胞实验结果与体内实验结果一致。.科学意义:慢性应激导致神经元损伤进而引发抑郁的发生，这可能与谷氨酸代谢紊乱有关。逍遥散可通过改善谷氨酸介导的兴奋性毒性引起的CUMS大鼠海马神经元损伤而发挥抗抑郁作用，其潜在途径可能是通过NR2B和PI3K/Akt信号通路。