基于“BDNF-mTOR信号通路”的逍遥散有效部位抗抑郁机制研究

Xiaoyao san is a famous anti-depression TCM compound drug with good clinical effect. If the anti-depression regulating mechanism of active fraction of Xiaoyao san could be revealed using current medical methods, it will provide theoretical foundation for the prevention and treatment of depression. Previous studies showed that signaling pathway of "BDNF-mTOR" was the core of antidepressant pathway in hippocampus and prefrontal cortex. It can trigger protein translation to maintain protein synthesis-dependent neural synaptic plasticity, and meanwhile, it was found that depression is always accompanied by repressive activation of this signaling pathway. Activation of this pathway can reverse sustained activation of HPA axis to the normal range. Based on these findings, the applicant proposes the following hypothesis: active fraction of Xiaoyao san is likely to treat depression through activating this pathway, and curative effect may drop deeply if blocking the pathway. To verify this hypothesis, experiments will be designed by activating and blocking the pathway based on building the rat model of "liver depression and spleen deficiency syndrome" by chronic restraint stress(CRS) method. Using buried pipe trace microinjection technology, this pathway will be activated by targeted microinjection of BDNF, while pathway will be blocked by targeted injection of LVshBDNF. The anti-depression regulation mechanism of Xiaoyao san will be investigated and revealed in many ways from the macroscopic behavior to microcosmic gene and protein level. Key indices including colocalization NR2B and PSD-95, BDNF, mTOR, GSK3β, hormonal level of HPA axis and neural morphology will be detected with key technology such as double-labelling immunofluorescence and electron microscope, etc. The success of this project will provide solid theoretical basis and experimental proof for revealing the neural regulation mechanism of this sinaling pathway underlying the anti-depression function of active fraction of Xiaoyao san.

名方逍遥散抗抑郁机制研究意义重大。研究表明，通过激活"BDNF-mTOR信号通路"，启动合成维持突触可塑性所必需的蛋白，可以逆转HPA轴持续活化的病理损害，重塑突触可塑性。因此提出以下假说：逍遥散有效部位通过激活该通路重塑突触可塑性，而阻断通路疗效可能会明显下降。为验证假说，本项目以"BDNF-mTOR信号通路"为理论依据，以海马和前额皮质为研究脑区，以通路关键节点蛋白为切入点，以激活和阻断通路为不同层次设计实验。在肝郁脾虚证模型基础上，采用靶向注射BDNF和LVshBDNF分别激活和阻断通路，以检测共定位表达的NR2B和PSD-95、通路节点蛋白及HPA轴活性为指标，以LVshBDNF靶向沉默BDNF表达、免疫荧光双标和电镜等为关键技术，从宏观行为学到微观基因蛋白水平，从形态学定性到指标定量多层面进行，探清逍遥散对通路效应的影响。有机结合实验结果和中医理论，揭示逍遥散有效部位抗抑郁机制。

中医药抗抑郁是近年来研究的热点。名方逍遥散调节抑郁临床疗效显著。研究表明，通过激活海马和前额皮质“BDNF-mTOR信号通路”，启动合成维持突触可塑性所必需的蛋白，可以逆转HPA轴持续活化的病理损害，重塑突触可塑性。结合国内外中西医专家和本人前期研究成果，提出如下假说：逍遥散有效部位通过激活海马和前额皮质“BDNF-mTOR信号通路”，抑制GSK3β活性，逆转HPA轴持续活化，重塑突触可塑性；而阻断该通路逍遥散抗抑郁疗效可能会明显下降。申请人以“激活BDNF-mTOR信号通路逆转HPA轴持续活化”为理论依据，以通路中关键节点蛋白为切入点，以激活和阻断通路为不同层次进行实验。在采用慢性束缚应激法造模和成功验证慢病毒载体LVshBDNF动态沉默BDNF的特异性的基础上，以海马DG区靶向注射BDNF和LVshBDNF分别激活和阻断通路，正反二方面探测逍遥散有效部位对突触可塑性（通路效应）、通路关键节点蛋白（BDNF、mTOR、GSK3β）及HPA轴活性的影响。实验从宏观行为学到微观基因蛋白水平，从形态学定性到指标定量多层面进行，实验结果基本验证了假说的合理性。行为学论证了肝郁脾虚证大鼠模型的成功建立。在造模结束时，与正常对照组比较，激活通路组群的宏观微观指标（行为学各项指标、HPA轴活性相关激素水平、电镜观察突触和线粒体的结果、RT-PCR、West-blot、尼氏染色、免疫组化、免疫荧光双标各项指标）基本无统计学差异（P0.05），而阻断通路组群的上述各项指标有统计学意义（P0.01或P0.05），截然相反的实验结果提示：BDNF-mTOR信号通路可能是逍遥散抗抑郁的一条核心通路，有机结合实验结果和中医理论，可能揭示逍遥散有效部位抗抑郁机理。