基于miRNA-144-3p介导GABA合成/转运/释放研究百合地黄汤（仲景原方）调控前额叶皮层GABA能神经元活性发挥抗抑郁作用的机制

Extensive research efforts about depression have been done in past decades; however, the etiology of depression is still indefinable, its diagnosis unclear and the pharmacotherapy ineffective. Recently, perpetual extensive studies to search for herbal formula from traditional Chinese medicine (TCM) are carried out. Indeed, some TCM formulas are highly potent and work on multi-targets with low toxicity for the treatment of depression. The clinical manifestations of depression are very similar to the symptoms of lily disease in the Golden Cabinet by Zhang Zhongjing in the early third century. The classical herbal formula Lily Bulb and Rehmannia Decoction (LBRD) has long been used as tonics for nourishing heart and lung, clearing heat and cooling blood in the treatment of depression. In previous studies, we found that the dysfunction of GABAergic neurons in the prefrontal cortex was related to the process of miRNA-144-3p mediated GABA synthesis, transport and release; while LBRD can significantly improve depression by increasing the release of GABA from the prefrontal cortex. Therefore, we hypothesis LBRD may regulate the prefrontal GABAergic neurons activity, promotes the release of GABA, rebalance of compatibility among subcellular compartments and finally enhance the synaptic transmission efficiency. In this project, we follow the principle of using the original volume of the classical traditional Chinese medicine prescriptions. The genetically modified animals was used to as a animal model of depression. We will use electrophysiology, brain stereotactic microinjection, RNA interference etc to investigate the antidepressant mechanism of LBRD on the body behavior, synapse structure and function, epigenetic. Lily Bulb and Rehmannia Decoction can rescue compatibility among the GABAergic neuron subcellular compartments and increase synaptic transmission efficiency through negatively affecting the level of prefrontal cortex GAD-67 and VGAT protein expression in which mediated by miRNA-144-3p.

抑郁症病因复杂，发病机制存在多种假说，据此研发的抗抑郁药物疗效欠佳。辨证论治指导的中药复方在抑郁症防治方面具有独特优势。抑郁症临床表现与《金匮要略》记载百合病症状相似，治当清心润肺、滋阴安神，方用百合地黄汤。我们前期研究发现，抑郁症前额叶GABA能神经元功能失调与miRNA-144-3p介导GABA合成、转运及释放相关；而百合地黄汤（仲景原方）通过增加前额叶GABA释放，明显改善抑郁症状。由此我们推测，百合地黄汤（仲景原方）改变前额叶miRNA-144-3p表达，促进GABA释放，恢复GABA能神经元编码输出信息功能，发挥抗抑郁作用。为此本项目拟利用慢性应激转基因抑郁模型，从机体行为、突触结构和功能、分子机制等层面，运用电生理、脑立体定位显微注射、RNA干扰等技术，正反两方面阐述百合地黄汤抗抑郁功效，及其调控miRNA-144-3p介导GABA能神经元功能活性，促进GABA释放的药理机制。

百合地黄汤为心肺阴虚内热之百合病的经典名方，而《金匮要略》中百合病的情志失调导致的行为表现与现代抑郁症的临床症状基本相似。其中，百合地黄汤对阴虚内热型抑郁有明显的治疗效果，可有效改善大便干、盗汗、悲伤易哭、失眠等症状。然而，国内外尚缺乏百合地黄汤干预阴虚内热型抑郁中枢神经生物学基础的深入研究。项目以百合病作为切入点，提出“多情交织暗耗阴液致心肺阴虚而渐生内热”科学问题，阐明百合病虚热与郁闷不舒躯体化症状可归因于阴虚内热情志失调。通过审证求因、审证求机、以证验方、以方佐证，构建百合病情志失调表征的阴虚内热型抑郁（慢性应激联合甲状腺激素）动物证候模型及评价体系；考证百合地黄汤本草关键信息，完成仲景原方基准样品制备。采用指纹/特征图谱结合多成份指标表征基准样品的化学物质组成，确定薯蓣皂苷、阿魏酸、毛蕊花糖苷及梓醇作为物质基准的关键质量属性；对药材、饮片、制剂开展相关性研究，确定制备工艺关键参数，明确阿魏酸、毛蕊花糖苷作为全过程质量控制体系的标记物；采用多项指标联合分析、综合评价百合地黄汤（仲景原方）缓解虚热与抑郁样行为与上调单胺类神经递质，降低外周与中枢炎症水平，拮抗HPA轴兴奋，纠正兴奋/抑制性神经递质失衡，减轻应激诱导的皮层神经细胞损伤相关；基于动物与细胞抑郁模型交叉佐证实验揭示百合地黄汤多种组分可改变一系列微小RNAs介导GABA/谷氨酸能突触、突触可塑性及轴突引导、昼夜节律、神经免疫与炎症等信号通路，实现机体多系统生理功能的协调与统一。项目的研究成果丰富百合地黄汤多途径治疗阴虚内热型抑郁的中枢神经细胞与分子机制，为从表观遗传修饰角度解析该方中医治则治法的生物学基础及中药新药研发提供充分科学依据，实现百合地黄汤的中医药源头创新。