基于miR-124—神经发生调控机制探讨中医调肝治法抗抑郁“求衡”思想的科学内涵

A new academic point of view is proposed theoretically as following: in connection with pathogenesis features of imbalance between obstruction and hyperactivity of liver-qi dispersing in the actual pathogenic process of depression, scientific connotation of "Seeking balance" idea of "Tiaogan " therapy and prescriptions is reflected in regulating relative balance between obstruction and hyperactivity of liver-qi dispersing which makes emotion normal. By taking miR-124—neurogenesis regulatory mechanism as the research focus, utilizing in vivo and in vitro pharmacological model to study demolition party of "Tiaogan " prescriptions, investigating the mechanisms of "Tiaogan " prescriptions of regulating double negative feedback loop between miR-124 and REST complex to regulate the level of miR-124 in hippocampal dentate gyrus, which could maintain the relative balance between neural stem cell proliferation and neuronal cell differentiation to exert antidepressant effect, the scientific connotation of "Seeking balance" idea of "Tiaogan " therapy and prescriptions would be revealed and illustrated in depth. In addition, the research on the mechanisms of "Tiaogan " therapy and prescriptions would provide new perspectives and ideas for treatment of psychophysiological diseases related to abnormal liver-qi dispersing. Meanwhile, some of the new targets, links or pathways of "Tiaogan " therapy and prescriptions will be revealed and clarified, which maybe provide adequate scientific basis for further research and development of antidepressant of "Tiaogan " prescriptions' in translational medicine.

本研究在理论方面提出新的学术观点：针对抑郁症实际发病过程中存在着肝疏不及与太过失衡的病机特点，调肝治法抗抑郁“求衡”思想的科学内涵在于通过调控肝疏不及与太过之间的相对平衡，使肝气敷和、情志调畅。以miR-124—神经发生调控机制为研究切入点，借助于整体实验和体外实验对调肝方药进行拆方研究；通过探讨调肝治法方药调控miR-124和REST复合物间的双重负反馈环路，调节海马齿状回miR-124水平，维持海马齿状回神经干细胞增殖与神经元细胞分化之间相对平衡，发挥抗抑郁效应的作用机理，深入揭示和阐释调肝治法方药抗抑郁“求衡”思想的科学内涵。通过对调肝治法方药机理的研究，不仅为今后中医临床有关肝失疏泄所致情志疾病的防治提供新的视角和思路，同时可以发现调肝方药一些新的药理学作用靶点、环节或通路，从而为调肝方药抗抑郁的转化医学研究提供充分的科学依据。

抑郁症的发生发展常具有肝疏不及与太过失衡的病机特点。平衡是肝主疏泄功能正常发挥的重要前提，因此以中医“求衡”思想指导下的调肝治法，即兼顾疏肝、清平肝，可能在抗抑郁治疗中更具优势。我们提出科学假说，抑郁症个体中海马miR-124 -神经发生调控机制的紊乱可能是抑郁症发生发展过程中肝疏不及与太过失衡的枢纽，而RE1-沉默转录阻遏物（REST）和miR-124间双重负反馈环路机制又是维持海马miR-124的平衡表达关键因素。在以上基础上，我们通过体外实验构建神经干细胞的miR-124功能获得型（gain of function，GOF）及miR-124功能缺失型(loss of function，LOF)模型，明确了神经干细胞中miR-124异常表达对神经干细胞增殖分化的影响，并观察加味四逆散（JWSNS）在因miR-124异常表达致神经干细胞增殖分化抑制时，对神经干细胞增殖分化的影响；在整体实验中复制CUMS抑郁症模型，动态观察应激致抑郁过程中抑郁样行为、海马miR-124表达、神经发生的动态变化，并在因miR-124表达降低的神经发生障碍时，观察JWSNS及其拆方疏肝方（SGF）、清平肝（QPF）对抑郁症状、海马miR-124、神经发生障碍及海马REST复合物表达的影响。结果显示：1.体外研究表明，神经干细胞miR-124 GOF模型的增殖分化能力无变化，但神经干细胞miR-124 LOF模型增殖分化能力显著减弱，JWSNS干预可显著增强神经干细胞miR-124 LOF模型增殖分化能力。2.整体实验表明， CUMS致抑郁过程中，会出现不同的病理阶段，伴随抑郁样行为逐步出现并稳定，海马miR-124呈先升高后降低的双向变化，且海马miR-124表达降低时神经发生障碍出现，此时海马REST复合物表达也显著上升，经JWSNS治疗，抑郁症状、认知障碍及神经发生障碍均显著改善，应激大鼠海马miR-124显著上调、REST复合物表达显著下降，SGF、QPF虽对以上病理改变也有一定治疗作用，但对于某些症状疗效不佳或疗效显著不及JWSNS。以上结果提示，JWSNS、SGF及QPF可能通过下调REST复合物的表达，改善CUMS大鼠海马miR-124的低表达，修复神经发生抑制，达到抗抑郁的疗效。另外，相比单一疏肝或清平肝治法，调肝治法抗抑郁疗效更佳，这可能为临床中医药抗抑郁提供一定线索。