基于青少期应激抑郁中缝背核突触前膜5-HTT/5-HT1AR通路探讨调肝治法方药的中枢作用机制

Changes of 5-HT system have been proved to be the important pathways of central mechanism of emotional pathopoiesis and the treatment based on the liver, but the dorsal raphe nucleus which acts as a key brain region of its synthesis and release has not been studied. The dorsal raphe nucleus also controls the negative feedback effects of 5-HT1A receptor itself on 5-HT releasing in presynaptic membrane and its interaction with the reuptake of 5-HT. More importantly than all of that,traditional Chinese medicine holds that "the liver is often enough" is the physiological and pathological characteristics of the youth. The level of 5-HT in adolescence is already mature and maintaines to adulthood, and it is the only relatively sure access to stress-induced depression in youth period. All of these suggest that begin with the research of regulating liver therapy and prescription on anti-stress damage mechanism of central 5 - HT from the 5-HT pathway in adolescence may be a more "stable" way. Based on our previous studies on 5-HT pathways in hippocampal, we try to carry out the changes of presynaptic membrane 5- HT1A receptor and 5-HTT in adolescent rats under stress and intervention mechanism of Jiaweisinisan (Gan-regulating prescription) from the form, content, transmission of information and other aspects by the way such as cell culture and animal experiments. We expect to give a full interpretation of the 5-HT central mechanism of regulating liver therapy form the aspects of “dorsal raphe nucleus - hippocampus pathways and synthesis -release – regulation”. The study may also provide a basis for seeking effective interventions of TCM in adolescent sentiment abnormality.

5-HT系统改变已证实是情志致病及其从肝论治中枢机制的重要通路，但作为其合成释放的关键脑区中缝背核、并控制5-HT释放效应的突触前膜上5-HT1A自身受体负反馈作用及其与5-HT重摄取间的相互作用尚未得到深入研究。更重要的是，5-HT是目前青少期应激抑郁发生的唯一较为肯定通路并影响至成年，这且与中医学青少期脏腑“肝常有余”生理病理特性相吻合，提示从青少期5-HT通路入手研究调肝治法方药抗应激损伤中枢5-HT机制将是一个更加“明确”的途径。由此，本课题在前期海马5-HT通路研究的基础上，通过动物实验及细胞培养等方法，拟从形态、含量、信号传导等方面开展青少期应激大鼠突触前膜5-HT1A受体与5-HTT改变及调肝方药加味四逆散干预机制的研究，以期从“中缝背核-海马作用通路；合成-释放-调控环节”完整阐释调肝治法5-HT中枢机制。本研究亦为寻求青少期情志异常的中医有效干预手段提供依据。

5-HT系统是目前青少期应激抑郁发生的唯一较为肯定通路并影响至成年，与中医学青少期脏腑“肝常有余”生理病理特性相吻合。5-HT1AR可能通过激活某些信号通路在神经发生和突触形成中发挥重要作用，并与行为和认知的某种缺陷有关。脑源性神经营养因子（BDNF）在海马神经发生、神经可塑性和神经细胞存活中发挥重要作用，与抑郁症发病机制密切相关。本研究采用早年母婴分离及青少期不可预知慢性应激抑郁模型，探索调肝治法方药对抑郁关键脑区的5-HT系统、其关通路及突触可塑性相关蛋白的作用机制研究。结果提示：1）加味四逆散可以显著提高青少期应激大鼠的抑郁表现，显著增加其海马和前额叶皮质中NE、DA、5-HT、5-HIAA及单胺类神经递质。2）青少期应激抑郁大鼠在经历成年后刺激下，可能导致抑郁症的复发与加重；加味四逆散干预对应激大鼠的一般状况、海马CA1区细胞形态、5-HT1AR、及5-HTT蛋白表达产生显著的逆转作用。3）早年母婴分离诱导大鼠在生长发育不同阶段均出现抑郁样行为，四逆散干预可能通过上调海马5-HT1AR-CREB-BDNF 信号通路发挥抗抑郁作用。4）母婴分离联合青少期慢性应激能够诱发大鼠抑郁样行为，四逆散干预可上调大鼠中缝背核中CaSR、BDNF、5-HT1R、5-HTT蛋白表达、改善海马和皮质Nissl体损伤、增加突触可塑性相关蛋白突触素、GAP-43和PSD-95表达，从而发挥抗抑郁效果。5）体外实验中四逆散含药血清对皮质酮损伤的PC12细胞具有一定的神经保护作用，其机制还在进一步研究中。综上，调肝治法方药可能通过调节应激大鼠海马及中缝背核5-HT系统功能紊乱、调节海马5-HT1AR-CREB-BDNF表达以修复并促进海马神经元突触可塑性，从而发挥抗抑郁效果。负性应激源对健康个体的认知模式和行为模式的影响相当持久，即使抑郁症患者脱离应激源且症状改善，他们仍会残留之前的消极认知方式，这就增加了抑郁症复发的风险。本研究成功模拟生长发育关键时期应激及复合应激下的肝失疏泄、情志内伤抑郁；丰富并完善了四逆散抗抑郁机制研究，为临床上治疗各个年龄阶段的抑郁症提供更可靠的实验依据。