Cav1.2介导的CaM/CaMKⅡ钙信号通路在PMS肝气郁证中的作用及舒郁胶囊干预机制研究

Premenstrual syndrome（PMS）is a typical TCM emotional disease，liver-qi stagnation is its major syndrome，abnormal liver catharsis is its pathogenesis but the specific mechanism needs to study. Calcium regulation disorder may be one of the pathogenesis of PMS, research showed that voltage - gated calcium channels（Cav）is important channel to regulation the intracellular Ca2+ concentration. Ca2+ binding to calmodulin（CaM）can activate the calmodulin dependent protein kinase ⅡCaMK Ⅱ）, and its substrates involved in synthesis and release of 5-HT, DA and Glu, which are closely related to the pathogenesis of PMS. We have found increased protein expression level of Cav1.2 subtypeα1C subunit（CACNA1C）in hippocampus of liver-qi stagnation of PMS model rats , the regulating liver formula Shu Yu capsule can correct this unusual expression level. The CACNA1C gene has been show is one of the candidate susceptibility genes of affective disorders. So these suggested that Cav1.2 mediated CaM/CaMk calium signal pathway may play an important role in the pathogenesis of PMS liver-qi stagnation. But lack of correlative research information until now. Based on our previous researches, it is the effective way to elucidate the essence of liver in taking charge of dispersion and regulating emotion by revealing mechanism of CaM/CaMKⅡ calium signal pathway mediated by Cav1.2 in the development of PMS liver-qi stagnation and ShuYu Capsule intervention. PMS liver-qi stagnation rat model, patch clam, fluorescence labeling to measure intracellular calcium concentration and microdialysis etc technologies were used to validate this speculation and TCM theory.

经前期综合征（PMS）是典型中医情志病证，肝气郁证为主要证型，肝疏泄失常为其关键病机，具体机制有待深入探索。已有研究显示Ca2+调节紊乱与PMS密切相关。电压门控钙通道（Cav）是细胞内Ca2+浓度调节的重要通道，Ca2+与CaM结合可激活CaMKⅡ，其作用底物参与调控与PMS发生有关的5-HT、DA和Glu等神经递质的合成和释放。我们前期研究发现PMS肝气郁证大鼠海马Cav1.2亚型α1C亚基CACNA1C蛋白表达增加，调肝方药舒郁胶囊可纠正，而CACNA1C是多种精神障碍性疾病的易感基因，提示Cav1.2介导的CaM/CaMKⅡ钙通路在PMS肝气郁证中发挥重要作用，国内外未见报道。本课题拟利用病证结合大鼠模型，借助膜片钳、荧光标记测钙和微透析等技术，探索Cav1.2介导的CaM/CaMKⅡ钙通路与PMS肝气郁证的关系及舒郁胶囊的干预机制。为揭示“肝主疏泄调畅情志”本质提供新的科学依据。

经前期综合征（PMS）病因病机复杂，钙离子调节紊乱可能是PMS发病机制之一。L型钙通道（LTCC）是细胞内Ca2+浓度调节的重要通道，Ca2+与CaM结合可激活CaMKⅡ，其作用底物包括TH、TPH、SYPⅠ等，参与5-HT、DA和Glu等神经递质的合成和释放，而这些神经递质与PMS发病机制密切相关。本课题在前期研究的基础上推测Cav1.2介导的CaM/CaMKⅡ钙信号通路可能与PMS的发生相关。因此本项目借助PMS肝气郁证大鼠模型，利用膜片钳、荧光标记测钙、免疫荧光、免疫印迹和微透析等技术研究了Cav1.2介导的CaM/CaMKⅡ钙通路与PMS肝气郁证的关系及舒郁胶囊的干预机制。首先复制了PMS 肝气郁证病症结合动物模型，利用宏观行为学、分子生物学技术明确了Cav1.2 介导的CaM/CaMKⅡ钙通路异常活化与PMS肝气郁证的发生有关，舒郁胶囊可通过纠正该通路关键基因的表达异常发挥其治疗作用。其次，结合中药血清药理学和相关的电生理技术，揭示了舒郁胶囊及其主要有效成分单体芍药苷可抑制Cav1.2 介导的CaM/CaMKⅡ钙信号通路发挥对神经元的保护作用。最后本项目利用L型钙通道工具药Bayk8644和尼莫地平，通过体内外两个层次的研究揭示了芍药苷可通过Cav1.2钙通路抑制细胞外液GABA浓度产生抗焦虑抑郁作用。通过上述多层次、系统深入的研究对本课题的假说进行了阐释，完成了本项目的研究目标。为明确PMS肝气郁证的脑中枢作用机制和舒郁胶囊作用靶点提供了新的科学依据。同时为研制PMS肝气郁证新药和临床治疗提供了实验基础和新的治疗靶标。