钩吻素子抗焦虑作用及其与脑内TSPO-神经甾体-GABA(A)受体通路关联机制

It has been urged to develop fast-acting, effective and safe anxiolytic drugs to improve the commonly prescribed antianxiety agents, which exist defects on side effects or acting promptly acting. A promising anxiolytic drug is supposed to be capable of affecting GABA(A) receptor through mediating neurosteroids production by activating TSPO in recent researches. Our previous study potentially suggested that the mechanism of koumine with anxiolytic activity might be connected to TSPO mediating pathway. In the present project, the anxiolytic properties of koumine will be evaluated through two anxiety animal models including social interaction test in mouse and CCK-4 induced panic-like responses model in rat. Further, the connection between koumine and TSPO-neurosteroids-GABA(A) receptor pathway in brain will be explored and analysed through isotope-labelling method which measures the affinity of TSPO with koumine, HPLC-MS measurement which detects the neurosteroids level in brain, whole-cell patch clamp techniques which tests the activity of GABA(A) receptor, and receptors blocking or inhibiting effect in an anxiety animal model which associates the mechanism mentioned above with anxiolytic effect of koumine. After that, the expectation of this project is to confirm the anxiolytic effect of koumine, elucidate its neural malecular pathway, and further understand the mechanism of anxiety disorder in neuroscience.

安全有效的新型抗焦虑药亟待研究。我们前期工作提示，钩吻素子可能具有抗焦虑作用，其机制可能与脑内TSPO介导的通路有关。本项目首先在小鼠社会交往试验和大鼠胆囊收缩素-4诱导模型上，进一步明确钩吻素子抗焦虑作用及其特征。在此基础上，以放射受体分析法分析钩吻素子与脑内TSPO的亲和力，并通过行为药理学效应的拮抗作用，推断钩吻素子抗焦虑作用与TSPO的关系；进而以HPLC-MS测定脑内神经甾体的含量，也通过行为药理学效应的拮抗作用，推断钩吻素子抗焦虑作用与TSPO调控神经甾体的关系；最后通过膜片钳全细胞纪录技术以及行为药理学效应的拮抗作用，分析钩吻素子抗焦虑作用与TSPO以及神经甾体调控GABAA受体激动作用的关系，以期探明钩吻素子抗焦虑作用与脑内TSPO-神经甾体-GABAA受体通路的关联机制。本研究将提出钩吻素子抗焦虑作用的可能新功效及其作用途径，并有助于深化理解焦虑症的神经生物学机制。

钩吻素子是一种潜在的新型抗焦虑药物，其抗焦虑效应及其作用机制亟待进一步的研究。本项目通过小鼠功能观察组合试验、小鼠旷场试验、大鼠Vogel饮水冲突试验、大鼠高架十字迷宫试验，以及孤立大鼠模型和天敌声音诱导大鼠焦虑症模型，进一步确认钩吻素子的抗焦虑作用；并在此基础上探讨其作用机制。结果显示，钩吻素子在以上多个动物模型中均有显著的抗焦虑疗效。荧光免疫组化结果显示钩吻素子的抗焦虑作用与大鼠海马的TSPO[translocator protein (18 kDa)]表达无关；放射受体分析法提示钩吻素子可能作用于TSPO变构调节位点；HPLC-MS和酶联免疫吸附法（ELISA）对多个焦虑模型的检测结果提示，钩吻素子的抗焦虑作用与增加大鼠海马、前额叶皮质和杏仁核的神经甾体（别孕烯醇酮和孕酮）的含量有关，但钩吻素子对大鼠海马区的γ-氨基丁酸（GABA）水平未见显著变化；此外，钩吻素子能抑制天敌声音诱导焦虑大鼠的血浆中异常升高的促肾上腺皮质激素（ACTH）和皮质酮水平。以上结果证明，钩吻素子可能通过脑内的TSPO，调控别孕烯醇酮和孕酮等神经甾体的含量，并进一步调节下丘脑-垂体-肾上腺轴（HPA）轴的异常活动而产生抗焦虑作用，提示钩吻素子的抗焦虑作用与脑内TSPO-神经甾体-GABA(A)受体-HPA轴通路密切相关。本研究为抗焦虑新药的研发提供新的药物作用靶标与作用途径，并有助于深化理解焦虑的分子神经生物学机制。