静宁方调控ADHD多巴胺D1、D2类受体拮抗作用及其下游cAMP/PKA、Ca2+/CaMKⅡ信号通路的机制研究

Attention Deficit Hyperactivity Disorder (ADHD) is a common and often persistent neurodevelopmental disorder. Recently the abnormal DA homeostasis is as the primary biochemical defect underlying the main hypothesis of the pathogenesis of ADHD. Clinical studies have found that JingNing formula can effectively alleviate the symptoms of hyperactivity. While, the mechanism that abnormity of DA synthesis ,intake and inactivation during the upstream path of DA cannot well explain the pathogenesis of ADHD. Based on dopamine D1, D2 receptors working against each other in the regulation of the downstream pathways, combining with the preliminary basis, we put forward the hypothesis that mechanism of ADHD symptoms improved by JingNing formula dues to regulating the percentage of D1 / D2 receptors. Considering that imbalance of DA receptors which resulting in suppressing of PKA, CaMKⅡbiological effects may lead to ADHD. This project intends to select SHR rats model, by using the HPLC method to detect the contents of DA in striatum of model rat; Western Blot, RT-PCR are used to detect contents of DA receptor and IP3, PKA, CaMKⅡexpression, Elisa method to detect expression of cAMP, Flow cytometry testing striatal cells Ca2+concentration. We are aiming to explore the mechanisms of JingNing formula regulating D1 / D2 receptors imbalance, as well as AC/cAMP/PKA, Ca2 + / CaM - PK signal pathways from multiple perspectives and methods. We plan to explore that JingNing formula, based on the theory of "balance of Yin and Yang", using the method of reinforcing qi and nourishing yin to treat ADHD, to further study the mechanism and the scientific connotation of traditional Chinese medicine in the treatment of ADHD.

前期研究发现，注意力缺陷多动障碍（ADHD）发病机制中多巴胺（DA）上游信号通路中DA合成、重摄取、灭活途径的异常，不能完全阐释本病发病机制。本研究基于D1、D2受体家族在调控下游信号通路中相互拮抗的作用机制，结合前期基础，认为静宁方改善ADHD症状可能与调节DA受体比例密切相关；故提出DA受体比例失衡进而抑制其下游信号通路中蛋白激酶PKA、CaMKⅡ生物效应，是导致ADHD发病原因的假说；拟选取SHR大鼠，用高效液相法检测其纹状体DA含量，Western Blot、RT-PCR技术检测D1、D2受体家族及IP3、PKA、CaMKⅡ的表达，Elisa法检测cAMP表达，流式细胞仪检测纹状体细胞Ca2+浓度；从多角度探讨静宁方对ADHD模型大鼠D1、D2受体家族及AC/cAMP/PKA、Ca2+/CaM-PK信号通路的调控机制，揭示静宁方基于“阴阳平衡”理论治疗ADHD的科学内涵。

注意缺陷多动障碍（ADHD）是一种儿童时期常见的以与发育水平不相符的注意缺陷、多动和冲动为主要临床表现的神经发育障碍，本病发病率高、共患病多、危害大，是全球公共卫生领域研究的热点问题。多巴胺的合成、分泌、再摄取和灭活等多巴胺上游信号通路的异常并不能阐明本病的发病机制。现代研究表明，多巴胺受体分为D1受体家族和D2受体家族，两类受体相互拮抗，共同调控下游信号通路，影响蛋白激酶如PKA和CaMKⅡ的生物活性，多巴胺D1、D2类受体比例失常与ADHD的发生发展密切相关。静宁方是经多年临床应用的专利处方，可以有效改善ADHD核心症状。我们推测静宁方改善ADHD症状可能与调节多巴胺受体比例密切相关，故提出多巴胺受体比例失衡进而抑制其下游信号通路中蛋白激酶PKA、CaMKⅡ生物效应，是导致ADHD发病原因的假说，采用自发性高血压大鼠作为ADHD动物模型，以不同剂量的静宁方进行干预，用哌甲酯作为阳性对照药物，通过高效液相、Western Blot、PCR、流式细胞技术等方法检测各组大鼠纹状体中多巴胺含量，多巴胺D1、D2类受体的表达情况及不同干预措施对多巴胺下游信号通路cAMP/PKA和Ca2+/CaMKⅡ的影响，揭示静宁方基于“阴阳平衡”理论治疗ADHD的科学内涵。经研究发现：（1）ADHD大鼠纹状体多巴胺水平降低，多巴胺D1、D2类受体水平均降低，D2/D1显著降低，纹状体多巴胺受体比值D2/D1下降与ADHD的发生、发展密切相关。（2）静宁方能够调节纹状体的多巴胺D1、D2类受体平衡，降低多巴胺下游信号通路cAMP/PKA和Ca2+/CaMKⅡ的兴奋性，从而改善ADHD症状。研究结果验证了我们提出的假说，为阐明ADHD的发病机制及相关途径，明确静宁方治疗ADHD的药效机制提供了新的科学依据。