健脾止动汤对TS共患焦虑大鼠CSTC通路兴奋/抑制性神经递质失衡的调节机制研究

Anxiety is a common comorbidity of Tourette syndrome. Anxiety is related to the severity of tics and often causes more damage than TS itself. For TS and anxiety, One of the main pathogenesis is thought to be an imbalance in neurotransmitters located in CSTC circuit. Clinical practice found that Jian-Pi-Zhi-Dong Decoction (JPZDD) could inhibit not only the tics of children but also the symptoms of anxiety. We hypothesized that JPZDD involved in regulating imbalance of 5-HT , glutamate and γ-aminobutyric acid and their receptors expression in striatum of comorbidity of TS and anxiety rats model. In this project, our research group will build up comorbidity of TS and anxiety rats model by intraperitoneal injecting IDPN combined with empty bottle stimulation, and detect the behavioral tests including stereotyped movement, open field test, high plus maze tests. And we will detect the morphological changes of the cortex , the striatum and the hypothalamus. Then 5-HT、Glu and GABA content and the ratio change in striatum of model rat were detected by the means of microdialysis with high-performance liquid chromatogram –electrochemical detection. At last the expression of 5-HT receptor, Glu receptor and GABA receptor in striatum of comorbidity of TS and anxiety will be examined by immunohistochemical and RT-PCR. In this project, we want to explore the mechanisms of JPZDD regulating imbalance of neurotransmitters from protein and molecular level and reveal the scientific connotation of treating different diseases with same method, providing new method and new supports for further applications in treating comorbidity of TS and anxiety.

焦虑是多发性抽动症（TS）常见的共患病，与抽动的严重程度呈正相关，常造成比TS本身更严重的损害。CSTC回路中神经递质失衡是TS和焦虑共同的发病机制。临床实践表明，健脾止动汤可有效抑制患儿抽动行为并改善焦虑症状，课题组提出该方通过调节纹状体5-HT、Glu、GABA受体的表达，恢复神经递质的平衡而达到治疗效果的科研假说。本项目拟以亚氨基二丙腈（IDPN）腹腔注射联合空瓶刺激法建立TS共患焦虑大鼠模型，采用刻板运动、旷场实验和高架十字迷宫进行行为学测试；电镜观察大鼠皮质、纹状体、下丘脑的形态学改变；活体微透析联合高效液相检测纹状体内5-HT、Glu、GABA含量及其比值变化；采用免疫组化、RT-PCR技术检测纹状体内5-HT、Glu及GABA受体的表达。本项目拟从蛋白、分子水平探索健脾止动汤对脑内神经递质的动态平衡调节作用，诠释异病同治的内在机理，提出和发展治疗TS共患焦虑的新思路和新途径。

TS共患焦虑的发生不仅体现在遗传心理因素等方面，还受到来自CSTC回路中神经递质的影响。其中5-HT、Glu及GABA是调节兴奋性/抑制性信号的主导，同时也是维持整个皮层运动区及生物运动兴奋性强弱的基础。课题组提出TS共患焦虑可能与脑内神经递质5-HT、Glu、GABA的失衡有关。健脾止动汤通过调节纹状体5-HT、Glu、GABA受体的表达，恢复神经递质的平衡而达到治疗效果的科研假说。针对以上假说，我们进行了深入的探索和研究，并探寻健脾止动汤抗抽动、抗焦虑的作用靶点和机制。. 首先，选用IDPN腹腔注射250mg/（kg•d）联合不确定空瓶刺激建立TS共患焦虑大鼠模型。随后，通过刻板运动、旷场实验和高架十字迷宫对该模型的表面效度进行了检验；采用高效液相法（HPLC）检测了海马和纹状体两个部位神经递质含量情况，来验证该模型的结构效度，结果显示，IDPN腹腔注射联合不确定空瓶刺激的模型，有着较理想的表面效度和结构效度。. 随后课题组在该模型的基础上进行了后续的系列研究。行为学检测结果显示，健脾止动汤能减轻大鼠的刻板运动评分，提高旷场实验总分，延长开放臂停留时间，具有较好的抗抽动、抗焦虑效果。采用光镜、电镜对大鼠的皮质、纹状体进行了形态学观察，光镜下各组间差异不明显，电镜结果发现模型组存在形态学的部分改变，主要表现为双层核膜的断裂、线粒体肿胀、排列紊乱等，而健脾止动汤可以修复部分受损的机能。血清Elisa结果显示，模型组兴奋性/抑制性神经递质失衡，表现为GABA、5-HT含量下降，Glu含量上升，经中药治疗后，可使神经递质趋于平衡。而在对相应受体的蛋白、基因表达检测中，我们发现健脾止动汤主要通过抑制皮质、纹状体内5-HT2CmRNA的表达，增加皮质、纹状体GABAA受体、NMDA受体的表达，进而改善5-HT含量相对不足，使5-HT、GABA系统兴奋性进一步增强，从而发挥积极的抗抽动、抗焦虑效应。. 本项目主要实验内容已完成，实验结论将陆续发表于国际及国内各大核心期刊。