健脾止动汤抑制TS大鼠纹状体cAMP/PKA系统介导DARPP-32(Thr-34)磷酸化的机制研究

One of the main pathogenesis is thought to be an imbalance in neurotransmitters located in CSTC loop in Tourette syndrome (TS). Preliminary studies on monoamines and amino acids have shown that abnormal expressions of these neurotransmitters accompanied with their receptors promote the direct pathway mediately, which further induce the occurrence of stereotyped behavior in TS models. Considering the specific neuropeptides and enzyme systems in the direct pathway provide the material basis for the facilitatory effect, we put forward the hypothesis that Jianpizhidong Decoction involves in regulating the phosphorylation of DARPP-32 mediated by the cAMP/PKA System in the striatum of TS models. In this project, we will build up TS model rats by intraperitoneal injecting IDPN, detect the contents of cAMP, PKA, DARPP-32 and DARPP-32 (Thr-34) in the striatum by means of ELISA and Western blot; test expressions of pDYNmRNA and DARPP-32mRNA applying in situ hybridization and RT-PCR. We are aiming to explore the suppression mechanisms of Jianpizhidong Decoction related to overpression of pDYN and the phosphorylation of DARPP-32 (Thr-34) mediated by the cAMP/PKA System, provide experimental and theoretical supports for further applications in clinic.

皮质-纹状体-丘脑-皮质（CSTC）回路神经递质失衡是多发性抽动症（TS）的主要病机，而CSTC中直接通路易化作用增强扮演十分重要的角色。前期对单胺及氨基酸类递质的研究发现，其表达异常可间接兴奋直接通路，诱导TS动物模型刻板行为发生。基于该通路中特异性神经肽和酶系为易化作用提供物质基础，本课题提出健脾止动汤抗抽动机制与抑制纹状体cAMP/PKA系统中DARPP-32（Thr-34）过磷酸化有关。综上，拟以IDPN建立TS大鼠模型；采用ELISA、Western blot检测大鼠纹状体cAMP、PKA、DARPP-32和DARPP-32（Thr-34）含量；原位杂交、RT-PCR检测pDYNmRNA、DARPP-32mRNA表达。从行为学、蛋白、核酸水平探讨健脾止动汤对纹状体pDYN过表达及cAMP/PKA系统介导DARPP-32（Thr-34）位点磷酸化的阻抑机制，为临床应用提供实验依据。

皮质-纹状体-丘脑-皮质（CSTC）回路神经递质失衡是多发性抽动症（TS）的主要病机，其回路中直接通路易化作用增强扮演十分重要的角色。基于该通路中特异性神经肽和酶系为易化作用提供物质基础，我们针对以上假说进行了比较深入的研究，并重点研究cAMP/PKA系统中DARPP-32（Thr-34）过磷酸化与动物刻板行为发生的联系。首先，我们对TS大鼠纹状体cAMP/PKA系统中AC、cAMP、PKA蛋白表达进行了检测。结果显示模型组这三种蛋白含量较空白组显著升高，而给药后三种蛋白含量均有所下降，表明直接通路易化作用增强与此三种蛋白含量高表达有关。其次，针对纹状体强啡肽（Dyn）及其前体物质强啡肽原（PDyn）的研究，我们发现模型组纹状体Dyn与PDyn mRNA含量显著增加，中西药组干预后，二者含量呈下降趋势，表明健脾止动汤对纹状体Dyn过表达具有抑制作用。再次，对DARPP-32蛋白表达研究中，我们发现健脾止动汤干预靶点集中在该蛋白上Thr-34位点，药物对该位点蛋白的抑制作用较为突出。归纳起来看，健脾止动汤对直接通路易化作用的干预作用主要体现在以下三点，即：（1）抑制AC、cAMP、PKA蛋白表达；（2）抑制Dyn与PDyn mRNA表达；（3）抑制DARPP-32蛋白中Thr-34位点磷酸化。本项目主要实验内容基本完成，实验结论陆续发表于国内各大核心期刊。我们还将继续对TS的发病机制以及健脾止动汤的药效机理做更进一步研究。