基于MiR-133b/PINK1/ERK反馈环路调控酪氨酸羟化酶探索疏肝理脾方对TD的干预机制

Dopamine (DA) overexpression is an important pathological mechanism of tic disorder (TD), so DA production inhibition is the key to treating TD. Basic research indicates that miR-133b can negatively regulate DA production. Its mechanism is related to inhibition of target gene PINK1 expression, regulation target pathway Ras/Raf/MEK/ERK transduction, and ultimately reduction DA rate-limiting enzyme tyrosine hydroxylase (TH). Previous studies have shown that Shugan Lipi Prescription can improve tic symptoms, nutritional status and constitution of TD children. Therefore, we put forward the hypothesis: Does Shugan Lipi Prescription regulate the Ras/Raf/MEK/ERK transduction by affecting miR-133b, inhibiting TH phosphorylation and gene expression, ultimately reduce DA production? Based on this, this study aims to carry out in vivo experiments and use miR-133b gene overexpression and silencing techniques to carry out in vitro experiment, use RTCA instrument and confocal microscope to observe the cell growth, structure and protein, use ELISA, Western-blot and qPCR technique to analyze the difference expression of Ras, Raf-1, MEK1/2, ERK1/2, p-CREB, p-TH and its mRNA and miR-133b, PINK1. By this study, we can explore the mechanism of Shugan Lipi Prescription in inhibition of DA generation, and ultimately provide objective basis for the treatments of TD.

多巴胺（DA）过表达是导致抽动障碍（TD）的重要病理机制，故抑制DA生成是治疗TD关键。MiR-133b可负反馈调节DA，其机制与抑制靶基因PINK1表达、进而调节靶通路Ras/Raf/MEK/ERK转导、最终降低DA限速酶酪氨酸羟化酶（TH）相关。前期研究表明疏肝理脾方可改善患儿抽动症状、营养状况及体质。故我们提出假说：疏肝理脾方是否通过影响miR-133b来调控Ras/Raf/MEK/ERK转导，抑制TH磷酸化及基因表达，降低DA生成？基于此，本研究拟通过体内实验和miR-133b过表达及沉默技术开展体外实验；利用RTCA仪、共聚焦显微镜观察细胞生长及结构、蛋白等，WB、qPCR等检测Ras、Raf-1、p-MEK、p-ERK及下游p-CREB、p-TH蛋白及其基因和上游miR-133b、PINK1表达；探索疏肝理脾方抑制DA生成的作用机制，最终为该方治疗TD提供客观依据。

项目背景：多巴胺（DA）过表达是导致抽动障碍的重要病理机制，故抑制DA生成是治疗TD关键。MiR-133b可负反馈调节DA，其机制与抑制靶基因PINK1表达、进而调节靶通路Ras/Raf/MEK/ERK转导、最终降低DA限速酶酪氨酸羟化酶（TH）相关。故我们提出假说：疏肝理脾方是否通过影响miR-133b来调控Ras/Raf/MEK/ERK转导，抑制TH磷酸化及基因表达，降低DA生成？.研究内容：拟通过体内实验和miR-133b过表达及沉默技术开展体外实验，主要开展两个研究，①疏肝理脾方通过调控miR-133b/PINK1/ERK反馈环路抑制TH磷酸化及mRNA表达干预抽动障碍的机制研究，②疏肝理脾方对miR-133b过表达和沉默的SH-SY5Y细胞中Ras/Raf/MEK/ERK通路的干预机制。.重要结果、关键数据：动物实验表明：抽动障碍可导致体重抑制，并出现行为学改变。泰必利及中药各剂量干预后体重抑制及行为学改变可缓解，中药高剂量组最为明显，并随药物口服时间延长，抽动症状及体重抑制优势更突出。模型制备后，大鼠Ras、Raf-1、MEK、ERK、CREB、TH、PINK1 蛋白及mRNA表达增高，泰必利及中药各剂量可下调表达，中药高剂量组最明显；miR-133b蛋白及 mRNA表达降低，泰必利及中药各剂量可上调表达，中药高剂量组最明显。细胞实验表明：EGF最佳作用浓度为10ng/ml，EGF最佳培养时间为12h，空白组含药血清最佳作用浓度5%，中药和泰必利含药血清最佳作用浓度均为10%，10%中药和10%泰必利含药血清最佳培养时间为均24h；疏肝理脾方和泰必利对下调Ras、Raf-1、MEK、ERK、CREB、TH、PINK1 mRNA表达，以及上调miR-133b mRNA表达均有效，其中中药更明显。.科学意义：以Ras/Raf/MEK/ERK信号转导过程中关键因子为主要切入点，初步阐明疏肝理脾方可通过调控miR-133b/PINK1/ERK反馈环路抑制大鼠CSTC回路纹状体区DA限速酶TH磷酸化及mRNA表达，从而达到治疗TD作用，进一步验证抽动障碍“脾虚肝亢、风痰内扰”的发病理论，为疏肝理脾方治疗TD的临床应用提供客观依据。