基于 miRNA-134 探讨强志组方干预模型大鼠 TS 复合恐惧行为障碍的 cAMP-PKA 效应靶点研究

The treatment of Tourette's syndrome is stubborn and often accompanied by a variety of behavioral disorders. It is urgent to find a safe and effective treatment method. Based on the "kidney deficiency" theory will be a variety of behavior change strong Zhi prescription "same treatment for different diseases", has verified the clinical curative effect of multi direction. Research has confirmed that tyrosine hydroxylase regulation of dopamine synthesis, pre experiment found strong prescription intervention the expression of tyrosine hydroxylase mRNA records, but its effect and precise target pathway is unclear, according to the study of cAMP-PKA signaling pathway plays a key role in the frontier theory affected the DA synthesis rate limiting enzyme of TH phosphorylation balance process, through the establishment of Tourette syndrome the model in multiple behavioral disorders in rats, observe the strong function of model rats before and after prescription records THmRNA, cAMP-PKA signal molecule expression changes, simultaneous observation of the efficacy and safety of tic and comorbid behavioral disorders, strong Zhi cAMP-PKA signaling pathway induced by prescription, DA expression of the rate limiting enzyme of TH synthesis, thereby regulating the function and the molecular mechanism of TS and its composite behavior disorder. To provide new targets and approaches for the study of five drugs for the treatment of mental and behavioral disorders.

多发性抽动症的治疗难点是抽动危害严重且伴发多种行为障碍，临床急需发掘同时兼及抽动和行为异常的安全、高效治疗方法和制剂。基于“肾志不足”理论的强志组方将多种行为改变“异病同治”，临床疗效已验证。研究热点证实酪氨酸羟化酶调控多巴胺合成 ，预实验发现强志组方通过调控脑区DA/TH比值，有效改善了TS模型大鼠及其复合的恐惧行为障碍，但其效用通路和精准靶点尚不清楚，本研究根据cAMP-PKA信号通路在影响DA合成限速酶TH磷酸化平衡过程中发挥关键作用前沿理论，通过建立多发性抽动症复合恐惧行为障碍大鼠模型，从整体、细胞、分子水平，观察强志组方作用前后模型大鼠纹状体DA、TH、THmRNA、cAMP-PKA、miR-134、CREB信号表达变化，明确强志组方影响DA/TH比值，调控TS复合行为障碍的效应靶点与作用机制，丰富中医学“肾藏志”与“肾志不足”证治理论，为治疗提供新的干预途径和有效制剂。

Tourette综合征（TS）是儿童时期常见的精神行为疾病。目前，发病机制未明，临床常合并强迫、恐惧等精神障碍，治疗难度大，为探究cAMP-PKA信号通路在影响DA合成限速酶TH磷酸化平衡的理论，本项目做了以下工作：.一、.构建抽动复合恐惧模型：亚氨基二丙腈（IDPN）诱导产生TS模型大鼠，造模后采用声光电刺激，诱导建立抽动复合恐惧模型。.二、.观察模型大鼠动作行为的改变及中药的作用影响。抽动复合恐惧模型大鼠诱导成功后，予强志组方干预，采用旷场行为学观察大鼠行为改变。结果显示：与模型组相比，强志组方干预后可有效减少模型大鼠的冻结时间和刻板行为次数，验证了强志组方治疗抽动合并恐惧的疗效。.三、.检测miR-134基因表达及中药的影响。抽动复合恐惧大鼠模型诱导后，检测各组miR-134在海马体、纹状体的表达，结果显示：强志组方可有效降低miR-134在纹状体表达，提高miR-134在海马体的表达，验证了miR-134在抽动复合恐惧中的作用机理。.四、.检测cAMP-PKA信号通路的相关蛋白的表达水平及中药的影响。模型大鼠取海马体、纹状体，采用Western Blot、酶联免疫法、PCR法检测各组cAMP-PKA通路蛋白表达水平，结果显示：强志组方可调控Th基因mRNA表达升高，调节DA在海马体中的表达增高，DA/TH含量趋于一致，进一步说明强志组方调控cAMP-PKA信号通路，其作用机制并非单一升高或降低DA与TH的水平，也非单一体现在海马体或纹状体，而是通过干预THmRNA的表达，使得同一脑组织的TH与DA水平相一致，从而起到改善刻板行为的作用。.结论：.1.实现多发性抽动复合行为障碍大鼠模型的构建，强志组方实现了“异病同治”、“一方多能”的干预机制。.2.强志组方可通过cAMP-PKA信号通路影响DA合成限速酶TH磷酸化平衡，影响多巴胺合成限速酶酪氨酸氢化酶的表达，调节DA/TH比值，改善了抽动、恐惧行为，明确作用机制和效应靶点。