帕金森病阴虚动风证大鼠黑质-纹状体中Wnt信号通路的动态演变及复方地黄颗粒的干预效应

Parkinson's disease （PD）belong to Chanzheng category by traditional Chinese medicine,endogenous wind syndrome caused by deficiency of YIN is the main syndrome type for PD,but the biological basis of the syndrome is not clear.In recent years, the study found that Wnt signaling pathway is closely related with PD.We assume that is expected to explore biological basis of PD syndrome with endogenous wind syndrome caused by deficiency of YIN with the help of network control mechanism in Wnt signal pathway.Our previous study have shown that compound rehmannia granule can obviously alleviate the symptom of PD, and its mechanism might be related to regulation of Wnt signaling pathway. Therefore, we believe that the regulatory network of Wnt signaling pathway may be the biological basis of PD with endogenous wind syndrome caused by deficiency of YIN and the mechanism by compound rehmannia Granule prevent and treat of PD. Based on this, this project intends to use molecular biology and pathology of experimental methodsto carry out the following research:（1）With the establishment of animal models of disease and syndrome,with the biological effect index in the regulation of Wnt signaling pathway,Exploration of biological basis of PD syndrome of endogenous wind syndrome caused by deficiency of YIN; (2)Follow the scientific concept of diseases, syndromes, prescriptions, from the angle of Wnt signal pathway regulation,to reveal the mechanism of compound rehmannia granule in the treatment of PD. We hope that through this research project, to provide a new perspective for the interpretation of the scientific connotation of TCM syndrome, to provide support for innovation and research of Chinese medicine in the treatment of PD.

帕金森病（PD）属中医“颤证”范畴，阴虚动风证是其发病的主要证型，但该证型的生物学基础尚不清楚。近年来研究发现，Wnt信号通路与PD密切相关。我们设想 借助Wnt信号通路的网络化调控机制可望探讨PD阴虚动风证的生物学基础。课题组前期研究表明复方地黄颗粒对PD有明显的缓解作用，其机制与Wnt信号通路的调控有关。由此，我们认为Wnt信号通路调控网络可能是PD阴虚动风证的生物学基础及复方地黄颗粒防治PD的机制所在。基于此，本项目拟采用分子生物学及病理形态学等实验方法，开展以下研究：（1）建立病证结合动物模型，围绕Wnt信号通路调控相关的生物效应指标，探索PD阴虚动风证的证候生物学基础；（2）遵循病-证-方对应的科学理念，从Wnt信号通路调控环节的角度，全面、动态揭示复方地黄颗粒治疗PD的作用机制。我们希望通过本项目的研究，为阐释中医证候的科学内涵提供新视角，为治疗PD的创新中药研究提供支撑。

帕金森病（PD）属中医“颤证”范畴，阴虚动风证是其发病的主要证型，但该证型的生物学基础尚不清楚。近年来研究发现，Wnt信号通路与PD密切相关。基于此，本项目开展了以下研究：（1）制备阴虚动风证PD 6-OHDA大鼠模型，将造模成功的55只大鼠随机分为模型组、美多芭组、复方地黄颗粒组(低、中、高剂量)。另取不造模大鼠11只为正常对照组，只注射抗坏血酸大鼠为假手术组。美多芭组给予美多芭150mg/kg，复方地黄颗粒低、中、高剂量组分别给颗粒剂1.75g/kg、3.5g/kg、7g/kg灌胃。正常对照组、假手术组和模型组以等体积蒸馏水2ml/只灌胃，每日1次，连续用药4周。观察大鼠神经行为学变化，分别采用RT-PCR法、Western blot法、免疫组化法检测Wnt1、LRP6、β-catenin、Fzd1、GSK3β的mRNA、蛋白及阳性细胞在损毁侧纹状体的表达。（2）造模方法及动物分组同上。基于前期研究，复方地黄颗粒低、中、高剂量组分别调整为3.5g/kg、7g/kg、14g/kg灌胃。其余组灌胃同上，每日1次，连续用药6周。分别采用RT-PCR法、Western blot法检测NGF、BDNF、GDNF、TrkA、TrkB、GFR-α1、Ret的mRNA、蛋白在损毁侧纹状体的表达，TH mRNA、蛋白在损毁侧黑质及纹状体的表达, 化学比色法检测MDA、GSH、GSH-Px、SOD在损毁侧黑质的含量。结果：（1）与正常对照组比较，6-OHDA模型组大鼠出现了旋转行为（P<0.01），Wntl、LRP6、β-catenin、Fzd1的mRNA、蛋白及阳性细胞表达降低（P<0.01），GSK3β的mRNA、蛋白及阳性细胞表达升高（P<0.05）。美多芭组和复方地黄颗粒高剂量组改善了模型组的旋转行为及相关指标的表达（P<0.05）。（2）与正常对照组比较，6-OHDA模型组大鼠NGF、BDNF、GDNF、TrkA、TrkB、GFR-α1、Ret、TH的mRNA、蛋白表达降低（P<0.01），氧化应激指标异常（P<0.001）。美多芭组和复方地黄颗粒中剂量组改善了模型组相关指标的表达（P<0.01）。科学意义：Wnt信号通路的改变是6-OHDA诱导的PD阴虚动风证大鼠模型的生物学基础，也是复方地黄颗粒保护DA能神经元的作用靶点。