补益肝肾法干预帕金森病多巴胺能通路的PKC信号转导机制

Parkinson's Disease (PD) is one of the three major disabled disease in the aged. Its pathogenesis has not been elucidated, and there is no effective therapy to PD. The incidence of PD is increasing year by year. PD has greatly burdened the patients, their families and the whole society. The previous research of the applicants has confirmed that the medicine of nourishing liver and kidney could regulate the metabolite level of dopamine (DA) and the activity of TH in the rats' corpus striatum. Other research has reported that the phosphorylation of Protein Kinasee C (PKC) may be an important pathogenic site of the DA pathway neurons' apoptosis of PD. But the apoptosis mechanism of the PKC signal pathway is still unclear, And whether there are new medicines which could act on PKC signal pathway are also unknown. This project will aim at the PKC signal pathway. The experimental animals will be given medicine of nourishing liver and kidney, in vitro and in vivo experiments will beundergone using the technologies of microscopic imaging, immunohistochemistry and molecular biology. The possible mechanisms of PKC signal pathway in the DA pathway neurons' apoptosis will be discussed underlain by the DA neurons' morphology and characters, the gene-related analysis of PCKδ, PKCε, ERK1 /2, NF-κB and Cyt c. The mechanism of the medicine of nourishing liver and kidney on PD will be explored. The results of this project may be provide objective basis for broadening the thinking of PD's treatment. This project may prompt the process of themodernization of Chinese medicine theorectically and practically.

帕金森病（PD）是目前中老年人三大致残疾病之一，其发病机制尚未阐明，临床尚无特效疗法，发病率却逐年上升，给个人、家庭和社会造成极大负担。申请者在前期基金项目支持下，已证实补益肝肾中药可调节纹状体多巴胺（DA）代谢水平及TH活性。研究发现，蛋白激酶 C（PKC）的磷酸化激活可能是PD多巴胺能通路DA神经元调亡的一个重要致病位点，但是PKC信号通路调控凋亡的机制未明，此信号通路是否存在新的药物治疗途径和靶点未知。本课题以PKC信号通路为切入点，运用补益肝肾中药，采用显微成像、免疫组化、分子生物学等技术，进行体外及整体动物实验，从DA神经元形态性状与PCKδ、PKCε、ERK1/2、NF-κB、Cyt c等基因相关性分析等方面，探讨PKC信号通路在DA神经元细胞凋亡过程中的可能机制，研究补益肝肾中药对其的作用机理，为拓宽PD治疗思路提供客观依据，对于推动中药现代化有着理论和现实的双重价值。

针对帕金森病（PD）这一中老年人三大致残疾病之一，本研究设立蛋白激酶 C（PKC）的磷酸化激活可能是PD多巴胺能通路DA神经元调亡的一个重要致病位点，补益肝肾中药通过调控PKC信号通路起到治疗作用的假说，并设计研究技术路线。自2014年本研究立项至今，现完成了课题的体外及整体动物实验研究内容：以PKC信号通路为切入点，运用补益肝肾中药，采用显微成像、免疫组化、分子生物学等技术，进行体外及整体动物实验，从DA神经元形态性状与PCKδ、PKCε、ERK1/2、NF-κB、Cyt c等基因相关性分析等方面，探讨PKC信号通路在DA神经元细胞凋亡过程中的可能机制，研究补益肝肾中药对其的作用机理。同时，设计完成临床研究，采用国内外公认的统一帕金森病评定量表（UPDRS）、中医证候量表、健康调查简表（SF-36）及安全性观察等指标，对治疗前后的患者进行监测，评价补益肝肾中药联合西药对肝肾阴虚型PD患者的疗效。结果显示：模型组TH免疫阳性细胞明显减少（P＜0.05），PCKδ、Cyt c、Caspase-3及NF-κB基因mRNA及蛋白水平显著升高（P＜0.05），PKCε、ERK1/2、多巴胺D1及D2受体基因mRNA及蛋白水平显著降低（P＜0.05）；补益肝肾中药组TH免疫阳性细胞明显增多（P＜0.05），PCKδ、Cyt c、Caspase-3及NF-κB基因mRNA及蛋白水平显著降低（P＜0.05），PKCε、ERK1/2、多巴胺D1及D2受体基因mRNA及蛋白水平显著升高（P＜0.05）；PD 大鼠损毁侧线粒体跨膜电位显著低于假手术组及对侧（P＜0.05）；模型组损毁侧线粒体跨膜电位显著低于美多芭组及中药组（P＜0.05），且与中药剂量呈正相关。各组未损毁侧线粒体跨膜电位无明显差异（P＞0.05）。提示中药可通过调控PD黑质-纹状体通路中多巴胺能PKC信号转导通路，降低DA 能神经元凋亡，从而发挥神经元保护机制。而临床研究进一步证实补益肝肾中药联合西药治疗肝肾阴虚型PD患者具有减毒增效疗效。研究结果为补益肝肾中药治疗帕金森病提供了临床及实验室依据，对其进一步推广应用具有重要的临床意义。