基于调节线粒体动态平衡探讨大补阴丸合牵正散防治PD保护DA神经元的机制

Parkinson’s disease(PD) is a common progressive neurodegenerative disorder. Mitochondria are now recognized to be dynamic and mobile reticular organelles that constantly undergo membrane remodeling through repeated cycles of fusion and fission. Strong evidence has emerged to implicate disturbed mitochondrial dynamics as central pathological components underpinning PD characterized by progressive degeneration of dopaminergic neurons. In Traditional Chinese Medicine (TCM), clinical manifestations of mitochondrial dysfunction are consistent with that of "yin deficiency of liver and kidney", which is believed as the key pathogenesis of PD. In the completed projects, our studies have demonstrated neuroprotective effects of DBYW and QZS, which are "enriching yin and nourishing kidney, calming the liver and suppressing yang, expelling wind and resolving phlegm", on both reducing brain mitochondrial gene damages in mouse models of PD and alleviating mitochondrial fragmentation in cellular PD models. Based on these results, we plan to explore the drug actions of DBYW and QZS on fusion/fission factors regulating mitochondrial dynamics in both cell and animal models of PD. Then, by establishing the cell models of PD with overexpression and knockdown of the fission gene Drp1 or the fusion gene OPA1, we will further elucidate the regulatory sites on mitochondrial dynamics from the two aspects of mitochondrial fission and fusion. And then, in the terms of Ca2+homeostasis, we will investigate the drug targets of DBYW and QZS upon dopaminergic neuron via regulating mitochondrial dynamics. The study aims to explore the molecular mechanisms of the above-mentioned formulas on regulating mitochondrial dynamics, which will enrich modern scientific connotation for the combination of formulas in TCM and lay the foundation for the track for effective anti-PD drugs from ancient formulas.

帕金森病（PD）是常见神经退行性疾病。研究发现线粒体是运动的细胞器，呈管网状、处于不断分裂融合的平衡状态，其动态失衡可致中脑多巴胺神经元丢失引发PD。现代中医理论认为PD中线粒体异常导致的临床表现与“肝肾阴虚”病机证候相符。我们前期研究的确发现，临床治疗PD的滋阴补肾、平肝潜阳、祛风化痰有效方药大补阴丸合牵正散可减轻PD动物脑线粒体基因损伤、减少PD细胞线粒体碎片化。在此基础上，通过PD动物和细胞模型进一步研究该合方对维持线粒体动态平衡相关分子的调节作用；又采用分裂/融合基因Drp1、Opa1敲减和过表达等技术，从分裂和融合两个方面探讨该合方调节线粒体动态平衡的环节；再从Ca2+稳态角度更深入地找寻调控线粒体动态平衡保护多巴胺神经元的靶点。旨在从线粒体动态平衡的角度探索该合方的分子机制，为中医临床的合方应用提供科学内涵，也为从古方中找寻临床有效抗PD药物奠定基础。

帕金森病（PD）是常见神经退行性疾病。研究发现线粒体是运动的细胞器，呈管网状、处于不断分裂融合的平衡状态，其动态失衡可致中脑多巴胺神经元丢失引发PD。现代中医理论认为PD中线粒体异常导致的临床表现与“肝肾阴虚”病机证候相符。我们前期研究的确发现，临床治疗PD的滋阴补肾、平肝潜阳、祛风化痰有效方药大补阴丸合牵正散可减轻PD动物脑线粒体基因损伤、减少PD细胞线粒体碎片化。在此基础上，通过PD动物和细胞模型进一步研究该合方对维持线粒体动态平衡相关分子的调节作用，采用分裂/融合基因DRP1、OPA1敲减和过表达等技术，从分裂和融合两个方面探讨该合方调节线粒体动态平衡的环节及钙稳态角度更深入地找寻调控线粒体动态平衡保护多巴胺神经元的靶点。 . 本研究发现在DRP1敲减和OPA1基因过表达的水平下，DBYW合QZS能明显改善PD小鼠的行为学，减少黑质多巴胺能神经元丢失，减轻黑质神经元的损伤，并提高多巴胺及其代谢产物DOPAC、HVA含量；可提高线粒体形态因子并明显改善线粒体形态和活性；提高脑线粒体酶复合物I的活性、线粒体膜电位及ATP水平；提高线粒体融合蛋白的表达，降低线粒体分裂蛋白的表达。在DRP1过表达和OPA1基因敲减的水平下，DBYW合QZS的治疗效果尚不明显。另一方面DBYW合QZS可以改善线粒体钙超载进而维持线粒体分裂融合的动态平衡，更好的发挥对线粒体形态和功能的保护作用，发挥治疗PD的作用。提示DBYW合QZS治疗PD的作用机制可能是通过调控DRP1和OPA1基因改善线粒动态平衡有密切的关系，为DBYW合QZS临床治疗PD的作用环节和机制研究提供了进一步的实验依据。