丹栀逍遥散调控Notch信号通路干预广泛性焦虑症神经血管再生的机制研究

Generalized anxiety disease(GAD) is the most common metal disease with long process, high recurrent rate and severe social harmfulness. Traditional Chinese Medicine has an advantage in treating GAD, and Danzhi Xiaoyao powder is a classic Chinese medicine prescription for GAD of Fire Derived from Stagnation of Liver-QI Type.. In our previous study, we established GAD rats model of Fire Derived from Stagnation of Liver-QI Type and we found that  Danzhi Xiaoyao powder could alleviate anxious behaviours and associated symptoms of GAD rats, decrease apoptosis  and enhance proliferation and differentiation of endogenic neural stem cells(NSCs) in hippocampus of GAD rats. However, further theory and deeper mechanism still needs to be studied.. This study is focused on the hot topic of neurological disease and the proliferation and differentiation of NSCs and angiogenesis in hippocampus will be tested dynamically to supplement the theory of neuroplasticity in hippocampus of GAD rats. Furthermore, the expression of Notch signaling pathway factors will be tested to explore the inner mechanism of Danzhi Xiaoyao powder promoting.neurogenesis and angiogenesis from the perspective of signal transdution. And we expect to confirm the effect targets and consummate the theory of Danzhi Xiaoyao powder treating GAD and finally serve clinical therapy.

广泛性焦虑障碍（GAD）是目前患病率最高的精神类疾病，且病情缠绵，容易复发，社会危害性大。中医药治疗GAD有独到的优势，其中，丹栀逍遥散是临床上治疗肝郁化火型GAD的经典方剂。. 前期的研究成功的制备了肝郁化火型GAD大鼠模型，并发现丹栀逍遥散能够减轻GAD大鼠的焦虑状态，减轻大鼠海马内的细胞凋亡，改善海马内神经干细胞（NSCs）的增殖与分化，但更深层机制尚未明了。. 本研究拟在前期研究结果的基础上，增加观察时点，以进一步动态的评价GAD大鼠海马内神经再生演变规律，并观察相应脑区血管新生的情况，以丰富神经血管重塑的内容。同时检测Notch信号通路相关因子蛋白和基因的表达水平变化，从信号转导的角度揭示丹栀逍遥散促进神经重塑的内在机理，明确丹栀逍遥散的作用靶点，完善丹栀逍遥散治疗GAD的理论学说，更好的为临床服务。

本研究通过不确定空瓶刺激法联合束缚法制备广泛性焦虑（GAD）大鼠（肝郁化火脾虚证）的“病证结合”动物模型，并设置了对照组、模型组、丹栀逍遥散（DZXYS）组、DZXYS+DAPT组和丁螺环酮组5组，分别在DZXYS干预第7d、14d时，观察海马神经干细胞（NSCs）增殖分化情况、海马内血管新生情况以及海马内notch信号通路表达情况。结果：①海马内NCSs增殖与分化能力检测：药物干预7天、14天后，模型组较对照组BrdU+/Nestin+数量显著增加，DZXYS组BrdU+/Nestin+数量较模型组降低；模型组较对照组BrdU+/Neun+数量显著降低，DZXYS组BrdU+/Neun+数量较模型组显著升高；模型组较对照组BrdU+/GFAP+数量显著增高，DZXYS组BrdU+/GFAP+数量较模型组显著降低。②海马内血管新生能力检测：CD34免疫组化及HE染色均示模型组海马血管数量较对照组无统计学差异，DZXYS组干预7天、14天后DG区血管数量较模型组显著升高，但均无显著时间效应。模型组DG区VEGF IOD值较对照组显著降低，DZXYS干预7、14天后VEGF IOD值较模型组显著升高；VEGFR2 IOD值模型组较对照组显著降低，DZXYS组干预7天后较模型组显著升高，14天后降低；模型组DG区Ang1表达量较对照组无统计学差异，干预7天后，DZXYS组Ang1表达量较模型组显著降低，14天后较模型组显著升高；模型组DG区Tie2表达量较对照组无统计学差异，DZXYS组干预后第7天、第14天较模型组显著升高。③海马区Notch信号通路关键靶点变化情况：rtPCR显示模型组较对照组Notch1mRNA、Hes1mRNA、Hes5mRNA、DLL4mRNA表达量显著上升，干预7天、14天后，DZXYS组Notch1mRNA、Hes1mRNA、Hes5mRNA表达量较模型组显著降低；DZXYS组DLL4mRNA表达量在干预7天后较模型组显著降低，干预14天后较模型组明显升高。WB蛋白表达情况与以上PCR结果基本一致。本项目通过研究Notch信号通路与GAD大鼠神经血管重塑的关系，揭示了Notch信号通路在GAD发病中的作用。也进一步揭示了丹栀逍遥散的作用机制和靶点。