基于血管生成开关-Nrf2/ARE通路调控的促血管新生与土家医"理阴化毒"干预血管性痴呆作用机理研究

According to the theory of 'Yin excess' inducing senescence and 'gas' inducing diseases in Tujia medicine, the treatment principle of 'Adjusting Yin and Eliminating Toxicity' is proposed to treat vascular dementia(VD). Based on 'adjusting Yin-regulating Angiogemic Switch- restoring the brain microvascular' and'eliminating toxicity-correcting Nrf2-ARE pathway and cascade regulating imbalance of GSH/GSSG' , the influence of TianZhuSan(TZS) on the cerbral vascular aging and the angiogenesis of natural aging rat models with common carotid artery( VD rats) will be evaluated and the effects of Angiogemic Swith will be detected then. The effects of TZS medicated serum on ROS and GSH/GSSG system in the hypoxic and aging endothelial cells will indacate the mechanism of redox balance. The weight distribution method is used to optimize TZS，The cerbral angiogenesis and the expression of promoting/inhibiting angiogenesisi factors of VD rats will be influenced by new TZS, and the regulating mechanism of TGF-β/TSP-1 affected by new TZS will be analyzed. In the hypoxic and aging endothelial cells, the relationship between Nrf2-ARE network cascade regualting, the balance of GSH/GSSG system, the angiogenesis and the cell proliferation and migration influenced by TZS will be observed. The mechanism of the angiogenesis and correcting redox imbalance regulated by Nrf2-ARE network,which is influenced by TZS, will be concluded. This project will benefit to iluminate the scientific connotation of adjusting Yin excess and eliminating Gadu in the methods of 'Adjusting Yin and Eliminating Toxicity', and improve the prescription system to treat VD in Tujia medicine.

据土家族医学"阴盛"致衰、"毒气"致病理论，提出血管性痴呆（VD）"理阴化毒"治法原则。以"理阴-调控血管生成开关-复健脑筋"、"化毒-纠正Nrf2-ARE级联GSH/GSSG失衡-祛除玍毒"为指引，评价天珠散（TZS）对自然衰老慢性低灌注型VD大鼠脑血管老化与血管新生影响，测定"血管生成开关"效应；以TZS含药血清对缺氧衰老内皮细胞（EC）ROS及GSH/GSSG系统作用反映氧化还原平衡参与机制。"权重配方法"优化TZS组成，分析其对VD大鼠脑血管新生、促/抑血管生成因子表达及TGF-β/TSP-1开关调控机制；在缺氧衰老脑EC上，观察TZS促细胞增殖、迁移及血管新生与GSH/GSSG系统平衡、Nrf2-ARE网络级联调控关系，明确TZS调控Nrf2-ARE网络纠正氧化还原失衡及促血管新生机制，进而阐明"理阴化毒"治法"理阴盛"、"祛玍毒"科学内涵，丰富土家族医学防治VD理法方药体系。

本项目以土家医在防治脑缺血性疾病上的“理阴化毒”治则的科学阐释为抓手，从天珠散原方、源头设计优化方、优化组分三个层面聚焦血管性痴呆的脑血管、神经保护及血管新生、神经发生等关键病机环节。天珠散能上调缺血后脑海马组织的Sirt-1和Bcl-2表达，抑制下游caspase-3的活化来发挥其神经元保护作用和抑制细胞凋亡；天珠散通过干预血管性痴呆后的NOS3、APOA1、INS、MMP2、MMP9、JAG1、IL1B、IGF1等关键靶点，适度调控VD大鼠的血管新生。由基线等比设计获得的天麻素、薯蓣皂苷元组分能抑制缺氧HUVECs 的凋亡，降低缺氧细胞内 iNOS 表达，增加eNOS，保持细胞内氧化还原平衡；通过降低VEGF、HIF-1α表达控制血管新生。其中，薯蓣皂苷元通过降低细胞线粒体的脱氢酶活性，减少细胞内 ROS 保护缺氧的内皮细胞；天麻素则能显著促进血管新生，提高微血管缺失模型斑马鱼的肠下血管面积和肠下血管分支数；天麻素增加NeuN的表达而促进神经干细胞向成熟神经元分化的潜能，促进神经再生，与血管新生相辅相成；采用权重配方设计的天珠增冰片新方，能更有效的减小缺血脑梗死体积，改善梗死区域脑组织结构和保护神经细胞。据此初步阐释了土家医“理阴化毒”治法内涵是以恢复脑血管内皮细胞氧化还原平衡、抗细胞凋亡与血管新生调控为表征的血管性痴呆干预机制：以神经元、血管内皮细胞凋亡、坏死及功能丧失为核心的缺血缺氧脑损伤改变。对天麻在中（汉）医方剂中的配伍规律的数据挖掘和德尔菲法调查，获得针对脑缺血及后遗症的天麻-钩藤、天麻-白芍、天麻-川芎等；延龄草在土家医的核心配伍有延龄草-天麻、延龄草-独活等。基于本项目实践，形成了对民族药研究的基本原则和策略，以及源于设计的民族药组方优化研究策略。本项目共发表标注有项目号的学术论文16篇，其中SCI来源论文1篇，中文核心期刊论文13篇；申请国家发明专利2项，获得授权1项。