黄杞苷制备及改善脑缺血损伤合并2型糖尿病脑血管新生作用机制

Type 2 Diabetes Mellitus (T2DM) is a major contributor to stroke. Angiogenesis in type 2 diabetic stroke are different to non-diabetic stroke. Abnormal angiogenesis delays neurological function recovery, especially in diabetic stroke. Rational (Non-anomalous) re-establishment of functional microvasculature promotes neurological function recovery in type 2 diabetic stroke. Engelitin, an active flavonoid compound, is isolated from the rhizome of Smilax china L. The primary results showed that Engelitin protected brain against cerebral injury in T2DM and improved angiogenesis during type 2 diabetic stroke. Its improvement of angiogenesis might modulate Vasohibin1 and Ang2/Ang1 ratio. Firstly, the objectives of this study are to prepare high pure and industrialized Engelitin. Secondly, the effects of Engelitin in a cultured rat BMEC using cell proliferation and tube formation will be determined, and its anti-apoptotic role will be investigated. Migrate and proliferation of the cultured rat vascular smooth muscle cells (VSMCs) will be investigated in vitro. Meanwhile, synergistic effects of BMEC and VSMC will investigated in vitro. In addition, it will investigated in experimental ischemic type 2 diabetic stroke model in vivo. It will be demonstrated MBEC and VSMC how to play synergistic role in angiogenesis. All experiments will be elucidated whether Vasohibin 1/VEGFR2 and Angs/Tie2 signaling pathways are directly related to its improvement of angiogenesis, and the angiogenesis is directly related to its modulation of synaptic plasticity. These studies will not only clarify Engelitin protect brain against type 2 diabetic cerebral injury and elucidate its molecular mechanism of angiogenesis, but also provide a theoretical basis to develop Engelitin as a first class new drug. In addition, it will provide and develop cell and molecular screening system of type 2 diabetic cerebral ischemia.

2型糖尿病(T2DM) 脑卒中发病率高，预后差。其预后差与缺血后异常的脑血管新生密切相关。适度的而非异常的血管新生是T2DM脑卒中患者神经功能恢复的基础和保证。前期研究显示黄杞苷改善实验性脑缺血合并T2DM脑血管新生，初步明确与改善Vasohibin1/VEGFR2和Ang2/Ang1比值有关。本项目拟优化制备高纯度黄杞苷的工艺，实现可产业化，并研究其对T2DM大鼠MBEC增殖、成管和凋亡及对血管平滑肌细胞（VSMC）增殖和迁移作用，证明黄杞苷如何促进MBEC和VSMC发挥协同作用以改善血管新生，探讨与Vasohibin 1/VEGFR2和Angs/Tie2信号通路的相关性，以及改善脑血管新生后可否诱导突触可塑性。上述研究不仅阐明黄杞苷改善大鼠脑缺血合并T2DM血管新生的分子机制，为黄杞苷的成药性提供理论依据，还可建立脑缺血合并T2DM损伤后脑血管新生和诱导突触可塑性的细胞与分子筛选平台。

本课题比较不同产地土茯苓中黄杞苷含量，优选土茯苓中黄杞苷的提取、精制和纯化方法。前期发现黄杞苷具有对db/db糖尿病小鼠和GK糖尿病大鼠脑缺血损伤有神经保护和促血管新生作用。本课题研究黄杞苷对糖尿病大鼠脑缺血损伤的保护作用、促血管新生作用及诱导突触可塑性，原代培养血管内皮/平滑肌细胞促血管新生过程与VEGF/VASH-1/VEGFR2/VCAM-1和Ang1/Tie2/PI3K/Akt/Bad信号通路的关系。.1.黄杞苷制备：确认越南产的黄杞苷含量最高（0.0575%）；提取方法是常温超声波辅助提取法提取效率最高，且70%乙醇具有最高的提取效率，黄杞苷含量为0.0684%；精制方法是大孔树脂AB-8对黄杞苷的富集效果最佳，得到的黄杞苷主要集中在75%乙醇洗脱部位，纯度高、损耗少；纯化方法是聚酰胺正相柱层析对黄杞苷目标物的分离效果较好，可以得到较为单一的目标成分，得到黄杞苷的纯度达67%。最终确定的方法，兼顾成本、能耗与制备周期，所得黄杞苷纯品的总收率为：47.1%。.2.药理实验体外研究：糖尿病大鼠原代培养脑血管内皮细胞和平滑肌细胞，研究黄杞苷对的细胞增殖、粘附、迁移和微管形成作用。通过使用VEFG、PI3K抑制剂及Ang1/2、VASH-1、VCAM-1 SiRNA，探讨黄杞苷的作用及促血管新生机制。结果：①黄杞苷能够增加BMEc细胞增殖、迁移和粘附能力，促进毛细血管新生。②抑制细胞LDH泄漏，降低Caspase-3活性，抑制细胞凋亡；③ 黄杞苷增加VEGF、p-VEGFR2，VCAM-1、Ang1、p-Tie2、p-Akt、p-Bad的表达，抑制VASH-1和Ang2的表达，其作用可被VEGF剂和PI3Kt抑制剂逆转。.3.药理实验整体动物研究：成年SD雄性大鼠，高脂饲料和STZ诱导2型糖尿病成模后，线栓法建立MCAO模型，进行相关指标检测。结果：①黄杞苷剂量依赖性减轻MCAO大鼠缺血面积、脑含水量、凋亡细胞数量，促进神经功能恢复；②治疗7天后，显著降低组织渗出、增加缺血区脑血流、增加CD31表达；显著增加VEGF、VEGFR2和VCAM-1表达，被Suramin逆转；增加p-Bad和p-Akt表达，被Wortmannin逆转。.黄杞苷通过VEGF/VASH-1/VEGFR2/VCAM-1和Ang1/Tie2/PI3K/Akt/Bad信号通路促进血管新生。