基于HIF-1-VEGF-Notch信号通路探讨锦鸡儿总黄酮对脑缺血后血管新生的影响及其调控机制

Ischemic stroke is a kind of common disease with high morbidity, high disability rate and high recurrence rate. Isehemia can irreversibly result in substantial neuronal degeneration and necrosis and cause severe neurological deficits. Promoting angiogenesis in ischemic brain to increase the number of new collateral circulation will increase the blood supply and improve the ischemic brain function. Studies have shown that HIF-1-VEGF-NOTCH signaling pathway is the main regulator of angiogenesis after Ischemia and hypoxia. Total flavones of Caragana（TFC）, a major active flavonoid isolated from Herba Caragana root, possesses wide pharmacological actions, especially in protecting cerebral ischemic injury. Previous studies have found that TFC have protective effects against cerebral ischemic injury by scavenging oxygen free radicals. So we speculate that TFC may be involved in the process of regulating angiogenesis and regulate angiogenesis by HIF-1-VEGF-Notch signaling pathway. However, it has not been reported that whether TFC can promote angiogenesis in ischemic brain. Therefore, this study will establish focal ischemia reperfusion model in rats, to explore the effect of TFC on angiogenesis after cerebral ischemia and the possible molecular regulation mechanism from the perspective of regulating the angiogenesis of HIF-1-VEGF-Notch signaling pathways. From the study, we could judge whether TFC influence angiogenesis through HIF-1-VEGF-Notch signaling pathway. And this experiment will provide theoretical and experimental basis for the development and clinical application of TFC.

缺血性脑卒中是一类发病率高、致残率高、复发率高的常见病。缺血可致大量神经元变性坏死，导致严重神经功能缺损。促进缺血区新生血管形成，可改善局部脑血流供应，挽救缺血半暗带内濒临死亡的神经细胞，促进神经功能恢复，这对脑卒中的治疗有重要作用。已有研究证明，脑缺血可诱导血管新生，HIF-1-VEGF-Notch通路可调节缺血缺氧后血管新生。锦鸡儿总黄酮是从锦鸡儿中提取的黄酮类成分，既往研究表明锦鸡儿总黄酮对脑缺血损伤有保护作用。据此，我们推测其作用靶点可能在血管，其作用机制可能通过HIF-1-VEGF-Notch调控脑缺血后血管新生过程。然而，锦鸡儿总黄酮对脑卒中后能否促进缺血区血管新生，尚未见报道。因此，本研究拟建立大鼠脑缺血/再灌注模型，明确锦鸡儿总黄酮抗脑梗死的有效性，从调控血管新生角度，探讨锦鸡儿总黄酮对脑缺血后血管新生的影响及可能的分子机制，为锦鸡儿总黄酮的开发及临床应用提供理论基础和实验依据。

目的：探讨锦鸡儿总黄酮对脑缺血后脑保护的影响及其改善脑血流灌注的机制。方法：采用线栓法制备大鼠局灶性脑缺血再灌注损伤模型，参照Zea Longa 评分法在缺血再灌注24h后行神经功能缺损评分，用TTC染色计算脑梗死面积，测定伊文思兰的含量评估血脑屏障的通透性，并用免疫荧光法和Western blot检测缺血脑组织中CD31、Ang-1、VEGF、Dll4、HIF-1α、Notch1、基质金属蛋白酶-9 /-2、claudin-5和occluding等蛋白和基因的表达。结果：锦鸡儿总黄酮对缺氧复氧诱导的脑微血管内皮细胞损伤具有保护作用，促进HIF- 1α-VEGF-Notch 1信号通路的表达。锦鸡儿总黄酮可改善脑缺血大鼠的神经功能缺损，能减少脑梗死面积，抑制脑缺血后基质金属蛋白酶-9/-2（MMP-9/-2）的表达，上调claudin-5和occluding的表达。模型组大鼠脑缺血后缺血半暗带区域CD31、Ang-1 及 VEGF 的mRNA表达与假手术组相比均明显升高，且TFC 60mg/kg治疗组中CD31、Ang-1 及 VEGF 的 mRNA 表达明显高于模型组(**P <0.01)。TFC 60mg/kg 治疗后，Ang1 和 VEGF蛋白表达也被上调。Western blot结果统计分析显示，模型组和治疗组与假手术组相比差异有统计学意义(**P <0.05,***P <0.01)。免疫荧光分析进一步证明TFC能促进 CD31、Ang-1 及 VEGF 的表达，高剂量组具有显著差异。与模型组相比，TFC 60 mg/kg 组脑组织缺血周边区Dll4、HIF-1和Notch1表达量增多。Western blot结果显示，与模型组相比，TFC 60mg/kg治疗组脑组织缺血周边区Dll4、HIF-1、Notch1蛋白表达显著上调，差异具有统计学意义（* P <0.05，＃P <0.05，**P<0.01）。结论：锦鸡儿总黄酮可改善神经功能缺损，减少脑梗死面积，对脑缺血后血脑屏障损伤具有保护作用，其作用机制可能与抑制脑缺血后MMP-9/-2的表达，促进claudin-5、 occluding、CD31、Ang-1、VEGF、Dll4、HIF-1、Notch1蛋白的表达，促进血管新生有关。