基于Nrf2/Keap1信号通路探讨丹龙醒脑方促血管性痴呆大鼠血管新生的作用机理

The Mechanism of Danlong Xingnao Formula Promoting Angiogenesis in Rats with vascular dementia Based on Nrf2/Keap1 Pathway

关键词:  丹龙醒脑方  血管性痴呆  Nrf2/Keap1  血管新生  血管内皮生长因子

Key words:  Danlong Xingnao Formula  Vascular dementia  Nrf2 /Keap1  Angiogenesis  Vascular endothelial growth factor

摘要:

目的 基于核因子E2相关因子2(Nrf2)/ Kelch样环氧氯丙烷相关蛋白-1(Keap1)信号通路探讨丹龙醒脑方对血管性痴呆(VD)大鼠血管新生的作用及机制。方法 采用改良2-VO法制备VD大鼠模型,随机将60只SD大鼠分为假手术组、模型组、尼莫地平组、丹龙醒脑方高剂量组(14.8/g·kg-1)、丹龙醒脑方中剂量组(7.4/g·kg-1)、丹龙醒脑方低剂量组(3.7/g·kg-1)。Morris水迷宫实验检测各组逃避潜伏期、跨越平台次数；苏木素-伊红(HE)染色法观察海马区病理形态学变化；采用免疫组化法检测海马组织微血管密度(MVD)、血管内皮生长因子(VEGF)表达；应用生化法检测大鼠海马组织中超氧化物歧化酶(SOD)、还原型谷胱甘肽(GSH) 活性和丙二醛(MDA)含量；Real-time PCR法检测海马组织Nrf2、Keap1、血红素加氧酶 1(HO-1)m RNA的表达；Western blotting检测海马组织Nrf2、Keap1、HO-1蛋白表达。结果 与模型组比较,丹龙醒脑方干预后可明显提高模型大鼠的学习、空间记忆能力(P<0.05,P<0.01),减轻海马组织的病理形态学损伤,增高MVD、VEGF表达(P<0.05,P<0.01),升高SOD、GSH 活性,降低MDA含量(P<0.05,P<0.01),上调Nrf2、HO-1 m RNA及Nrf2、HO-1蛋白表达(P<0.05,P<0.01),降低Keap1 m RNA及Keap1蛋白表达(P<0.05,P<0.01)。结论 丹龙醒脑方通过激活Nrf2/Keap1信号通路减轻氧化应激、促进血管新生,进而改善VD大鼠的学习记忆能力。

Abstract:

ABSTRACT: OBJECTIVE To investigate the effect and mechanism of Danlong Xingnao Formula on angiogenesis in vascular dementia (VD) rats based on the nuclear factor E2 related factor 2 (Nrf2)/Kelch like epichlorohydrin related protein-1 (Keap1) signaling pathway. METHODS The VD rat model was established by the modified 2-VO method and 60 SD rats were randomly divided into sham operation group, model group, Nimodipine group,high-dose Danlong Xingnao Formula group(14.8/g·kg-1), middle-dose Danlong Xingnao Formula group(7.4/g·kg-1) and low-dose Danlong Xingnao Formula group(3.7/g·kg-1) . Morris water maze test to detect escape latency and cross-platform times. The pathomorphological changes of rat hippocampal neurons by hematoxylin-eosin(HE)staining. Detection of microvessel density (MVD), vascular endothelial growth factor (VEGF)expression in hippocampal tissue by using immunohistochemistry.The activity of SOD, GSH and the content of MDA in liver tissue of mice were detected by biochemical method.Real time PCR method for detecting the expression of Nrf2,Keap1and HO-1 mRNA in hippocampal tissue. Western blotting detection of Nrf2,Keap1and HO-1 protein expression in hippocampal tissue.RESULTS Compared with the model group, the intervention of Danlong Xingnao Formula can significantly improve the learning and spatial memory abilities of model rats (P<0.05, P<0.01), alleviated pathological and morphological damage to hippocampal tissue, increased MVD and VEGF expression (P<0.05, P<0.01), increased SOD and GSH activity, reduced MDA content (P<0.05, P<0.01), and upregulated Nrf2, HO-1 mRNA and Nrf2, HO-1 protein expression (P<0.05, P<0.01), Reduced the expression of Keap1 mRNA and Keap1 protein (P<0.05, P<0.01).CONCLUSION The Danlong Xingnao Formula reduce oxidative stress and promotes angiogenesis by activate the Nrf2/Keap1 signaling pathway, thereby improve the learning and memory abilities of VD rats.