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    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制

    Mechanism of Danlong Xingnao Formula Promoting Angiogenesis in Rats with Vascular Dementia Based on Nrf2/Keap1 Pathway

      摘要
    • 摘要:
      目的 基于核因子E2相关因子2(nuclear factor erythroid 2-related factor 2，Nrf2)/Kelch 样环氧氯丙烷相关蛋白1(Kelch like epichlorohydrin related protein-1，Keap1)信号通路探讨丹龙醒脑方对血管性痴呆(vascular dementia，VD)大鼠血管新生的作用及机制。
      方法 SD大鼠采用改良双侧颈总动脉结扎法制备VD大鼠模型，随机分为模型组、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)，每组10只，分别给予相应药物灌胃。同时设置假手术组，等量生理盐水灌胃，共给药4周。Morris水迷宫试验检测各组逃避潜伏期、跨越平台次数；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(microvessel density，MVD)、血管内皮生长因子(vascular endothelial growth factor，VEGF)表达；应用生化法检测大鼠海马组织中还原型谷胱甘肽(glutathione，GSH)活性、丙二醛(malondialdehyde，MDA)和超氧化物歧化酶(superoxide dismutase，SOD)含量；Real-time PCR法检测海马组织Nrf2、Keap1、血红素加氧酶 1(heme oxygenase 1，HO-1) mRNA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GSH、SOD活性，降低MDA含量(P<0.05，P<0.01)，上调Nrf2、HO-1 mRNA及Nrf2、HO-1蛋白表达(P<0.05，P<0.01)，降低Keap1 mRNA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:
      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  SD rats were give modified bilateral common carotid artery occlusion to establish VD rat model. SD rats were randomly divided into model group, Nimodipine group, Danlong Xingnao Formula high-dose(14.8 g·kg−1) group, Danlong Xingnao Formula medium-dose(7.4 g·kg−1) group and Danlong Xingnao Formula low-dose(3.7 g·kg−1) group with 10 rats in each group. Administer corresponding medication by gavage separately, at the same time, the sham operation group was given the same amount of normal saline, all rats were treated for 4 weeks. Morris water maze test to detect escape latency and cross-platform times. The pathomorphological changes of rat hippocampal neurons by HE staining. Detection of microvessel density(MVD), vascular endothelial growth factor(VEGF) expression in hippocampal tissue by using immunohistochemistry. Biochemical method were used to measure the levels of glutathione(GSH), malondialdehyde(MDA), superoxidedismutase(SOD) in the hippocampal tissue. Real-time PCR method for detecting the expression of Nrf2, Keap1 and heme oxygenase 1(HO-1) mRNA in hippocampal tissue. Western blotting detection of Nrf2, Keap1 and HO-1 protein expression in hippocampal tissue.
      RESULTS  Compared with the model group, the intervention of Danlong Xingnao Formula could significantly improve the learning and spatial memory abilities of model rats(P<0.05, P<0.01), alleviate pathological and morphological damage to hippocampal tissue, increase MVD and VEGF expression(P<0.05, P<0.01), increase SOD and GSH activity, reduce MDA content(P<0.05, P<0.01), and upregulate Nrf2, HO-1 mRNA and Nrf2, HO-1 protein expression(P<0.05, P<0.01), reduce the expression of Keap1 mRNA and Keap1 protein(P<0.05, P<0.01).
      CONCLUSION  The Danlong Xingnao Formula reduces oxidative stress and promotes angiogenesis by activating the Nrf2/Keap1 signaling pathway, thereby improves the learning and memory abilities of VD rats.

       

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