中国神经再生研究(英文版) ›› 2022, Vol. 17 ›› Issue (3): 577-586.doi: 10.4103/1673-5374.314326

• 原著:脑损伤修复保护与再生 • 上一篇    下一篇

下调miR-491-5p表达可促进创伤性脑损伤后的血管生成及其分子机制探讨

  

  • 出版日期:2022-03-15 发布日期:2021-10-15

Downregulation of miR-491-5p promotes neovascularization after traumatic brain injury

Wei Tang1, Zong-Duo Guo1, Wei-Na Chai1, Dong-Lin Du1, Xiao-Min Yang1, Lang Cao2, Hong Chen1, Chao Zhou1, Chong-Jie Cheng1, Xiao-Chuan Sun1, Zhi-Jian Huang1, *, Jian-Jun Zhong1, *   

  1. 1Department of Neurosurgery, First Affiliated Hospital of Chongqing Medical University, Chongqing, China; 2Department of Ophthalmology, First Affiliated Hospital of Chongqing Medical University, Chongqing, China
  • Online:2022-03-15 Published:2021-10-15
  • Contact: Zhi-Jian Huang, MD, PhD, zhijian@cqmu.edu.cn; Jian-Jun Zhong, MD, PhD, jianjun@hospital.cqmu.edu.cn.
  • Supported by:
    This work was supported by the National Natural Science Foundation of China, Nos. 82071397 (to XCS), 82071332 (to ZDG); the Youth Fund of the National Natural Science Foundation of China, No. 81801230 (to JJZ); and the Excellent Scientific Research Talents Fund of the First Affiliated Hospital of Chongqing Medical University, China (to JJZ).

摘要:

miR-491-5p在调节细胞增殖和迁移中起着重要的作用,但关于其在调节创伤性脑损伤后血管新生的作用却未见报道。(1)实验构建了C57BL/6小鼠控制性皮质损伤模型,并利用小鼠脑微血管内皮细胞构建氧糖剥夺细胞模型;(2)在体内实验中,通过qRT-PCR检测发现在小鼠侧脑室注射miR-491-5p agomir或antagomir能使小鼠创伤性脑损伤后脑组织中miR-491-5p的表达增加或降低,且miR-491-5p的表达在创伤性脑损伤后轻微下调;(3)为探究miR-491-5p在创伤性脑损伤后的神经保护作用,通过神经功能缺损评分、莫氏水迷宫实验、激光散斑技术和免疫荧光染色发现,下调miR-491-5p的表达,见创伤性脑损伤后神经运动功能恢复和创伤周围脑血流恢复,增加lectin阳染的微血管数量和神经元的存活数量;(4)体外实验中,为探究miR-491-5p对调节血管新生的机制,实验通过qRT-PCR检测发现在脑微血管内皮细胞内转染miR-491-5p mimic或inhibitor能使miR-491-5p的表达增加或降低,双荧光素酶实验和Western blot检测证实金属硫蛋白2是miR-491-5p的靶基因,通过CCK-8实验、流式细胞仪和DCFH-DA探针法检测发现,下调miR-491-5p能够增加氧糖剥夺干预后脑微血管内皮细胞活力,减轻氧糖剥夺干预后的脑微血管内皮细胞的凋亡和氧化应激反应;通过细胞划痕实验、Transwell实验、管腔形成实验和Western blot实验检测发现,下调miR-491-5p能够通过金属硫蛋白2依赖性缺氧诱导因子1α/血管内皮生长因子通路促进脑微血管内皮细胞增殖、迁移和管腔形成;(5)上述数据证实,下调miR-491-5p的表达能够促进创伤性脑损伤后新生血管的形成,脑血流恢复和神经功能的恢复,激活金属硫蛋白2依赖性缺氧诱导因子1α/血管内皮生长因子通路和减轻氧化应激是实现其作用的可能机制。

https://orcid.org/0000-0002-8241-0761 (Zhi-Jian Huang); https://orcid.org/0000-0002-9481-5088 (Jian-Jun Zhong)  

关键词: 脑损伤, 细胞迁移, 细胞增殖, 内皮细胞, 缺氧诱导因子1α, 神经元, 新生血管, microRNA, 金属硫蛋白2, 血管内皮生长因子

Abstract: MicroRNA-491-5p (miR-491-5p) plays an important role in regulating cell proliferation and migration; however, the effect of miR-491-5p on neovascularization after traumatic brain injury remains poorly understood. In this study, a controlled cortical injury model in C57BL/6 mice and an oxygen-glucose deprivation model in microvascular endothelial cells derived from mouse brain were established to simulate traumatic brain injury in vivo and in vitro, respectively. In the in vivo model, quantitative real-time-polymerase chain reaction results showed that the expression of miR-491-5p increased or decreased following the intracerebroventricular injection of an miR-491-5p agomir or antagomir, respectively, and the expression of miR-491-5p decreased slightly after traumatic brain injury. To detect the neuroprotective effects of miR-491-p, neurological severity scores, Morris water maze test, laser speckle techniques, and immunofluorescence staining were assessed, and the results revealed that miR-491-5p downregulation alleviated neurological dysfunction, promoted the recovery of regional cerebral blood flow, increased the number of lectin-stained microvessels, and increased the survival of neurons after traumatic brain injury. During the in vitro experiments, the potential mechanism of miR-491-5p on neovascularization was explored through quantitative real-time-polymerase chain reaction, which showed that miR-491-5p expression increased or decreased in brain microvascular endothelial cells after transfection with an miR-491-5p mimic or inhibitor, respectively. Dual-luciferase reporter and western blot assays verified that metallothionein-2 was a target gene for miR-491-5p. Cell counting kit 8 (CCK-8) assay, flow cytometry, and 2ʹ,7ʹ-dichlorofluorescein diacetate (DCFH-DA) assay results confirmed that the downregulation of miR-491-5p increased brain microvascular endothelial cell viability, reduced cell apoptosis, and alleviated oxidative stress under oxygen-glucose deprivation conditions. Cell scratch assay, Transwell assay, tube formation assay, and western blot assay results demonstrated that miR-491-5p downregulation promoted the migration, proliferation, and tube formation of brain microvascular endothelial cells through a metallothionein-2-dependent hypoxia-inducible factor-1α/vascular endothelial growth factor pathway. These findings confirmed that miR-491-5p downregulation promotes neovascularization, restores cerebral blood flow, and improves the recovery of neurological function after traumatic brain injury. The mechanism may be mediated through a metallothionein-2-dependent hypoxia-inducible factor-1α/vascular endothelial growth factor signaling pathway and the alleviation of oxidative stress. All procedures were approved by Ethics Committee of the First Affiliated Hospital of Chongqing Medical University, China (approval No. 2020-304) on June 22, 2020. 

Key words: brain injury, cell migration, cell proliferation, endothelial cell, hypoxia-inducible factor-1 alpha, metallothionein 2, microRNA, neovascularization, neurons, vascular endothelial growth factor

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