中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2021, Vol. 16 ›› Issue (2): 325-332.doi: 10.4103/1673-5374.290904

• 原著:脑损伤修复保护与再生 • 上一篇    下一篇

蛛网膜下腔出血后MAP4K4可引起早期血脑屏障损害

  

  • 出版日期:2021-02-15 发布日期:2020-12-03
  • 基金资助:
    国家自然科学基金（81971133，81671313）、辽宁省科技计划（20180550504）、中国人民解放军医学科学青年育种项目（17QNP053）；中国博士后科学基金（2016M592951）。

MAP4K4 induces early blood-brain barrier damage in a murine subarachnoid hemorrhage model

Zheng Zou1, 2, #, Yu-Shu Dong2, #, Dong-Dong Liu2, 3, Gen Li2, 3, Guang-Zhi Hao2, Xu Gao2, Peng-Yu Pan2, *, Guo-Biao Liang2, *   

  1. 1 Department of Neurosurgery, General Hospital of Northern Theater Command (General Hospital of Shenyang Military Command), The Graduate Training Base of Liaoning Medical College, Shenyang, Liaoning Province, China;  2 Department of Neurosurgery, General Hospital of Northern Theater Command (General Hospital of Shenyang Military Command), Shenyang, Liaoning Province, China;   3 Dalian Medical University, Dalian, Liaoning Proivnce, China
  • Online:2021-02-15 Published:2020-12-03
  • Contact: Peng-Yu Pan, MD, PhD, panpengyu09@sina.com; Guo-Biao Liang, MD, PhD, liangguobiao6708@163.com.
  • Supported by:
    This work was supported by the National Natural Science Foundation of China, Nos. 81971133 (to GBL), 81671313 (to YSD); the Science and Technology Project of Liaoning Province of China, No. 20180550504 (to PYP); the Medical Science Youth Breeding Project of Chinese People’s Liberation Army, No. 17QNP053 (to YSD); the China Postdoctoral Science Foundation, No. 2016M592951 (to YSD).

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摘要：

有研究发现丝裂原激活蛋白激酶4(MAP4K4)蛋白在内皮细胞中的表达并促进炎症性血管损伤。内皮细胞是血脑屏障重要组分。为深入揭示MAP4K4在蛛网膜下腔出血后的病理生理学作用，实验评估了MAP4K4在大脑蛛网膜下腔出血后的时空表达。通过线穿法建立同侧/患侧蛛网膜下腔出血小鼠模型，建模后分4组，分别经脑室内注射MAP4K4重组蛋白、对照小干扰RNA和MAP4K4小干扰RNA，灌胃MAP4K4小分子抑制剂PF-06260933。采用神经功能评分法、脑水含量评分法、组织学染色、免疫荧光法、免疫印迹法、明胶酶谱法分析神经功能变化及血管损伤机制发现：(1) 蛛网膜下腔出血后24 h，MAP4K4在损伤侧大脑皮质中表达升高，并与内皮标志物凝集素共定位；(2) MAP4K4重组蛋白组神经功能评分降低，脑水肿程度加重，血脑屏障破坏，并且上调磷酸化核转录因子κB(p-p65)和MMP9蛋白表达，降解紧密连接蛋白(ZO-1和cluadin 5)，而采用MAP4K4小干扰RNA干预后可逆转上述结果；(3)采用MAP4K4抑制剂PF-06260933干预可减轻小鼠血脑屏障损伤，促进神经功能恢复，减少p-p65和MMP9蛋白表达；(4)实验结果进一步说明了MAP4K4引起蛛网膜下腔出血后的早期血脑屏障损害，其机制可通过抑制MAP4K4/NF-κB/MMP9途径来验证。

https://orcid.org/0000-0001-8694-5483 (Peng-Yu Pan); 

https://orcid.org/0000-0001-6332-190X (Guo-Biao Liang)

关键词: 中枢神经系统, 脑, 大鼠, 损伤, 修复, 恢复, 再生, 途径

Abstract: Sterile-20-like mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) is expressed in endothelial cells and activates inflammatory vascular damage. Endothelial cells are important components of the blood-brain barrier. To investigate whether MAP4K4 plays a role in the pathophysiology of subarachnoid hemorrhage, we evaluated the time-course expression of MAP4K4 after subarachnoid hemorrhage. A subarachnoid hemorrhage model was established using the intravascular perforation method. The model mice were assigned to four groups: MAP4K4 recombinant protein, scramble small interfering RNA, and MAP4K4 small interfering RNA were delivered by intracerebroventricular injection, while PF-06260933, a small-molecule inhibitor of MAP4K4, was administrated orally. Neurological score assessments, brain water assessments, Evans blue extravasation, immunofluorescence, western blot assay, and gelatin zymography were performed to analyze neurological outcomes and mechanisms of vascular damage. MAP4K4 expression was elevated in the cortex at 24 hours after subarachnoid hemorrhage, and colocalized with endothelial markers. MAP4K4 recombinant protein aggravated neurological impairment, brain edema, and blood-brain barrier damage; upregulated the expression of phosphorylated nuclear factor kappa B (p-p65) and matrix metalloproteinase 9 (MMP9); and degraded tight junction proteins (ZO-1 and claudin 5). Injection with MAP4K4 small interfering RNA reversed these effects. Furthermore, administration of the MAP4K4 inhibitor PF-06260933 reduced blood-brain barrier damage in mice, promoted the recovery of neurological function, and reduced p-p65 and MMP9 protein expression. Taken together, the results further illustrate that MAP4K4 causes early blood-brain barrier damage after subarachnoid hemorrhage. The mechanism can be confirmed by inhibiting the MAP4K4/NF-κB/MMP9 pathway. All experimental procedures and protocols were approved by the Experimental Animal Ethics Committee of General Hospital of Northern Theater Command (No. 2018002) on January 15, 2018.

Key words: brain, central nervous system, injury, pathways, rat, recovery, regeneration, repair