《中国康复理论与实践》

中国康复理论与实践 >

2019 , Vol. 25 >Issue 11: 1303 - 1308

DOI: https://doi.org/10.3969/j.issn.1006-9771.2019.00.013

基础研究

清开灵对小胶质细胞缺氧活化诱导的脑微血管内皮细胞Toll样受体4、gp91phox和闭锁小带蛋白-1表达的影响

  • 田超 ,
  • 玛娜璐璐 ,
  • 袁梦晨 ,
  • 王丽琴 ,
  • 安娜 ,
  • 张涵莱 ,
  • 邢雁伟 ,
  • 孙逸坤 ,
  • 高永红
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  • 1.北京中医药大学东直门医院中医内科学教育部和北京市重点实验室，北京市 100700
    2.中国中医科学院广安门医院，北京市 100053
田超(1994-)，男，汉族，陕西西安市人，硕士研究生，主要研究方向：中医药防治心脑血管疾病应用基础研究。

收稿日期: 2019-05-23

  修回日期: 2019-07-12

  网络出版日期: 2019-11-21

基金资助

国家自然科学基金项目(No. 81173229; No. 81673899)

收起

Effect of Qingkailing on Expression of Toll-like Receptor 4, gp91phox and Zonula Occludens-1 in Cerebrovascular Endothelial Cells Induced by Hypoxia Activating Microglias

  • TIAN Chao ,
  • MANA Lu-lu ,
  • YUAN Meng-chen ,
  • WANG Li-qin ,
  • AN Na ,
  • ZHANG Han-lai ,
  • XING Yan-wei ,
  • SUN Yi-kun ,
  • GAO Yong-hong
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  • 1.Key Laboratory of Chinese Internal Medicine of Ministry of Education and Beijing, Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing 100700, China
    2.Guang’anmen Hospital, Chinese Academy of Chinese Medical Sciences, Beijing 100053, China

Received date: 2019-05-23

  Revised date: 2019-07-12

  Online published: 2019-11-21

Supported by

Supported by National Natural Science Foundation of China (No. 81173229; No. 81673899)

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摘要

目的 探讨清开灵注射液抑制小胶质细胞缺氧活化后，脑微血管内皮细胞Toll样受体4 (TLR4)、gp91phox和闭锁小带蛋白-1 (ZO-1)的表达。方法 小鼠BV2小胶质细胞分为6组。正常组和模型组用无血清DMEM培养基培养，低、中、高浓度清开灵组用含清开灵注射液0.0625%、0.125%和0.25%的无血清DMEM培养基培养，米诺环素组用含米诺环素200 nmol/L的无血清DMEM培养基培养。除正常组外，其余各组缺氧24 h后复氧24 h，各组培养液分别加入Balb/c内皮细胞培养液中培养24 h。CCK-8检测内皮细胞活力，比色法检测一氧化氮浓度，Western blotting检测共培养模型内皮细胞TLR4、gp91phox和ZO-1表达。结果 与正常组比较，模型组细胞存活率和ZO-1蛋白的表达明显降低(P < 0.01)，一氧化氮浓度、TLR4和gp91phox蛋白表达升高(P < 0.05)；与模型组比较，各清开灵组和米诺环素组细胞存活率和ZO-1蛋白表达升高(P < 0.05)，一氧化氮浓度、gp91phox和TLR4蛋白表达降低(P < 0.05)。结论 清开灵注射液可通过抑制小胶质细胞缺氧活化，降低脑微血管内皮细胞TLR4和gp91phox蛋白表达，提高ZO-1蛋白表达，提高脑微血管内皮细胞的生存和功能。

关键词： 缺血性脑卒中; 清开灵注射液; 血脑屏障; 小胶质细胞; 内皮细胞

本文引用格式

田超 , 玛娜璐璐 , 袁梦晨 , 王丽琴 , 安娜 , 张涵莱 , 邢雁伟 , 孙逸坤 , 高永红 . 清开灵对小胶质细胞缺氧活化诱导的脑微血管内皮细胞Toll样受体4、gp91phox和闭锁小带蛋白-1表达的影响[J]. 中国康复理论与实践, 2019 , 25(11) : 1303 -1308 . DOI: 10.3969/j.issn.1006-9771.2019.00.013

Abstract

Objetive To investigate the effect of Qingkailing injection on the expression of Toll-like receptor 4 (TLR4), gp91phox and zonula occludens-1 (ZO-1) in cerebrovascular endothelial cells induced by hypoxia activation of microglias.Methods BV2 microglia cells were divided into six groups. They were cultured in serum-free DMEM, while the Qingkailing groups of low, middle and high dosages were cultured with 0.0625%, 0.125% and 0.25% Qingkailing injection, respectively, and minocycline group with minocycline of 200 nmol/L. The groups other than control group underwent hypoxia for 24 hours and reoxygenation for 24 hours. Then, the medium of microglia was put into the medium of Balb/c endothelial cells for 24 hours. The cell viability of endothelial cells was measured with CCK-8, the concentration of nitric oxide (NO) was detected with colorimetry, and the experission of TLR4, gp91phox and ZO-1 was detected with Western blotting. Results Compared with the control group, the cell viability and the expression of ZO-1 decreased in the model group (P < 0.01), while the concentration of NO and the expression of TLR4 and gp91phox increased (P < 0.05). Compared with the model group, the cell viability and the expression of ZO-1 increased in the Qingkailing groups and the minocycline group (P < 0.05), while the concentration of NO and the expression of TLR4 and gp91phox decreased (P < 0.05). Conclusion Qingkailing injection may enhance the survival and function of cerebrovascular endothelial cells by inhibiting the hypoxia activation of microglias, reducing the expression of TLR4 and gp91phox, and increasing the expression of ZO-1.

Key words： ischemic stroke; Qingkailing injection; blood-brain barrier; microglia; endothelial cell

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