中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2019, Vol. 14 ›› Issue (5): 783-793.doi: 10.4103/1673-5374.249226

• 原著:脑损伤修复保护与再生 • 上一篇    下一篇

疏血通注射液保护氧糖剥夺再灌注损伤的脑微血管内皮细胞

  

  • 出版日期:2019-05-15 发布日期:2019-05-15
  • 基金资助:

    国家自然科学基金项目（81573644, 81573733）；天津市131创新团队项目；国家科技重大专项课题（2012ZX09101201-004）；天津市科技计划项目（16PTSYJC00120）；天津市应用基础与前沿技术研究计划（一般项目）（14JCYBJC28900）；国家国际科技合作专项项目（2015DFA30430）；天津市自然科学基金重点项目（16ICZDJC36300）；广西科学研究与技术开发计划项目（14125008-2-5）

Shuxuetong injection protects cerebral microvascular endothelial cells against oxygen-glucose deprivation reperfusion

Zuo-Yan Sun 1, 2 , Fu-Jiang Wang 1 , Hong Guo 1 , Lu Chen 1 , Li-Juan Chai 1 , Rui-Lin Li 1 , Li-Min Hu 1 , Hong Wang 1 , Shao-Xia Wang 1, 3, 4   

  1. 1 Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin, China
    2 Department of Pharmacy, Linyi Central Hospital, Linyi, Shandong Province, China
    3 School of Integrative Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin, China
    4 Key Laboratory of Pharmacology of Traditional Chinese Medical Formula, Ministry of Education, Tianjin University of Traditional Chinese Medicine, Tianjin, China
  • Online:2019-05-15 Published:2019-05-15
  • Contact: Shao-Xia Wang, MD, wangshaoxia1@163.com.
  • Supported by:

    This study was supported in part by the National Natural Science Foundation of China, No. 81573644, 81573733; the Tianjin 131 Innovative Team Project, China; the National Major Science and Technology Project of China, No.2012ZX09101201-004; the Science and Technology Plan Project of Tianjin of China, No. 16PTSYJC00120; the Applied Foundation and Frontier Technology Research Program of Tianjin of China (General Project), No. 14JCYBJC28900; the National International Science and Technology Cooperation Project of China, No. 2015DFA30430; the Key Program of the Natural Science Foundation of Tianjin of China, No. 16ICZDJC36300 (to HW); the Scientific Research and Technology Development Plan Project of Guangxi Zhuang Autonomous Region of China, No. 14125008-2-5.

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摘要：

多年来，由水蛭和蚯蚓制成的疏血通注射液在中国已用于临床治疗急性脑卒中，但是其具体的保护机制尚不明确。因此实验将由美国ATCC提供的bEnd.3细胞置于不含葡萄糖的DMEM培养基95% N2/5% CO2环境培养6h，再以高糖培养基95％空气和5％CO2湿润环境培养18h建立氧糖剥夺再灌注模型，然后以稀释32，64，128倍的疏血通注射液在氧糖剥夺再灌注期间进行干预。（1）实验使用CCK-8评估细胞活性，荧光法检测受损细胞释放的LDH情况，荧光酶标仪测量细胞内活性氧的水平，荧光探针测量线粒体超氧化物水平，细胞电阻仪测量跨上皮细胞电阻来说明单层细胞的完整性，FITC-葡聚糖测试血脑屏障渗透性，实时反转录PCR分析肿瘤坏死因子α、白细胞介素1β、白细胞介素6、诱导型一氧化氮合酶mRNA的表达水平，Western blot分析caspase3，细胞间黏附分子1，血管细胞黏附分子1，occludin，血管内皮生长因子，cleaved-caspase3，bcl2，p-ERK，ERK，NF-κBp65，p-IκBα，IκBα，IKK，p-IKK，Claudin5，ZO-1的表达；（2）实验结果表明，疏血通注射液可增加bEnd.3细胞的活性和bcl2的表达，降低cleaved-caspase3的表达，还可抑制活性氧和线粒体过氧化物的产生，抑制后肿瘤坏死因子α、白细胞介素1β、白细胞介素6、诱导型一氧化氮合酶mRNA的表达以及细胞间黏附分子1和血管细胞黏附分子1的表达，显著增加跨上皮细胞电阻，降低血脑屏障渗透性，并上调Claudin-5，Occludin及ZO-1的表达，减少NF-κBp65及血管内皮生长因子表达、IκBα，ERK1/2和IKK磷酸化水平。表明疏血通注射液对氧糖剥夺再灌注损伤的脑微血管内皮细胞具有保护作用，其保护作用与减少线粒体超氧化物的产生，抑制炎症反应，抑制血管内皮生长因子、ERK1/2及NF-κBp65信号通路等多途径有关。

orcid: 0000-0002-1762-8684(Shao-Xia Wang)

关键词: 疏血通注射液, 脑微血管内皮细胞, 氧糖剥夺再灌注, 紧密连接蛋白, 线粒体功能, 炎症因子, 血脑屏障, 神经保护

Abstract:

Shuxuetong injection composed of leech (Hirudo nipponica Whitman) and earthworm (Pheretima aspergillum) has been used for the clinical treatment of acute stroke for many years in China. However, the precise neuroprotective mechanism of Shuxuetong injection remains poorly understood. Here, cerebral microvascular endothelial cells (bEnd.3) were incubated in glucose-free Dulbecco’s modified Eagle’s medium containing 95% N2/5% CO2 for 6 hours, followed by high-glucose medium containing 95% O2 and 5% CO2 for 18 hours to establish an oxygen-glucose deprivation/reperfusion model. This in vitro cell model was administered Shuxuetong injection at 1/32, 1/64, and 1/128 con¬centrations (diluted 32-, 64-, and 128-times). Cell Counting Kit-8 assay was used to evaluate cell viability. A fluorescence method was used to measure lactate dehydrogenase, and a fluorescence microplate reader used to detect intracellular reactive oxygen species. A fluorescent probe was also used to measure mitochondrial superoxide production. A cell resistance meter was used to measure transepithelial resistance and examine integrity of monolayer cells. The fluorescein isothiocyanate-dextran test was performed to examine blood-brain barrier per¬meability. Real-time reverse transcription polymerase chain reaction was performed to analyze mRNA expression levels of tumor necrosis factor alpha, interleukin-1β, interleukin-6, and inducible nitric oxide synthase. Western blot assay was performed to analyze expression of caspase-3, intercellular adhesion molecule 1, vascular cell adhesion molecule 1, occludin, vascular endothelial growth factor, cleaved caspa se-3, B-cell lymphoma 2, phosphorylated extracellular signal-regulated protein kinase, extracellular signal-regulated protein kinase, nuclear factor-κB p65, I kappa B alpha, phosphorylated I kappa B alpha, I kappa B kinase, phosphorylated I kappa B kinase, claudin-5, and zonula oc¬cludens-1. Our results show that Shuxuetong injection increases bEnd.3 cell viability and B-cell lymphoma 2 expression, reduces cleaved caspa se-3 expression, inhibits production of reactive oxygen species and mitochondrial superoxide, suppresses expression of tumor necrosis factor alpha, interleukin-1β, interleukin-6, inducible nitric oxide synthase mRNA, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1, markedly increases transepithelial resistance, decreases blood-brain barrier permeability, upregulates claudin-5, occludin, and zonula occludens-1 expression, reduces nuclear factor-κB p65 and vascular endothelial growth factor expression, and reduces I kappa B alpha, extracellular signal-regulated protein kinase 1/2, and I kappa B kinase phosphorylation levels. Overall, these findings suggest that Shuxuetong injection has protective effects on brain microvascular endothelial cells after oxygen-glucose deprivation/reperfusion. Moreover, its protective effect is associated with reduction of mitochondrial superoxide production, inhibition of the inflammatory response, and inhibition of vascu¬lar endothelial growth factor, extracellular signal-regulated protein kinase 1/2, and the nuclear factor-κB p65 signaling pathway.

Key words: nerve regeneration, Shuxuetong injection, brain microvascular endothelial cells, oxygen-glucose deprivation/reperfusion, tight junction proteins, mitochondrial function, inflammatory factors, blood-brain barrier, neuroprotection, neural regeneration