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

doi:10.3969/j.issn.1006-9771.2019.00.013

《中国康复理论与实践》 ›› 2019, Vol. 25 ›› Issue (11): 1303-1308.doi: 10.3969/j.issn.1006-9771.2019.00.013

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、gp91^phox和闭锁小带蛋白-1表达的影响

田超¹, 玛娜璐璐¹, 袁梦晨¹, 王丽琴¹, 安娜¹, 张涵莱¹, 邢雁伟², 孙逸坤¹, 高永红¹

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

收稿日期:2019-05-23 修回日期:2019-07-12 出版日期:2019-11-25 发布日期:2019-11-21
通讯作者: 高永红，E-mail： gaoyh7088@163.com E-mail:gaoyh7088@163.com
作者简介: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。
基金资助:
国家自然科学基金项目(No. 81173229; No. 81673899)

Effect of Qingkailing on Expression of Toll-like Receptor 4, gp91^phox and Zonula Occludens-1 in Cerebrovascular Endothelial Cells Induced by Hypoxia Activating Microglias

TIAN Chao¹, MANA Lu-lu¹, YUAN Meng-chen¹, WANG Li-qin¹, AN Na¹, ZHANG Han-lai¹, XING Yan-wei², SUN Yi-kun¹, GAO Yong-hong¹

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:2019-05-23 Revised:2019-07-12 Published:2019-11-25 Online:2019-11-21
Contact: GAO Yong-hong, E-mail: gaoyh7088@163.com E-mail:gaoyh7088@163.com
Supported by:
Supported by National Natural Science Foundation of China (No. 81173229; No. 81673899)

摘要/Abstract

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)、gp91^phox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、gp91^phox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gp91^phox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、gp91^phox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gp91^phox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

Abstract: Objetive To investigate the effect of Qingkailing injection on the expression of Toll-like receptor 4 (TLR4), gp91^phox 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, gp91^phox 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 gp91^phox 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 gp91^phox 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 gp91^phox, and increasing the expression of ZO-1.

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

中图分类号:

R743.3

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、gp91^phox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.

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. Effect of Qingkailing on Expression of Toll-like Receptor 4, gp91^phox and Zonula Occludens-1 in Cerebrovascular Endothelial Cells Induced by Hypoxia Activating Microglias[J]. 《Chinese Journal of Rehabilitation Theory and Practice》, 2019, 25(11): 1303-1308.

参考文献

1 CorbynZ. Statistics: a growing global burden [J]. Nature, 2014, 510(7506): S2-S3.
2 SimonettiG, StefaniniM, KondaD, et al. Endovascular management of acute stroke [J]. J Cardiovasc Surg, 2013, 54(1): 101-114.
3 AlbersG W, GoyalM, JahanR, et al. Ischemic core and hypoperfusion volumes predict infarct size in SWIFT PRIME [J]. Ann Neurol, 2016, 79(1): 76-89.
4 LiuT J, ZhangJ C, GaoX Z, et al. Effect of sevoflurane on the ATPase activity of hippocampal neurons in a rat model of cerebral ischemia-reperfusion injury via the cAMP-PKA signaling pathway [J]. Kaohsiung J Med Sci, 2018, 34(1): 22-33.
5 LiW, PanR, QiZ, et al. Current progress in searching for clinically useful biomarkers of blood-brain barrier damage following cerebral ischemia [J]. Brain Circ, 2018, 4(4): 145-152.
6 StreitW J, CondeJ R, FendriekS E, et al. Role of microglia in the central nervous system's immune response [J]. Neurol Res, 2005, 27(7): 685-691.
7 LiuW, TangY, FengJ. Cross talk between activation of microglia and astrocytes in pathological conditions in the central nervous system [J]. Life Sci, 2011, 89(5-6): 141-146.
8 KettenmannH, HanischU K, NodaM, et al. Physiology of microglia [J]. Physiol Rev, 2011, 91(2): 461-553.
9 ParkhurstC N, GanW B. Microglia dynamics and function in the CNS [J]. Curr Opin Neurobiol, 2010, 20(5): 595-600.
10 ParadaE, CasasA I, Palomino-AntolinA, et al. Early toll-like receptor 4 blockade reduces ROS and inflammation triggered by microglial pro-inflammatory phenotype in rodent and human brain ischaemia models [J]. Br J Pharmacol, 2019, 176(15): 2764-2779.
11 JungY S, LeeS W, ParkJ H, et al. Electroacupuncture preconditioning reduces ROS generation with NOX4 down-regulation and ameliorates blood-brain barrier disruption after ischemic stroke [J]. J Biomed Sci, 2016, 23(1): 32.
12 GuoM, ZhangL, LiuH, et al. A metabolomic strategy to screen the prototype components and metabolites of Qingkailing injection in rat urine by high-performance liquid chromatography with tandem mass spectrometry [J]. J Sep Sci, 2014, 37(20): 2844-2850.
13 黄德仁,梁军寿,吕小亮,等. 清开灵联合脑蛋白水解物治疗脑梗死的临床观察[J]. 齐齐哈尔医学院学报, 2014, 35(9): 1264-1265.
14 孙良明,程发峰,王雪茜,等. 清开灵注射液治疗急性中风的系统评价和Meta分析[J]. 中国中医急症, 2016, 25(5): 772-776, 857.
15 SerlinY, OferJ, Ben-ArieG, et al. Blood-brain barrier leakage [J]. Stroke, 2019, 50(5): 1266-1269.
16 田子健,杨楠,杨蕾琪. 缺血再灌注后血脑屏障通透性改变的研究进展[J]. 中国医药指南, 2011, 9(23): 222-223.
17 AbdullahiW, TripathiD, RonaldsonP T. Blood-brain barrier dysfunction in ischemic stroke: targeting tight junctions and transporters for vascular protection [J]. Am J Physiol Cell Physiol, 2018, 315(3): C343-C356.
18 KimJ Y, KimN, YenariM A. Mechanisms and potential therapeutic applications of microglial activation after brain injury [J]. CNS Neurosci Ther, 2015, 21(4): 309-319.
19 PunP B, LuJ, MoochhalaS. Involvement of ROS in BBB dysfunction [J]. Free Radic Res, 2009, 43(4): 348-364.
20 ManaL, WangS, ZhuH, et al. Qingkailing suppresses the activation of BV2 microglial cells by inhibiting hypoxia/reoxygenation-induced inflammatory responses [J]. Evid Based Complement Alternat Med, 2014, 2014: 1-8.
21 KnottA B, Bossy-WetzelE. Nitric oxide in health and disease of the nervous system [J]. Antioxid Redox Signal, 2009, 11(3): 541-554.
22 GuY, ZhengG, XuM, et al. Caveolin-1 regulates nitric oxide mediated matrix metalloproteinases activity and blood-brain barrier permeability in focal cerebral ischemia [J]. J Neurochem, 2012, 120(1): 147-156.
23 MohammadiM T, DehghaniG A. Nitric oxide as a regulatory factor for aquaporin-1 and 4 gene expression following brain ischemia/reperfusion injury in rat [J]. Pathol Res Pract, 2015, 211(1): 43-49.
24 AnttilaJ E, WhitakerK W, WiresE S, et al. Role of microglia in ischemic focal stroke and recovery: focus on Toll-like receptors [J]. Prog Neuropsychopharmacol Biol Psychiatry, 2017, 79(PtA): 3-14.
25 Tajalli-NezhadS, KarimianM, BeyerC, et al. The regulatory role of Toll-like receptors after ischemic stroke: neurosteroids as TLR modulators with the focus on TLR2/4 [J]. Cell Mol Life Sci, 2019, 76(3): 523-537.
26 NalamoluK R, SmithN J, ChelluboinaB, et al. Prevention of the severity of post-ischemic inflammation and brain damage by simultaneous knockdown of toll-like receptors 2 and 4 [J]. Neuroscience, 2018, 373: 82-91.
27 GaoW, ZhaoZ, YuG, et al. VEGI attenuates the inflammatory injury and disruption of blood-brain barrier partly by suppressing the TLR4/NF-κB signaling pathway in experimental traumatic brain injury [J]. Brain Res, 2015, 1622: 230-239.
28 LvY, LiuW, RuanZ, et al. Myosin IIA regulated tight junction in oxygen glucose-deprived brain endothelial cells via activation of TLR4/PI3K/Akt/JNK1/2/14-3-3ε/NF-κB/MMP9 signal transduction pathway [J]. Cell Mol Neurobiol, 2019, 39(2): 301-319.
29 RochfortK D, CollinsL E, MurphyR P, et al. Downregulation of blood-brain barrier phenotype by proinflammatory cytokines involves NADPH oxidase-dependent ROS generation: consequences for interendothelial adherens and tight junctions [J]. PLoS One, 2014, 9(7): e101815.
30 WangY, FanX, TangT, et al. Rhein and rhubarb similarly protect the blood-brain barrier after experimental traumatic brain injury via gp91phox subunit of NADPH oxidase/ROS/ERK/MMP-9 signaling pathway [J]. Sci Rep, 2016, 6(1): 37098.
31 高永红,王珊,玛娜璐璐,等. 清开灵对小胶质细胞BV2缺氧再复氧损伤gp~(91phox)表达的影响[J]. 现代生物医学进展, 2014, 14(35): 6807-6809, 6817.
32 ItohM, FuruseM, MoritaK, et al. Direct binding of three tight junction-associated MAGUKs, ZO-1, ZO-2, and ZO-3, with the COOH termini of claudins [J]. J Cell Biol, 1999, 147(6): 1351-1363.
33 JiaoH, WangZ, LiuY, et al. Specific role of tight junction proteins claudin-5, occludin, and ZO-1 of the blood-brain barrier in a focal cerebral ischemic insult [J]. J Mol Neurosci, 2011, 44(2): 130-139.

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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、gp91^phox和闭锁小带蛋白-1表达的影响

Effect of Qingkailing on Expression of Toll-like Receptor 4, gp91^phox and Zonula Occludens-1 in Cerebrovascular Endothelial Cells Induced by Hypoxia Activating Microglias

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Effect of Qingkailing on Expression of Toll-like Receptor 4, gp91^phox and Zonula Occludens-1 in Cerebrovascular Endothelial Cells Induced by Hypoxia Activating Microglias