白果内酯通过Nrf2/HO-1信号通路改善氧化应激所致体外血脑屏障损伤的研究
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摘要
目的研究白果内酯对过氧化氢(H_2O_2)所致体外血脑屏障损伤的保护作用及其机制。方法 hCMEC/d3与HEB细胞共培养建立体外血脑屏障模型,模型分为6组:空白对照组、H_2O_2模型组(500μmol/L)、白果内酯低中高剂量组(0.2、1、5μmol/L)、依达拉奉(10μmol/L)阳性组。给予不同浓度白果内酯及依达拉奉预处理体外血脑屏障模型24 h,利用H_2O_2(500μmol/L)诱导2 h模拟氧化应激损伤。采用CCK-8法检测细胞活性,同时检测血脑屏障模型一氧化氮(NO)、还原型谷胱甘肽(GSH)、乳酸脱氢酶(LDH)及总抗氧化能力;考察跨内皮细胞电阻值及荧光素钠(Na-F)透过率;蛋白免疫印迹检测紧密连接蛋白(claudin-5、occludin、VE-Cadherin)和抗氧化蛋白(Nrf2、HO-1)的表达水平。结果氧化应激会引起体外血脑屏障通透性的增加,下调紧密连接蛋白表达水平。白果内酯预处理可提高内皮细胞总抗氧化能力及NO、GSH水平,降低LDH水平,维持细胞正常的电阻值及较低的Na-F的透过,同时缓解氧化应激引起的紧密连接蛋白下调,并上调Nrf2/HO-1信号通路抗氧化蛋白表达。结论白果内酯可作用于抗氧化信号通路Nrf2/HO-1保护氧化应激所致血脑屏障通透性增加。
Objective To investigate the protective effect and mechanism of bilobalide on blood-brain barrier(BBB) injury induced by hydrogen peroxide(H_2O_2) in vitro. Methods The hCMEC/d3 cells and HEB cells were co-cultured to establish an in vitro BBB model. The model was divided into 6 groups,including the control group,H_2O_2 model group(500 μmol/L),three different doses of bilobalide groups(0.2,1 and 5 μmol/L),and Edaravone(10 μmol/L) positive control group. Different concentrations of bilobalide and edaravone were used to pretreat the BBB model for 24 h,and H_2O_2(500 μmol/L) was induced for 2 h to form an oxidative stress injury model. Cell viability assay was measured by cell counting Kit-8(CCK-8). The levels of nitric oxide(NO),glutathione(GSH),lactate dehydrogenase(LDH) and total antioxidant capacity were detected in the BBB model. Transendothelial electrical resistance(TEER) and sodium fluorescein(Na-F) permeability were used to investigate BBB permeability. Western blotting was used to detect the expression of tight junction-associated proteins(claudin-5,occludin and VE-Cadherin) and antioxidant proteins(Nrf2 and HO-1). Results Oxidative stress increased the permeability of the in vitro BBB and down-regulated the expression of tight junction proteins. Pretreatment with bilobalide increased the total antioxidant capacity of endothelial cells,and NO and GSH levels,reduced LDH levels,maintained the normal BBB resistance and the lower sodium fluorescein permeation,relieved the down-regulation of the tight junction proteins,and up-regulated Nrf2/HO-1 pathway protein expression. Conclusion Bilobalide can protect the BBB permeability from oxidative stress damage through the antioxidant action mediated by Nrf2/HO-1 pathway.
Objective To investigate the protective effect and mechanism of bilobalide on blood-brain barrier(BBB) injury induced by hydrogen peroxide(H_2O_2) in vitro. Methods The hCMEC/d3 cells and HEB cells were co-cultured to establish an in vitro BBB model. The model was divided into 6 groups,including the control group,H_2O_2 model group(500 μmol/L),three different doses of bilobalide groups(0.2,1 and 5 μmol/L),and Edaravone(10 μmol/L) positive control group. Different concentrations of bilobalide and edaravone were used to pretreat the BBB model for 24 h,and H_2O_2(500 μmol/L) was induced for 2 h to form an oxidative stress injury model. Cell viability assay was measured by cell counting Kit-8(CCK-8). The levels of nitric oxide(NO),glutathione(GSH),lactate dehydrogenase(LDH) and total antioxidant capacity were detected in the BBB model. Transendothelial electrical resistance(TEER) and sodium fluorescein(Na-F) permeability were used to investigate BBB permeability. Western blotting was used to detect the expression of tight junction-associated proteins(claudin-5,occludin and VE-Cadherin) and antioxidant proteins(Nrf2 and HO-1). Results Oxidative stress increased the permeability of the in vitro BBB and down-regulated the expression of tight junction proteins. Pretreatment with bilobalide increased the total antioxidant capacity of endothelial cells,and NO and GSH levels,reduced LDH levels,maintained the normal BBB resistance and the lower sodium fluorescein permeation,relieved the down-regulation of the tight junction proteins,and up-regulated Nrf2/HO-1 pathway protein expression. Conclusion Bilobalide can protect the BBB permeability from oxidative stress damage through the antioxidant action mediated by Nrf2/HO-1 pathway.
引文
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[24] 王朔,王振杰,赵峰. 丹参素对血管内皮细胞氧化应激损伤保护作用的实验研究 [J].现代中西医结合杂志,2011,20 (20):2493-2494.
[25] WANG J,FIELDS J,ZHAO C,et al. Role of Nrf2 in protection against intracerebral hemorrhage injury in mice [J]. Free Radic Biol Med,2007,43(3):408-414.
[26] SHAH Z A,LI R,THIMMULAPPA R K,et al. Role of reactive oxygen species in modulation of Nrf2 following ischemic reperfusion injury [J]. Neuroscience,2007,147(1):53-59.
[27] ZHUANG H,PIN S,CHRISTEN Y,et al. Induction of heme oxygenase 1 by Ginkgo biloba in neuronal cultures and potential implications in ischemia[J]. Cell Mol Biol (Noisy-le-grand),2002,48 (6):647-653.
[2] DEFEUDIS F V . Bilobalide and neuroprotection [J]. Pharmacol Res,2002,46 (6):565-568.
[3] 熊国营,张凌云,李晓君. 白果内酯耐缺氧作用研究 [J].当代医学,2015,21(36):11-12.
[4] PRIYANKA A,NISHA V M,ANUSREE S S,et al. Bilobalide attenuates hypoxia induced oxidative stress,inflammation,and mitochondrial dysfunctions in 3T3-L1 adipocytes via its antioxidant potential [J]. Free Radical Res,2014,48(10): 1206-1217.
[5] RODRIGUES S F,GRANGER D N . Blood cells and endothelial barrier function [J]. Tissue Barriers,2015,3(1/2):1-11.
[6] PERSIDSKY Y,RAMIRES S H,HAORAH J,et al. Blood-brain barrier: structural components and function under physiologic and pathologic conditions [J]. J Neuroimmune Pharm,2006,1(3):223-236.
[7] VARATHARAJ A,GALEA I. The blood-brain barrier in systemic inflammation [J]. Brain Behav Immun,2017,60:1-12.
[8] CHI O Z,MELLENDER S J,BARSOUM S,et al. Effects of rapamycin pretreatment on blood-brain barrier disruption in cerebral ischemia-reperfusion [J]. Neurosci Lett,2016,620 (4):132-136.
[9] LIU Weiye,WANG Zhibin,ZHANG Lichao,et al. Tight junction in blood-brain barrier: an overview of structure,regulation,and regulator substances [J]. CNS Neurosci Ther,2012,18(8):609-615.
[10] SALEEM S,ZHUANG H,BISWAL S,et al. Ginkgo biloba extract neuroprotective action is dependent on heme oxygenase 1 in ischemic reperfusion brain injury [J]. Stroke,2008,39(12): 3389-3396.
[11] 薛小燕,郭小华,李敏,等.神经退行性疾病发病机制研究进展[J].中国老年学杂志,2015,35(11):3149-3152.
[12] HSU Chiuling,WU Yuhlin,TANG Gaujun,et al. Ginkgo biloba extract confers protection from cigarette smoke extract-induced apoptosis in human lung endothelial cells: role of heme oxygenase-1 [J]. Pulm Pharmacol Ther,2009,22(4):286.
[13] LI Min,ZHANG Xiangjian,CUI Lili,et al. The neuroprotection of oxymatrine in cerebral ischemia/reperfusion is related to nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated antioxidant response: role of Nrf2 and Hemeoxygenase-1 expression [J]. Biol Pharm Bull,2011,34(5):595-601.
[14] WEKSLER B,ROMERO I A,COURAUD P O . The hCMEC/D3 cell line as a model of the human blood brain barrier [J]. Fluids Barriers CNS,2013,10(1):1-10.
[15] BURKHART A,THOMSEN L B,THOMSEN M S,et al. Transfection of brain capillary endothelial cells in primary culture with defined blood-brain barrier properties [J]. Fluids Barriers CNS,2015,12(1):1-14.
[16] 张水华,季龙凤,马璟. 非接触式共培养体外血脑屏障模型的跨膜电阻及通透性[J].中国药理学与毒理学杂志,2012,26 (6):882-887.
[17] RITCHIE R H,DRUMMOND G R,SOBEY C G,et al. The opposing roles of NO and oxidative stress in cardiovascular disease [J]. Pharmacol Res,2017,116 (12):57-69.
[18] NIEMANN B,ROHRBACH S,MILLER M R,et al. Oxidative stress and cardiovascular risk: obesity,diabetes,smoking,and pollution[J]. J Am Coll Cardiol,2017,70 (2):230-251.
[19] HENCHCLIFFE C,BEAL M F . Mitochondrial biology and oxidative stress in Parkinson disease pathogenesis [J]. Nat Rev Neurol,2008,4(11):600-609.
[20] 黄慧芬,郑丽云,方世记,等. 依达拉奉对过氧化氢诱导血管内皮细胞Nrf2/HO-1表达的影响[J].中国临床药学杂志,2017,26 (2):88-92.
[21] ABBOTT N J,RONNBACK L,HANSSON E,Astrocyte- endothelial interactions at the blood-brain barrier [J]. Nat Rev Neurosci,2006,7 (1):41-53.
[22] STANIMIROVIC D B,BANIYAGHOUB M,PERKINS M,et al. Blood-brain barrier models: in vitro to in vivo translation in preclinical development of CNS-targeting biotherapeutics [J]. 2014,10 (2):141-155.
[23] 乔丽杰,王延让,张明. Nrf2/HO-1通路在氧化损伤保护机制中研究进展 [J].中国职业医学,2013,40 (1):82-84.
[24] 王朔,王振杰,赵峰. 丹参素对血管内皮细胞氧化应激损伤保护作用的实验研究 [J].现代中西医结合杂志,2011,20 (20):2493-2494.
[25] WANG J,FIELDS J,ZHAO C,et al. Role of Nrf2 in protection against intracerebral hemorrhage injury in mice [J]. Free Radic Biol Med,2007,43(3):408-414.
[26] SHAH Z A,LI R,THIMMULAPPA R K,et al. Role of reactive oxygen species in modulation of Nrf2 following ischemic reperfusion injury [J]. Neuroscience,2007,147(1):53-59.
[27] ZHUANG H,PIN S,CHRISTEN Y,et al. Induction of heme oxygenase 1 by Ginkgo biloba in neuronal cultures and potential implications in ischemia[J]. Cell Mol Biol (Noisy-le-grand),2002,48 (6):647-653.