钒暴露对大鼠小胶质细胞炎症反应和迁移的影响
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摘要
目的观察偏钒酸钠(Na VO3·2H2O)暴露对小胶质细胞炎症反应和迁移的影响,探讨钒神经毒性的相关机制。方法偏钒酸钠孵育原代培养的SD大鼠小胶质细胞,免疫荧光技术显示小胶质细胞形态的变化及其特异性标志物Iba1的表达,免疫印迹技术检测i NOS、COX-2、ERK和p-ERK蛋白表达,酶联免疫吸附实验检测炎症因子TNF-α,IL-1β的释放水平;构建划痕迁移模型,免疫荧光技术记录偏钒酸钠对小胶质细胞迁移的影响。结果偏钒酸钠孵育小胶质细胞后,神经小胶质细胞形态由静息态的分支状向吞噬细胞样形态转变,其特异性标志物Iba1表达显著增加;i NOS和COX-2的蛋白表达和TNF-α,IL-1β释放水平与对照组相比较均显著升高;偏钒酸钠促进小胶质细胞的迁移。结论偏钒酸钠显著促进了小胶质细胞的炎症反应和迁移。
Objective To observe the changes of rat microglial inflammation and migration after exposure to sodium metavanadate( Na VO3·2H2O),and to analyze the possible mechanisms of vanadium neurotoxicity. Methods Primary cultured rat microglial cells were incubated with Na VO3·2H2O. Morphological changes and the Iba1 expression of microglia were tested by immunofluorescence assay. i NOS,Cox-2,ERK and p-ERK protein expressions were determined by western blotting. The levels of TNF-α and IL-1β in the culture medium were tested by enzyme-linked immunosorbent assay. The migration of microglia was tested by immunofluorescence staining using wound-healing assay. Results Microglia changed from resting state with ramous shape to round shape in activated state after Na VO3·2H2O exposure,and the expression of Iba1 increased obviously. The protein expressions of i NOS and COX-2 increased significantly compared with the control. The levels of TNF-α and IL-1β were also increased significantly. Na VO3·2H2O promotes the migration of microglia through ERK pathway. Conclusions Exposure to Na VO3·2H2O promotes primary cultured rat microglial inflammation and migration. These results suggest that the inflammatory reaction of microglia may be one of the possible mechanisms of neurotoxicity caused by vanadium exposure.
Objective To observe the changes of rat microglial inflammation and migration after exposure to sodium metavanadate( Na VO3·2H2O),and to analyze the possible mechanisms of vanadium neurotoxicity. Methods Primary cultured rat microglial cells were incubated with Na VO3·2H2O. Morphological changes and the Iba1 expression of microglia were tested by immunofluorescence assay. i NOS,Cox-2,ERK and p-ERK protein expressions were determined by western blotting. The levels of TNF-α and IL-1β in the culture medium were tested by enzyme-linked immunosorbent assay. The migration of microglia was tested by immunofluorescence staining using wound-healing assay. Results Microglia changed from resting state with ramous shape to round shape in activated state after Na VO3·2H2O exposure,and the expression of Iba1 increased obviously. The protein expressions of i NOS and COX-2 increased significantly compared with the control. The levels of TNF-α and IL-1β were also increased significantly. Na VO3·2H2O promotes the migration of microglia through ERK pathway. Conclusions Exposure to Na VO3·2H2O promotes primary cultured rat microglial inflammation and migration. These results suggest that the inflammatory reaction of microglia may be one of the possible mechanisms of neurotoxicity caused by vanadium exposure.
引文
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[4]Liu Y,Woodin MA,Smith TJ,et al.Exposure to fuel-oil ash and welding emissions during the overhaul of an oil-fired boiler[J].J Occup Environ Hyg,2005,2(9):435-443.
[5]崔鹂,张勤,兰亚佳.钒及其化合物的神经行为毒性研究进展[J].毒理学杂志,2013,27(01):64-67.
[6]Maciejczyk P,Chen LC.Effects of subchronic exposures to concentrated ambient particles(CAPs)in mice.VIII.Sourcerelated daily variations in in vitro responses to CAPs[J].Inhal Toxicol,2005,17(4-5):243-253.
[7]Nimmerjahn A,Kirchhoff F,Helmchen F.Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo[J].Science,2005,308(5726):1314-1318.
[8]Prinz M,Mildner A.Microglia in the CNS:immigrants from another world[J].Glia,2011,59(2):177-187.
[9]Graeber MB.Changing face of microglia[J].Science,2010,330(6005):783-788.
[10]Orre M,Kamphuis W,Osborn LM,et al.Isolation of glia from Alzheimer's mice reveals inflammation and dysfunction[J].Neurobiol Aging,2014,35(12):2746-2760.
[11]Prokop S,Miller KR,Heppner FL.Microglia actions in Alzheimer’s disease[J].Acta Neuropathol,2013,126(4):461-477.
[12]Luo J,Sun Y,Lin H,et al.Activation of JNK by vanadate induces a Fas-associated death domain(FADD)-dependent death of cerebellar granule progenitors in vitro[J].J Biol Chem,2003,278(7):4542-4551.
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[14]Todorich B,Olopade JO,Surguladze N,et al.The mechanism of vanadium-mediated developmental hypomyelination is related to destruction of oligodendrocyte progenitors through a relationship with ferritin and iron[J].Neurotox Res,2011,19(3):361-373.
[15]Cudaback E,Li X,Montine KS,et al.Apolipoprotein E isoform-dependent microglia migration[J].FASEB J,2011,25(6):2082-2091.
[16]Glass CK,Saijo K,Winner B,et al.Mechanisms underlying inflammation in neurodegeneration[J].Cell,2010,140(6):918-934.
[17]Innamorato NG,Lastres-Becker I,Cuadrado A.Role of microglial redox balance in modulation of neuroinflammation[J].Curr Opin Neurol,2009,22(3):308-314.
[18]Iadecola C,Anrather J.The immunology of stroke:from mechanisms to translation[J].Nat Med,2011,17(7):796-808.
[19]Harms AS,Barnum CJ,Ruhn KA,et al.Delayed dominant-negative TNF gene therapy halts progressive loss of nigral dopaminergic neurons in a rat model of Parkinson’s disease[J].Mol Ther,2011,19(1):46-52.
[20]Wake H,Moorhouse AJ,Nabekura J.Functions of microglia in the central nervous system—beyond the immune response[J].Neuron Glia Biol,2011,7(1):47-53.