二苯基碘和夹竹桃麻素通过降低早产儿外周血单个核细胞p47phox位移与水平减少活性氧的产生
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摘要
目的观察NADPH氧化酶抑制剂二苯基碘(DPI)和夹竹桃麻素(apocynin)对NADPH氧化酶亚基p47phox介导的活性氧(ROS)产生的影响,探讨细胞在高氧条件下,p47phox介导ROS产生的机制。方法 32周以下早产儿,尚未吸氧前取外周血2 m L,分离纯化外周血单个核细胞(PBMC)将所得细胞分为对照组、高氧组、高氧联合DPI处理组、高氧联合apocynin处理组进行培养。对照组置于37℃、50 m L/L的CO2培养箱中,高氧及相应处理组置于950 m L/L的O2与50 m L/L的CO2混合气体中培养48 h。采用Mitosox Red标记结合激光共聚焦显微镜检测PBMC内ROS的生成量、硫代巴比妥酸比色法检测培养液丙二醛含量、免疫荧光检测p47phox在细胞内的定位及移位率、Western blot法检测p47phox的蛋白水平。结果与高氧组相比,其余三组ROS和丙二醛明显减少,p47phox移位率与含量也显著降低;与对照组相比,高氧联合DPI处理组及高氧联合apocynin处理组ROS、丙二醛、p47phox移位率与含量并无显著差异。结论 DPI和apocynin能通过降低p47phox移位与含量来减少高氧诱导的ROS升高。
Objective To observe the effects of NADPH oxidase inhibitor diphenylene iodonium( DPI) and apocynin on the generation of reactive oxygen species( ROS) induced by p47 phox and the mechanism of p47phox-induced ROS production under hyperoxic conditions. Methods Peripheral blood mononuclear cells( PBMCs) were isolated from the peripheral blood( 2 m L) of premature infants of less than 32 weeks without oxygen uptake. The isolated cells were divided into four groups,control group,hyperoxia group,hyperoxia and DPI group,hyperoxia and apocynin group. The control group was cultured in incubator with 50 m L / L CO2 at 37℃,and the other groups were cultured in 950 m L / L O2 and 50 m L / L CO2 mixed gas. After 48 hours,ROS was detected by Mitosox Red staining under a confocal laser scanning microscope;malondialdehyde( MDA) was measured by thiobarbituric acid colorimetry; the location and translocation rate of p47 phox was observed by immunofluorescence staining; the level of p47 phox protein was tested by Western blotting. Results Compared with the hyperoxia group,the remaining three groups showed significantly decreased ROS and MDA levels and reduced translocation rate and level of p47 phox. Compared with the control group,both the hyperoxia and DPI group and the hyperoxia and apocynin group were not significantly different in the above indexes. Conclusion DPI and apocynin can reduce hyperoxia-induced ROS production by decreasing the translocation and level of p47 phox.
Objective To observe the effects of NADPH oxidase inhibitor diphenylene iodonium( DPI) and apocynin on the generation of reactive oxygen species( ROS) induced by p47 phox and the mechanism of p47phox-induced ROS production under hyperoxic conditions. Methods Peripheral blood mononuclear cells( PBMCs) were isolated from the peripheral blood( 2 m L) of premature infants of less than 32 weeks without oxygen uptake. The isolated cells were divided into four groups,control group,hyperoxia group,hyperoxia and DPI group,hyperoxia and apocynin group. The control group was cultured in incubator with 50 m L / L CO2 at 37℃,and the other groups were cultured in 950 m L / L O2 and 50 m L / L CO2 mixed gas. After 48 hours,ROS was detected by Mitosox Red staining under a confocal laser scanning microscope;malondialdehyde( MDA) was measured by thiobarbituric acid colorimetry; the location and translocation rate of p47 phox was observed by immunofluorescence staining; the level of p47 phox protein was tested by Western blotting. Results Compared with the hyperoxia group,the remaining three groups showed significantly decreased ROS and MDA levels and reduced translocation rate and level of p47 phox. Compared with the control group,both the hyperoxia and DPI group and the hyperoxia and apocynin group were not significantly different in the above indexes. Conclusion DPI and apocynin can reduce hyperoxia-induced ROS production by decreasing the translocation and level of p47 phox.
引文
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[16]赵帅,董文斌,张婵.慢病毒介导的shRNA对A549细胞Pin1表达的干扰作用[J].细胞与分子免疫学杂志,2014,30(3):245-249.
[17]张应金.早产儿视网膜病筛查及相关因素的临床分析[J].中华妇幼临床医学杂志,2012,8(2):187-189.
[18]Cavallaro G,Filippi L,Bagnoli P,et al.The pathophysiology of retinopathy of prematurity:an update of previous and recent knowledge[J].Acta Ophthalmol,2014,92(1):2-20.
[19]Lee J H,Lee J S,Kim Y R,et al.Hispidin isolated from Phellinus linteus protects against hydrogen peroxide-induced oxidative stress in pancreatic MIN6Nβ-cells[J].J Med Food,2011,14(11):1431-1438.
[20]Usatyuk P V,Singleton P A,Pendyala S,et al.Novel role for non-muscle myosin light chain kinase(MLCK)in hyperoxia-induced recruitment of cytoskeletal proteins,NADPH oxidase activation,and reactive oxygen species generation in lung endothelium[J].J Biol Chem,2012,287(12):9360-9370.
[21]Tsai K H,Wang W J,Lin C W,et al.NADPH oxidase-derived superoxide anion-induced apoptosis is mediated via the JNK-dependent activation of NF-κB in cardiomyocytes exposed to high glucose[J].J Cell Physiol,2012,227(4):1347-1357.
[22]Kleniewska P,Piechota A,Skibska B,et al.The NADPH oxidase family and its inhibitors[J].Arch Immunol Ther Exp,2012,60(4):277-294.
[2]Elnakish M T,Hassanain H H,Janssen P M,et al.Emerging role of oxidative stress in metabolic syndrome and cardiovascular diseases:important role of Rac/NADPH oxidase[J].J Pathol,2013,231(3):290-300.
[3]Bedard K,Krause K H.The NOX family of ROS-generating NADPH oxidases:physiology and pathophysiology[J].Physiol Rev,2007,87(1):245-313.
[4]Meng T,Yu J,Lei Z,et al.Propofol reduces lipopolysaccharide-induced,NADPH oxidase(NOX 2)mediated TNF-αand IL-6 production in macrophages[J/OL].Clin Dev Immunol,2013,2013:325481.doi:10.1155/2013/325481.Epub 2013 Nov 26.
[5]Casbon A J,Long M E,Dunn K W,et al.Effects of IFN-γon intracellular trafficking and activity of macrophage NADPH oxidase flavocytochrome b558[J].J Leokuc Biol,2012,92(4):869-882.
[6]Meijles D N,Fan L M,Howlin B J,et al.Molecular Insight of p47phox phosphorylation dynamics in the regulation of NADPH oxidase activation and superoxide production[J].J Biol Chem,2014,289(33):22759-22770.
[7]Jackman K A,Miller A A,De Silva T M,et al.Reduction of cerebral infarct volume by apocynin requires pretreatment and is absent in Nox2-deficient mice[J].Br J Pharmacol,2009,156(4):680-688.
[8]Zheng S,Zhong Z M,Qin S,et al.Advanced oxidation protein products induce inflammatory response in fibroblast-like synoviocytes throughNADPH oxidase-dependent activation of NF-κB[J].Cell Physiol Biochem,2013,32(4):972-985.
[9]De Minicis S,Seki E,Paik Y H,et al.Role and cellular source of nicotinamide adenine dinucleotide phosphate oxidase in hepatic fibrosis[J].Hepatology,2010,52(4):1420-1430.
[10]Wind S,Beuerlein K,Eucker T,et al.Comparative pharmacology of chemically distinct NADPH oxidase inhibitors[J].Br J Pharmacol,2010,161(4):885-898.
[11]徐洪涛,杨慧.高体积分数氧致A549细胞肺表面活性物质蛋白-A表达的变化[J].实用儿科临床杂志,2012,27(14):1101-1104.
[12]党嘉文,孙鸿燕,董文斌,等.高氧肺损伤形成过程中小窝蛋白-1、TGF-β1表达关系的体内外研究[J].重庆医学,2013,42(18):2067-2070.
[13]王蕾,吴和平,舒筱灿,等.p47phox向胞膜移位调节高糖导致的内皮细胞内活性氧升高[J].医学研究杂志,2011,40(7):65-67.
[14]付红敏,李利,汤春辉.降钙素基因相关肽调控细胞外信号调节激酶减轻高体积分数氧对胎鼠肺泡Ⅱ型上皮细胞的损伤作用[J].实用儿科临床杂志,2012,27(8):607-610.
[15]Brew N,Hooper S B,Zahra V,et al.Mechanical ventilation injury and repair in extremely and very preterm lungs[J/OL].PLoS One,2013,8(5):e63905.doi:10.1371/journal.pone.0063905.Print2013.
[16]赵帅,董文斌,张婵.慢病毒介导的shRNA对A549细胞Pin1表达的干扰作用[J].细胞与分子免疫学杂志,2014,30(3):245-249.
[17]张应金.早产儿视网膜病筛查及相关因素的临床分析[J].中华妇幼临床医学杂志,2012,8(2):187-189.
[18]Cavallaro G,Filippi L,Bagnoli P,et al.The pathophysiology of retinopathy of prematurity:an update of previous and recent knowledge[J].Acta Ophthalmol,2014,92(1):2-20.
[19]Lee J H,Lee J S,Kim Y R,et al.Hispidin isolated from Phellinus linteus protects against hydrogen peroxide-induced oxidative stress in pancreatic MIN6Nβ-cells[J].J Med Food,2011,14(11):1431-1438.
[20]Usatyuk P V,Singleton P A,Pendyala S,et al.Novel role for non-muscle myosin light chain kinase(MLCK)in hyperoxia-induced recruitment of cytoskeletal proteins,NADPH oxidase activation,and reactive oxygen species generation in lung endothelium[J].J Biol Chem,2012,287(12):9360-9370.
[21]Tsai K H,Wang W J,Lin C W,et al.NADPH oxidase-derived superoxide anion-induced apoptosis is mediated via the JNK-dependent activation of NF-κB in cardiomyocytes exposed to high glucose[J].J Cell Physiol,2012,227(4):1347-1357.
[22]Kleniewska P,Piechota A,Skibska B,et al.The NADPH oxidase family and its inhibitors[J].Arch Immunol Ther Exp,2012,60(4):277-294.