下调paralemmin-3表达对脂多糖诱导的肺泡上皮细胞核转录因子-κB活性的影响
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摘要
目的研究下调paralemmin-3(PALM3)表达对脂多糖(LPS)诱导的肺泡上皮A549细胞核转录因子-κB(NF-κB)活性的影响。方法将针对PALM3的小干扰核糖核苷酸(siRNA)转染A549细胞后(PALM3siRNA转染组),用RT-PCR法检测PALM3 mRNA表达水平,同时设立control siRNA转染组及正常细胞组作为对照。转染48 h后,对3组细胞同时给予LPS刺激12 h后,收集细胞核蛋白和培养上清液,用ELISA的方法检测各组细胞培养上清液中的TNF-α、IL-6和IL-1β水平,用凝胶电泳迁移率实验的方法检测各组细胞NF-κB活性。结果特异性siRNA转染后成功下调PALM3在A549细胞中的表达;给予LPS刺激后,与正常细胞组及control siRNA转染组相比,PALM3 siRNA转染组细胞释放炎性因子TNF-α、IL-6和IL-1β减少,同时PALM3 siRNA转染组细胞中NF-κB活性受抑。结论下调PALM3的表达可减轻A549细胞对LPS诱导的炎症反应,调控PALM3的表达有望成为治疗急性肺损伤/急性呼吸窘迫综合征等炎症失控性疾病的新靶点。
Objective To investigate the effects of down-regulation of paralemmin-3(PALM3) expression on activation of nuclear transcription factor-κB( NF-κB) induced by lipopolysaccharide( LPS) in alveolar epithelial cells(A549 cells).Methods The expression of PALM3 was down-regulated by PALM3 siRNA in A549 cells.At the 48 th hour after transfection,the mRNA level of PALM3 in A549 cells was determined by RT-PCR.Meanwhile,an empty vehicle was used as a negative control group and non-transfection A549 cell was used as a normal control group.LPS was administered into the cell culture medium at the 48 thhour post-transfection.The culture supernatant and nucleoprotein were collected at the 12 th hour after LPS-stimulation.The concentrations of TNF-α,IL-6 and IL-1β were detected by ELISA.The activity of NF-κB was measured by EMSA.Results The PALM3 mRNA expression was successfully suppressed by PALM3 siRNA.Compared with negative and normal control groups,silencing PALM3 could significantly inhibit the release of TNF-α,IL-6 and IL-1β in A549 cells after LPS-stimulation.And,the activity of NF-κB was also inhibited in PALM-suppressed A549 cells.Conclusion Down-regulation of PALM3 expression can attenuate the inflammatory reactions to LPS in A549 cells.Regulating the PALM3 expression may be a novel therapy target for the treatment of acute lung injury / acute respiratory distress syndrome and other inflammatory diseases.
Objective To investigate the effects of down-regulation of paralemmin-3(PALM3) expression on activation of nuclear transcription factor-κB( NF-κB) induced by lipopolysaccharide( LPS) in alveolar epithelial cells(A549 cells).Methods The expression of PALM3 was down-regulated by PALM3 siRNA in A549 cells.At the 48 th hour after transfection,the mRNA level of PALM3 in A549 cells was determined by RT-PCR.Meanwhile,an empty vehicle was used as a negative control group and non-transfection A549 cell was used as a normal control group.LPS was administered into the cell culture medium at the 48 thhour post-transfection.The culture supernatant and nucleoprotein were collected at the 12 th hour after LPS-stimulation.The concentrations of TNF-α,IL-6 and IL-1β were detected by ELISA.The activity of NF-κB was measured by EMSA.Results The PALM3 mRNA expression was successfully suppressed by PALM3 siRNA.Compared with negative and normal control groups,silencing PALM3 could significantly inhibit the release of TNF-α,IL-6 and IL-1β in A549 cells after LPS-stimulation.And,the activity of NF-κB was also inhibited in PALM-suppressed A549 cells.Conclusion Down-regulation of PALM3 expression can attenuate the inflammatory reactions to LPS in A549 cells.Regulating the PALM3 expression may be a novel therapy target for the treatment of acute lung injury / acute respiratory distress syndrome and other inflammatory diseases.
引文
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[2]ZHANG C,WU X,ZHAO Y,et al.SIGIRR inhibits toll-like receptor 4,5,9-mediated immune responses in human airway epithelial cells[J].Mol Biol Rep,2011,38(1):601-609.
[3]CHEN X,WU X,ZHAO Y,et al.A novel binding protein of single immunoglobulin IL-1 receptor-related molecule:Paralemmin-3[J].Biochem Biophys Res Commun,2011,404(4):1029-1033.
[4]TIAN F,NI Y F,ZHAO J B,et al.Single immunoglobulin IL-1 receptor-related protein attenuates the lipopolysaccharide-induced inflammatory response in A549 cells[J].Chem Biol Interact,2010,183(3):442-449.
[5]CORNISH J A,KLOC M,DECKER G L,et al.Xlcaax-1 is localized to the basolateral membrane of kidney tubule and other polarized epithelia during xenopus development[J].Dev Biol,1992,150(1):108-120.
[6]HU B,PETRASCH PARWEZ E,LAUE MM,et al.Molecular characterization and immunohistochemical localization of palmdelphin,a cytosolic isoform of the paralemmin protein family implicated in membrane dynamics[J].Eur J Cell Biol,2005,84(11):853-866.
[7]KUTZLEB C,SANDERS G,YAMAMOTO R,et al.Paralemmin,a prenyl-palmitoyl-anchored phosphoprotein abundant in neurons and implicated in plasma membrane dynamics and cell process formation[J].J Cell Biol,1998,143(3):795-813.
[8]陈旭昕,吴学玲,陈华萍,等.SIGIRR过表达对LPS诱导的H292细胞NF-κB活性的影响[J].重庆医学,2011,40(8):732-734.
[9]MUKHOPADHYAY S,HOIDAL J R,MUKHERJEE T K.Role of TNF-αin pulmonary pathophysiology[J].Respir Res,2006,7:125.
[10]KNEPLER J L,TALER I N,GUPTA M P,et al.Peroxynitrite causes endothelial cell monolayer barrier dysfunction[J].Am J Physiol Cell Physiol,2001,281(3):C1064-C1075.