α1抗胰蛋白酶抑制脂多糖诱导肺泡上皮细胞炎症因子的分泌
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摘要
目的探讨α1抗胰蛋白酶(AAT)对脂多糖(LPS)诱导肺泡上皮细胞炎症因子分泌的影响及其机制。方法选择具有肺泡上皮细胞特征的非小细胞肺癌细胞系A549为研究对象,将其分为对照组、AAT(0.5mg/mL)组、LPS(10μg/mL)组、AAT+LPS组4组。分别加入AAT和LPS进行干预和诱导,细胞培养10h后收集并提取蛋白,采用蛋白免疫印迹法(Western Blot)检测核转录因子-κB(NF-κB)抑制蛋白IκBα和NF-κB p65的表达水平;培养18h后分别收集细胞培养上清和细胞,采用酶联免疫吸附法(ELISA)检测细胞上清中肿瘤坏死因子-α(TNF-α)、白细胞介素-8(IL-8)和单核细胞趋化因子-1(MCP-1)的含量,采用实时荧光定量PCR检测细胞中白细胞介素-1β(IL-1β)的mRNA表达水平。结果与LPS组比较,AAT+LPS组细胞培养上清中TNF-α、IL-8、MCP-1的含量明显降低,细胞中IL-1βmRNA的水平降低,组间比较差异有统计学意义(P<0.05);Western blot结果显示,与LPS组比较,AAT+LPS组IκBα的磷酸化和细胞核内NF-κB p65的蛋白水平明显下降。结论 AAT能够有效地抑制LPS诱导肺泡上皮细胞炎症的因子的分泌,此过程可能是通过抑制NF-κB的激活和进入细胞核而发挥作用。
Objective To investigate the effects and underlying mechanisms ofα1-antitrypsin(AAT)on inflammatory cytokines production induced by lipopolysaccharide(LPS)in lung alveolar epithelial cells.Methods Human alveolar epithelial cells A549 were divided into control group,AAT(0.5 mg/mL)group,LPS(10μg/mL)group and AAT+LPS group,after 10 hincubation,the expression levels of phosphorylated IκBαand NF-κB p65 subunit were determined by Western Blot.And after 18 hincubation,TNF-α,IL-8and monocyte chemotactic factor-1(MCP-1)contents in cell supernatant were determined by ELISA,and the mRNA level of IL-1βwas quantified by quantitative real time PCR.Results α1-antitrypsin eliminated the phosphorylation of IκBαand the nuclear distribution of NF-κB induced by LPS.In addition,compared with the LPS-treated cells,the levels of TNF-α,IL-8,MCP-1in cell culture medium and the IL-1βmRNA level were significantly lower in LPS plus AAT treated cells(P <0.05).Western Blot showed that compared with the LPS group,the phosphorylation of IκBαand the nuclear translocation of NF-κB p65 were significantly reduced in LPS+AAT group.Conclusion α1-antitrypsin inhibits pro-inflammatory cytokines production induced by LPS via reducing the NF-κB nuclear translocation.
Objective To investigate the effects and underlying mechanisms ofα1-antitrypsin(AAT)on inflammatory cytokines production induced by lipopolysaccharide(LPS)in lung alveolar epithelial cells.Methods Human alveolar epithelial cells A549 were divided into control group,AAT(0.5 mg/mL)group,LPS(10μg/mL)group and AAT+LPS group,after 10 hincubation,the expression levels of phosphorylated IκBαand NF-κB p65 subunit were determined by Western Blot.And after 18 hincubation,TNF-α,IL-8and monocyte chemotactic factor-1(MCP-1)contents in cell supernatant were determined by ELISA,and the mRNA level of IL-1βwas quantified by quantitative real time PCR.Results α1-antitrypsin eliminated the phosphorylation of IκBαand the nuclear distribution of NF-κB induced by LPS.In addition,compared with the LPS-treated cells,the levels of TNF-α,IL-8,MCP-1in cell culture medium and the IL-1βmRNA level were significantly lower in LPS plus AAT treated cells(P <0.05).Western Blot showed that compared with the LPS group,the phosphorylation of IκBαand the nuclear translocation of NF-κB p65 were significantly reduced in LPS+AAT group.Conclusion α1-antitrypsin inhibits pro-inflammatory cytokines production induced by LPS via reducing the NF-κB nuclear translocation.
引文
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[16]GRIMSTEIN C,CHOI Y K,SATOH M,et al.Combination of alpha-1antitrypsin and doxycycline suppresses collagen-induced arthritis[J].J Gene Med,2010,12(1):35-44.
[17]ZHANG B,LU Y,CAMPBELL-THOMPSON M,et al.Alpha1-antitrypsin protects beta-cells from apoptosis[J].Diabetes,2007,56(5):1316-1323.
[18]JEDICKE N,STRUEVER N,AGGRAWAL N,et al.alpha-1-antitrypsin inhibits acute liver failure in mice[J].Hepatology,2014,59(6):2299-2308.
[2]WEIR G C,KOULAMNDA M.Control of inflammation with alpha1-antitrypsin:apotential treatment for islet transplantation and new-onset type 1diabetes[J].Curr Diab Rep,2009,9(2):100-102.
[3]GERAGHTY P,EDEN E,PILLAI M,et al.alpha1-Antitrypsin activates protein phosphatase 2Ato counter lung inflammatory responses[J].Am J Respir Crit Care Med,2014,190(11):1229-1242.
[4]ELSHIKHA A S,LU Y,CHEN M J,et al.Alpha 1 Antitrypsin Inhibits Dendritic Cell Activation and Attenuates Nephritis in a Mouse Model of Lupus[J].PLoS One,2016,11(5):e0156583.
[5]DOS SANTOS C C,HAN B,ANDRADE C F,et al.DNA microarray analysis of gene expression in alveolar epithelial cells in response to TNFalpha,LPS,and cyclic stretch[J].Physiol Genomics,2004,19(3):331-342.
[6]SUNIL V R,CONNOR A J,GUO Y,et al.Activation of type II alveolar epithelial cells during acute endotoxemia[J].Am J Physiol Lung Cell Mol Physiol,2002,282(4):872-880.
[7]SHARMA A K,FERNANDEZ L G,AWAD A S,et al.Proinflammatory response of alveolar epithelial cells is enhanced by alveolar macrophage-produced TNF-alpha during pulmonary ischemia-reperfusion injury[J].Am J Physiol Lung Cell Mol Physiol,2007,293(1):105-113.
[8]SORRELLS S,CAMPRUBI S,GRIFFIN R,et al.SPARTA clinical trial design:exploring the efficacy and safety of two dose regimens of alpha1-proteinase inhibitor augmentation therapy in alpha1-antitrypsin deficiency[J].Respir Med,2015,109(4):490-499.
[9]MULGREW A T,TAGGART C C,MCELVANEY N G.Alpha-1-antitrypsin deficiency:current concepts[J].Lung,2007,185(4):191-201.
[10]TWIGG M S,BROCKBANK S,LOWRY P,et al.The role of serine proteases and antiproteases in the cystic fibrosis lung[J].Mediators Inflamm,2015,2015:293053.
[11]SINDEN N J,BAKER M J,SMITH D J,et al.alpha-1-antitrypsin variants and the proteinase/antiproteinase imbalance in chronic obstructive pulmonary disease[J].Am J Physiol Lung Cell Mol Physiol,2015,308(2):179-190.
[12]张振军,陈菲,樊一笋,等.氧化型α1-抗胰蛋白酶对HBE细胞释放炎症因子的影响[J].中国病理生理杂志,2013,29(11):2054-2059.
[13]LI Z,ALAM S,WANG J,et al.Oxidized{alpha}1-antitrypsin stimulates the release of monocyte chemotactic protein-1from lung epithelial cells:potential role in emphysema[J].Am J Physiol Lung Cell Mol Physiol,2009,297(2):388-400.
[14]GOOPTU B,LOMAS D A.Conformational pathology of the serpins:themes,variations,and therapeutic strategies[J].Annu Rev Biochem,2009,78:147-176.
[15]GRIMSTEIN C,CHOI Y K,WASSERFALL C H,et al.Alpha-1antitrypsin protein and gene therapies decrease autoimmunity and delay arthritis development in mouse model[J].J Transl Med,2011,9:21.
[16]GRIMSTEIN C,CHOI Y K,SATOH M,et al.Combination of alpha-1antitrypsin and doxycycline suppresses collagen-induced arthritis[J].J Gene Med,2010,12(1):35-44.
[17]ZHANG B,LU Y,CAMPBELL-THOMPSON M,et al.Alpha1-antitrypsin protects beta-cells from apoptosis[J].Diabetes,2007,56(5):1316-1323.
[18]JEDICKE N,STRUEVER N,AGGRAWAL N,et al.alpha-1-antitrypsin inhibits acute liver failure in mice[J].Hepatology,2014,59(6):2299-2308.