全氟化碳对脂多糖诱导肺泡上皮细胞炎性反应及单免疫球蛋白白介素-1相关蛋白表达的影响
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摘要
目的探讨全氟化碳(PFC)对脂多糖(LPS)诱导人肺泡上皮A549细胞炎性反应及单免疫球蛋白白介素-1受体相关蛋白(SIGIRR)表达的影响。方法实验分为正常细胞组、全氟化碳+脂多糖(PFC+LPS)组、全氟化碳(PFC)组及脂多糖(LPS)组。各组细胞经相应处理24h后,用酶联免疫吸附法(ELISA)检测培养上清中的TNF-α、IL-6和IL-10含量,用蛋白免疫印迹法(Western-blot)检测核转录因子-κB(NF-κB)和SIGIRR的表达。结果 ELISA结果显示,LPS组TNF-α、IL-6水平较正常细胞组升高(P<0.05);PFC+LPS组TNF-α、IL-6水平较LPS组下降(P<0.05);与正常细胞组相比,PFC组IL-10水平升高(P<0.05),PFC+LPS组IL-10水平较LPS组升高(P<0.05)。Western-blot结果显示,与正常细胞组相比,LPS组核内磷酸化NF-κB p65表达上调(P<0.05),PFC+LPS组核内磷酸化NF-κB p65表达水平较LPS组降低(P<0.05);与正常细胞组相比,LPS组SIGIRR表达水平下调(P<0.05),PFC+LPS组SIGIRR表达水平较LPS组上调(P<0.05)。PFC本身对TNF-α、IL-6含量及NF-κB p65表达无影响,但PFC可促进A549细胞释放IL-10及上调SIGIRR表达(P<0.05)。结论 PFC可减轻A549细胞对LPS诱导的炎性反应,促进抗炎因子IL-10分泌及上调负调控分子SIGIRR表达是可能的分子机制。
Objective To investigate the Effects of perfluorocarbon on the inflammatory reaction induced by LPS and the expression of single immunoglobin IL-1receptor related protein(SIGIRR)in alveolar epithelial cells(A549cells).Methods The A549 cells were divided into four groups(normal cell group,PFC+LPS group,PFC group and LPS group).A549 cells of each group were incubated for 24 h,and then the culture supernatants,cellular total protein and cellular nuclear protein were collected.The levels of TNF-α,IL-6and IL-10 in the culture supernatants were detected by ELISA.The protein expression levels of NF-κB p65 in nuclear extracts and the protein expression levels of SIGIRR in A549 cells of each group were also measured by Western-blot analysis.Results The results of ELISA demonstrated that the levels of TNF-αand IL-6in LPS group were higher than those in normal cell group(P<0.05).Compared with LPS group,the levels of TNF-αand IL-6in PFC+LPS group were significantly decreased(P<0.05).Meanwhile,the results of ELISA also demonstrated that the levels of IL-10 in PFC+LPS group were higher than those in LPS group and PFC could promote IL-10 secretion in A549cells(P<0.05).The results of Western-blot analysis showed that the protein levels of phospho-NF-κB p65 in LPS group were higher than those in normal cell group(P<0.05).Compared with LPS group,the protein levels of phospho-NF-κB p65 in PFC+LPS group were significantly decreased(P<0.05).Moreover,the results of Western-blot analysis also showed that the protein levels of SIGIRR in PFC+LPS group were higher than those in LPS group and PFC could upregulate the expression of SIGIRR in A549cells(P<0.05),conversely,LPS suppressed the expression of SIGIRR in A549 cells.Conclusion PFC could attenuate the LPS-induced inflammation in alveolar epithelial cells.Promotion of IL-10 secretion and upregulaiton SIGIRR expression may the possible mechanism of PFC protection against LPS-induce inflammation in alveolar epithelial cells.
Objective To investigate the Effects of perfluorocarbon on the inflammatory reaction induced by LPS and the expression of single immunoglobin IL-1receptor related protein(SIGIRR)in alveolar epithelial cells(A549cells).Methods The A549 cells were divided into four groups(normal cell group,PFC+LPS group,PFC group and LPS group).A549 cells of each group were incubated for 24 h,and then the culture supernatants,cellular total protein and cellular nuclear protein were collected.The levels of TNF-α,IL-6and IL-10 in the culture supernatants were detected by ELISA.The protein expression levels of NF-κB p65 in nuclear extracts and the protein expression levels of SIGIRR in A549 cells of each group were also measured by Western-blot analysis.Results The results of ELISA demonstrated that the levels of TNF-αand IL-6in LPS group were higher than those in normal cell group(P<0.05).Compared with LPS group,the levels of TNF-αand IL-6in PFC+LPS group were significantly decreased(P<0.05).Meanwhile,the results of ELISA also demonstrated that the levels of IL-10 in PFC+LPS group were higher than those in LPS group and PFC could promote IL-10 secretion in A549cells(P<0.05).The results of Western-blot analysis showed that the protein levels of phospho-NF-κB p65 in LPS group were higher than those in normal cell group(P<0.05).Compared with LPS group,the protein levels of phospho-NF-κB p65 in PFC+LPS group were significantly decreased(P<0.05).Moreover,the results of Western-blot analysis also showed that the protein levels of SIGIRR in PFC+LPS group were higher than those in LPS group and PFC could upregulate the expression of SIGIRR in A549cells(P<0.05),conversely,LPS suppressed the expression of SIGIRR in A549 cells.Conclusion PFC could attenuate the LPS-induced inflammation in alveolar epithelial cells.Promotion of IL-10 secretion and upregulaiton SIGIRR expression may the possible mechanism of PFC protection against LPS-induce inflammation in alveolar epithelial cells.
引文
[1]ZhouZ,You Z.Mesenchymal Stem Cells Alleviate LPS-Induced Acute Lung Injury in Mice by MiR-142a-5p-Controlled Pulmonary Endothelial Cell Autophagy[J].Cell Physiol Biochem,2016,38(1):258-266.
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[7]陈旭昕,孟激光,韩志海,等.干扰paralemmin-3表达抑制脂多糖诱导的肺泡上皮细胞的炎性反应[J].西部医学,2015,27(7):964-966,970.
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[11]曹璐,李春笋,梁志欣,等.全氟化碳治疗ALI的细胞生物学研究进展[J].国际呼吸杂志,2015,35(14):1093-1097.
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[13]徐顺贵,吴国明,徐智,等.全氟化碳吸入治疗急性肺损伤的研究进展[J].国际呼吸杂志,2007,27(18):1434-1437.
[14]唐瑾,郭忠良.全氟化碳在治疗ALI/ARDS时对促炎因子影响的研究进展[J].同济大学学报:医学版,2014,(4):129-132.
[15]KochT,Ragaller M,Haufe D,et al.Perfluorohexane attenuates proinflammatory and procoagulatory response of activated monocytes and alveolar macrophages[J].Anesthesiology,2001,94(1):101-109.
[16]WoodsCM,Neslund G,Kornbrust E,et al.Perflubron attenuates neutrophil adhesion to activated endothelial cells in vitro[J].Am J Physiol Lung Cell Mol Physiol,2000,278(5):L1008-1017.
[17]McDonaghP,Cerney K,Hokama J,et al.Perflubron emulsion reduces inflammation during extracorporeal circulation[J].J Surg Res,2001,99(1):7-16.
[18]HashimotoN,Kawabe T,Imaizumi K,et al.CD40plays a crucial role in lipopolysaccharide-induced acute lung injury[J].Am J Respir Cell Mol Biol,2004,30(6):808-815.
[19]LiuLM,Tu WJ,Zhu T,et al.IRF3is an important molecule in the UII/UT system and mediates immune inflammatory injury in acute liver failure[J].Oncotarget,2016,7(31):49027-49041.
[20]ChenX,Wu X,Zhao Y,et al.A novel binding protein of single immunoglobulin IL-1receptor-related molecule:Paralemmin-3[J].Biochem Biophys Res Commun,2011,404(4):1029-1033.
[21]ChenX,Zhao Y,Wu X,et al.Enhanced expression of single immunoglobulin IL-1receptor-related molecule ameliorates LPSinduced acute lung injury in mice[J].Shock,2011,35(2):198-204.
[2]LiJW,Wu X.Mesenchymal stem cells ameliorate LPS-induced acute lung injury through KGF promoting alveolar fluid clearance of alveolar type II cells[J].Eur Rev Med Pharmacol Sci,2015,19(13):2368-2378.
[3]钱桂生,陈旭昕.急性肺损伤/急性呼吸窘迫综合征发病机制的研究进展[J].内科理论与实践,2010,5(6):460-463.
[4]陈旭昕,孟激光,张燕,等.干扰PALM3表达对小鼠内毒素性急性肺损伤的影响[J].广东医学,2015,(20):3101-3104.
[5]陈旭昕.SIGIRR与TLRs信号通路[J].四川医学,2011,32(3):428-430.
[6]付玉梅,陈旭昕,韩志海.Toll样受体4信号传导通路在急性肺损伤发病机制中的作用[J].转化医学杂志,2015,(4):236-239.
[7]陈旭昕,孟激光,韩志海,等.干扰paralemmin-3表达抑制脂多糖诱导的肺泡上皮细胞的炎性反应[J].西部医学,2015,27(7):964-966,970.
[8]BleylJU,Ragaller M,Tsch9 U,et al.Changes in pulmonary function and oxygenation during application of perfluorocarbon vapor in healthy and oleic acid-injured animals[J].Crit Care Med,2002,30(6):1340-1347.
[9]GalvinIM,Steel A,Pinto R,et al.Partial liquid ventilation for preventing death and morbidity in adults with acute lung injury and acute respiratory distress syndrome[J].Cochrane Database Syst Rev,2013,(7):CD003707.
[10]尹晓峰,钱国强,樊毫军.全氟化碳乳剂预处理对急性肺损伤大鼠的保护作用[J].中国急救医学,2014,34(1):53-56.
[11]曹璐,李春笋,梁志欣,等.全氟化碳治疗ALI的细胞生物学研究进展[J].国际呼吸杂志,2015,35(14):1093-1097.
[12]郭忠良,梁永杰.液体通气在急性呼吸窘迫综合征中的研究进展[J].国际呼吸杂志,2009,29(24):1500-1504.
[13]徐顺贵,吴国明,徐智,等.全氟化碳吸入治疗急性肺损伤的研究进展[J].国际呼吸杂志,2007,27(18):1434-1437.
[14]唐瑾,郭忠良.全氟化碳在治疗ALI/ARDS时对促炎因子影响的研究进展[J].同济大学学报:医学版,2014,(4):129-132.
[15]KochT,Ragaller M,Haufe D,et al.Perfluorohexane attenuates proinflammatory and procoagulatory response of activated monocytes and alveolar macrophages[J].Anesthesiology,2001,94(1):101-109.
[16]WoodsCM,Neslund G,Kornbrust E,et al.Perflubron attenuates neutrophil adhesion to activated endothelial cells in vitro[J].Am J Physiol Lung Cell Mol Physiol,2000,278(5):L1008-1017.
[17]McDonaghP,Cerney K,Hokama J,et al.Perflubron emulsion reduces inflammation during extracorporeal circulation[J].J Surg Res,2001,99(1):7-16.
[18]HashimotoN,Kawabe T,Imaizumi K,et al.CD40plays a crucial role in lipopolysaccharide-induced acute lung injury[J].Am J Respir Cell Mol Biol,2004,30(6):808-815.
[19]LiuLM,Tu WJ,Zhu T,et al.IRF3is an important molecule in the UII/UT system and mediates immune inflammatory injury in acute liver failure[J].Oncotarget,2016,7(31):49027-49041.
[20]ChenX,Wu X,Zhao Y,et al.A novel binding protein of single immunoglobulin IL-1receptor-related molecule:Paralemmin-3[J].Biochem Biophys Res Commun,2011,404(4):1029-1033.
[21]ChenX,Zhao Y,Wu X,et al.Enhanced expression of single immunoglobulin IL-1receptor-related molecule ameliorates LPSinduced acute lung injury in mice[J].Shock,2011,35(2):198-204.