摘要
[目的]探讨大气细颗粒物(PM_(2.5))对人肺腺癌A549细胞合成一氧化氮(NO)、诱导型一氧化氮合酶(iNOS)活性的影响及其机制。[方法]采用空气总悬浮颗粒物采样器采集南京市大厂区空气中PM_(2.5),并制成0、12、25、50、100、200、400μg/m L悬液,染毒A549细胞,采用MTT比色法测定细胞毒性;用硝酸还原酶法测NO;用一氧化氮合酶测定试剂盒测iNOS;用实时定量PCR法检测iNOS m RNA表达水平的改变;用蛋白质免疫印迹(Western blot)法检测iNOS、核转录因子-κB(NF-κB)和丝裂原活化蛋白激酶(p38-MAPK)蛋白表达水平,p65-NF-κB和p38-MAPK两条通路的抑制剂分别采用PDTC和SB203580。[结果]PM_(2.5)与A549细胞存活率呈剂量-反应关系(r=-0.971,P<0.05);NO和iNOS释放量均随剂量增加而升高(rNO=0.989,riNOS=0.950,均P<0.05),染毒剂量为100μg/m L时,NO释放量高达(97.40±11.11)μmol/L,是对照组的6.59倍;iNOS释放量为(16.16±0.75)U/m L,与对照组相比增加了约29%。经SB203580和PDTC两种抑制剂预处理后,iNOS释放量下降:抑制剂SB203580浓度为25μmol/L时,iNOS释放量为(3.149±0.139)U/m L;抑制剂PDTC浓度为25μmol/L时,iNOS释放量为(4.361±0.182)U/m L,约占对照组1/4;iNOSm RNA表达量下调,各染毒组与对照组相比,差异均有统计学意义(P<0.05)。两种抑制剂均能抑制PM_(2.5)诱导的A549细胞iNOS蛋白表达。[结论]PM_(2.5)引起A549细胞存活率降低,具有剂量-反应关系,且剂量≥25μg/m L的PM_(2.5)可刺激A549细胞NO和iNOS的合成,PM_(2.5)可能通过NF-κB和MAPK两条通路调控iNOS的合成。
[Objective]To investigate the effects and mechanism of PMz5 on change of nitric oxide(NO)and inducible nitric oxide synthase(iNOS)synthesis in human lung adenocarcinoma A549 cells.[Methods]PM2.5 was collected from Dachang District of Nanjing and made into different concentrations(0,12,25,50,100,200,and 400μg/mL)for exposure.MTT assay was used for detecting cytotoxicity.No and iNOS were assessed using nitrate reductase assay and nitric oxide synthase test kit.The expression level of iNOS mRNA was detected by real-time quantitative PCR;and the protein expressions of iNOS,nuclear factor kappa B(NF-κB),and p38 mitogen-activated protein kinases(p38-MAPK)were detected by Western blot.PDTC and SB203580 were used as inhibitors for p65-NF-κB and p38-MAPK pathways.[Results]PM2.5 affected the survival rate of A549 cells in a dose-response manner(r=-0.971,P〈0.05),and the emissions of NO and iNOS were increased with higher doses(rNo=0.989,riNOS=0.950,both Ps〈0.05).When PM2.5 was 100μg/mL,NO emission was(97.40±11.11)μmol/L,6.59 times as much as the control group,and iNOS emission was(16.16±0.75)U/mL,29%more than the control group.After preprocessed using SB203580 and PDTC,the iNOS emission declined.When SB203580 was 25μmol/L,the iNOS release was(3.149±0.139)U/mL;when PDTC was 25μmol/L,the iNOS release was(4.361±0.182)U/mL,approximately 1/4 of the control group.Inhibitors also down-regulated the expression of iNOS mRNA,and there was a significant difference between various exposure groups and the control group(P〈0.05).Both the inhibitors'inhibited the protein expressions of iNOS in A549 cells.[Conclusion]PM2.5 could decrease cell survival rate in a dose-response manner and stimulate the ceils to synthesize NO and iNOS with the PM2.5 dose of≥25μg/mL.PM2.5 may regulate the synthesis of iNOS through NF-κB and MAPK pathways.
作者
昂盛骏
熊丽林
唐萌
ANG Sheng-jun;XIONG Li-lin;TANG Meng(Jiangsu Key Laboratory for Biomaterials and Devices/Key Laboratory of Environmental Medicine Engineering of Ministry of Education/Suzhou Collaborative Innovation Center of Nano Science and Technology,School of Public Health,Southeast University,Nanjing,Jiangsu 210009,China)
出处
《环境与职业医学》
CAS
CSCD
北大核心
2016年第5期433-437,共5页
Journal of Environmental and Occupational Medicine
基金
国家重大科学研究计划(编号:2011CB933404)
