【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。更多还原

【Abstract】 [Objective] To investigate the effects and mechanism of PM_2.5 on change of nitric oxide（NO） and inducible nitric oxide synthase（iNOS） synthesis in human lung adenocarcinoma A549 cells. [Methods] PM_2.5 was collected from Dachang District of Nanjing and made into different concentrations（0, 12, 25, 50, 100, 200, and 400 μg/m L） 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 m RNA 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] PM_2.5 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 PM_2.5was 100 μg/m L, 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/m L, 29% more than the control group. After preprocessed using SB203580 and PDTC, the iNOS emission declined. When SB203580 was 25 μmol/L, the iNOS release was（3.149±0.139） U/m L; when PDTC was 25 μmol/L, the iNOS release was（4.361±0.182） U/m L, approximately 1/4 of the control group. Inhibitors also down-regulated the expression of iNOS m RNA, 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] PM_2.5 could decrease cell survival rate in a dose-response manner and stimulate the cells to synthesize NO and iNOS with the PM_2.5 dose of ≥25 μg/m L. PM_2.5 may regulate the synthesis of iNOS through NF-κB and MAPK pathways.更多还原