Effects of PM_2.5 on iNOS Synthesis in Human Lung Adenocarcinoma A549 Cells and Related Mechanism

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/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 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 (r_NO=0.989, r_iNOS=0.950, both Ps < 0.05). When PM_2.5 was 100 μg/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 25 μmol/L, the iNOS release was (3.149±0.139) U/mL; when PDTC was 25 μmol/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 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/mL. PM_2.5 may regulate the synthesis of iNOS through NF-κB and MAPK pathways.