Nuclear factor-kappaB regulates the transcription of NADPH oxidase 1 in human alveolar epithelial cells

Objective Acute lung injury (ALI) is characterized by inflammation and oxidative stress. Nuclear factor-kappaB (NF-κB) mediates the expression of various inflammation-related genes, including the NADPH oxidase family. This study aimed to identify the potential regulatory role of NF-κB on NADPH oxidases in tumor necrosis factor-α (TNF-α)-induced oxidative stress in human alveolar epithelial cells. Methods A549 cells were treated with TNF-α for 24 h to establish ALI cell models. RT-PCR, western blot, assessment of oxidative stress, Alibaba 2.1 online analysis, electrophoretic mobility shift assays and luciferase reporter analysis were employed to identify the potential regulatory role of NF-κB on NADPH oxidases in TNF-α-induced oxidative stress in human alveolar epithelial cells. Results The expression of NF-κB/p65 was notably upregulated in TNF-α-stimulated A549 cells. NF-κB knockdown by siRNA significantly inhibited the TNF-α-induced oxidative stress. Moreover, NF-κB/p65 siRNA could inhibit the activation of NOX1, NOX2 and NOX4 mRNA and protein expression in TNF-α-stimulated A549 cells. The next study demonstrated that NF-κB activated the transcription of NOX1 by binding to the -261 to -252 bp (NOX1/κB2, TAAAAATCCC) region of NOX1 promoter in TNF-α-stimulated A549 cells. Conclusion Our data demonstrated that NF-κB can aggravate TNF-α-induced ALI by regulating the oxidative stress response and the expression of NOX1, NOX2 and NOX4. Moreover, NF-κB could promote the NOX1 transcriptional activity via binding its promoter in TNF-α-stimulated A549 cells.

Synchrotron-based X-ray fluorescence microscopy reveals accumulation of polymyxins in single human alveolar epithelial cells

Intravenous administration of the last-line polymyxins results in poor drug exposure in the lungs and potential nephrotoxicity; while inhalation therapy offers better pharmacokinetics/pharmacodynamics for pulmonary infections by delivering the antibiotic to the infection site directly. However, polymyxin inhalation therapy has not been optimized and adverse effects can occur. This study aimed to quantitatively determine the intracellular accumulation and distribution of polymyxins in single human alveolar epithelial A549 cells. Cells were treated with an iodine-labeled polymyxin probe FADDI-096 (5.0 and 10.0 μM) for 1, 4, and 24 h. Concentrations of FADDI-096 in single A549 cells were determined by synchrotron-based X-ray fluorescence microscopy. Concentration- and time-dependent accumulation of FADDI-096 within A549 cells was observed. The intracellular concentrations (mean ± SEM, n ≥ 189) of FADDI-096 were 1.58 ± 0.11, 2.25 ± 0.10, and 2.46 ± 0.07 mM following 1, 4 and 24 h of treatment at 10 μM, respectively. The corresponding intracellular concentrations following the treatment at 5 μM were 0.05 ± 0.01, 0.24 ± 0.04, and 0.25 ± 0.02 mM (n ≥ 189). FADDI-096 was mainly localized throughout the cytoplasm and nuclear region over 24 h. The intracellular zinc concentration increased in a concentration- and time-dependent manner. This is the first study to quantitatively map the accumulation of polymyxins in human alveolar epithelial cells and provides crucial insights for deciphering the mechanisms of their pulmonary toxicity. Importantly, our results may shed light on the optimization of inhaled polymyxins in patients and the development of new-generation safer polymyxins.