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 TNF-α-induced oxidative stress in human alveolar epithelial cells. Methods Type II alveolar epithelial cell-derived A549 cells were treated with TNF-α for 24 hours to establish ALI cell models. RT-PCR, western blot, DCFH-DA ROS assay, 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 ROS generation. Moreover, NF-𝜅B/p65 siRNA could inhibite 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 activation of ROS generation 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.
Background: TNM stage is widely applied to classify lung cancer and the foundation of clinical decisions. However, increasing studies have pointed out that this staging system is not precise enough especially for the N status. In this study, we aim to build a convenient survival prediction model that incorporated the current items of lymph node status. Methods: We collected data of resectable NSCLC(IA-IIIB) patients from Surveillance, Epidemiology, and End Results (SEER) database (2006-2015). X-tile program was applied to calculate the optimal threshold of metastatic lymph nodes ratio (MLNR). Then, independent prognostic factors were determined by multivariable cox regression analysis and enrolled to build a nomogram model. The calibration curve as well as the concordance index(C-index ) were selected to evaluate the nomogram. Finally, patients were grouped based on their specified risk points and divided into three risk levels. The prognostic value of MLNR and examined lymph nodes number (ELNs) were presented in subgroups. Results: 40853 NSCLC patients after surgery were finally enrolled and analyzed. Age, metastatic lymph nodes ratio, histology type, adjuvant treatment, and AJCC 8th T stage were deemed as independent prognostic parameters after multivariable cox regression analysis. Nomogram was built using those variables and its efficiency in predicting patients’ survival was better than the conventional AJCC stage system after evaluation. Our new model has a significant higher concordance index(C-index) (training set,0.683 v 0.641, respectively; P<0.01; testing set, 0.676 v 0.638, respectively; p<0.05). Similarly, the calibration curve shows the nomogram was in better accordance with the actual observation in both cohorts. And then, after risk stratification, we found MLNR is more reliable than ELNs in predicting overall survival(OS). Conclusions: We developed a nomogram model for NSCLC patients after surgery. This novel and useful tool outperforms the widely used TNM staging system and could benefits clinicians in treatment options and cancer control.
Acute lung injury causes severe inflammation and oxidative stress in lung tissues. In this study, we analyzed the potential regulatory role of nuclear factor erythroid-2-related factor 2 (Nrf2) on NADPH oxidase 1 (NOX1) in tumor necrosis factor-α (TNF-α)-induced inflammation and oxidative stress in human type II alveolar epithelial cells. In this study, A549 cells were transfected with Nrf2 siRNA and overexpression vectors for 6 h before being induced by TNF-α for 24 h. TNF-α upregulated the expression of NOX1 and Nrf2 in A549 cells. Furthermore, overexpression of Nrf2 could reduce TNF-α-induced NF-κB mRNA and protein expression after transfection with the Nrf2 siRNA vector, and the levels of IL-6, IL-8, ROS, and malondialdehyde (MDA) in TNF-α-induced A549 cells increased, while the level of total antioxidation capability (T-AOC) decreased. On the other hand, the overexpression of Nrf2 decreased the levels of IL-6, IL-8, ROS, and MDA, while increasing T-AOC. The mRNA and protein levels of NOX1 were dramatically increased by TNF-α, while those changes were notably suppressed by Nrf2 overexpression. Further studies demonstrated that Nrf2 sup-pressed NOX1 transcription by binding to the -1199 to -1189 bp (ATTACACAGCA) region of the NOX1 promoter in TNF-α-stimulated A549 cells. Our study suggests that Nrf2 may bind to and regulate NOX1 expression to antagonize TNF-α-induced inflammatory reaction and oxidative stress in A549 cells.
Background: Cigarette smoking is a major risk factor for chronic obstructive pulmonary disease (COPD) and lung cancer. Epithelial-mesenchymal transition (EMT) is an essential pathophysiological process in COPD and plays an important role in airway remodeling, fibrosis, and malignant transformation of COPD. Previous studies have indicated FERMT3 is downregulated and plays a tumor suppressive role in lung cancer. However, the role of FERMT3 in COPD, including EMT, has not yet been investigated. Methods: The present study aimed to explore the potential role of FERMT3 in COPD and its underlying molecular mechanisms. Two GEO datasets were combined to identify FERMT3 involved in COPD. The expression of FERMT3 was identified in COPD from two GEO datasets. We then established EMT animal models and cell models through cigarette smoke (CS) or cigarette smoke extract (CSE) exposure to detect the expression of FERMT3 and EMT markers. RT-PCR, western blot, immunohistochemical, cell migration, and cell cycle were employed to investigate the potential regulatory effect of FERMT3 in CSE-induced EMT. Results: Based on the GEO dataset analysis, the expression of FERMT3 was downregulated in COPD-smoker bronchoalveolar lavage fluid than that in Non-smoker. Cigarette smoke exposure reduced the FERMT3 expression and induces EMT both in vivo and in vitro. The results showed that overexpression of FERMT3 could inhibit EMT induced by CSE in A549 cells. Furthermore, the CSE-induced cell migration and cell cycle progression were reversed by FERMT3 overexpression. Mechanistically, our study showed that overexpression of FERMT3 inhibited CSE-induced EMT through the Wnt/β-catenin signaling. Conclusions: In summary, these data suggest FERMT3 regulates cigarette smoke-induced epithelial-mesenchymal transition through Wnt/β-Catenin signaling. These findings indicated that FERMT3 was correlated with the development of COPD and may serve as a potential target for both COPD and lung cancer.
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