The involvement of Nrf۲-antioxidant response element signaling pathway in the oxidative stress induced by chronic cigarette smoke exposure in lung epithelial cells: The protective role of crocin

Introduction: Cigarette smoke (CS) as a major source of air pollution contains many free radicals, reactive oxygen, carcinogenic and toxic chemicals which have serious damaging effects on DNA, proteins, and cellular organelles. Nuclear erythroid-related factor ۲ (Nrf۲), a redox-sensitive transcription factor, is involved in transcriptional regulation of many antioxidant genes, including glutamate-cysteine ligase (GCL). CS is known to cause oxidative stress and deplete glutathione (GSH) levels in alveolar epithelial cells. We hypothesized that crocin as a carotenoid chemical compound has antioxidant signaling properties by inducing GSH biosynthesis via the activation of Nrf۲ and protects lung epithelial cells against CS-mediated oxidative stress.Methods: Human alveolar epithelial cells (A۵۴۹) were exposed to ۱, ۲.۵ and ۵% cigarette smoke extracts (CSE) with or without crocin (۵۰۰ μM) and the cells exposed to H۲O۲ was used as a positive control group. After ۴۸ h exposure, the flowcytometric analysis was performed to detect reactive oxygen intermediates in cells. Then, the oxidant/antioxidant parameters (MDA, GSH, SOD, CAT) and the Nrf۲ pathway modification (GCL activity and Nrf۲ gene expression and localization) were assayed in all groups. Finally, Differences between experimental groups were analyzed by one-way ANOVA and followed by post hoc Tukey’s test.Results: Treatment of human alveolar epithelial (A۵۴۹) cells with CS extract (CSE) dose dependently decreased GCL activity and GSH levels, effects that were associated with enhanced production of reactive oxygen species (ROS). Crocin restored CSE-depleted GSH levels by upregulation of GCL via activation of Nrf۲ and also quenched CSE-induced release of reactive oxygen species. Also, CSE caused to cytoplasmic accumulation of Nrf۲ in lung epithelial cells. Surprisingly, crocin attenuated CSE-mediated Nrf۲ cytoplasmic accumulation with induce nuclear translocation of Nrf۲ in A۵۴۹ cells which associated with increased GCL activity and GSH biosynthesis. Conclusions: In summary, Crocin by inducing GSH synthesis attenuates CSE-mediated GSH depletion and protects epithelial cells to CSE-induced oxidative stress via Nrf۲ pathway. These results may have implications in dietary modulation of natural antioxidants in treatment of pulmonary diseases induced by cigarette smoke.

Keywords:

Lung Epithelial Cell , Cigarette Smoke , Oxidative Stress , Nrf۲ , Crocin