Epoxyeicosatrienoic acids (EETs) are metabolic products of free arachidonic acid, which are produced through cytochrome P-450 (CYP) epoxygenases. EETs have anti-inflammatory, antiapoptotic, and antioxidative activities. However, the effect of EETs on cigarette smoke-induced lung inflammation is not clear. Autophagy is believed to be involved in the pathogenesis of chronic obstructive pulmonary disease. In addition, nuclear erythroid-related factor 2 (Nrf2), a transcription factor that regulates many antioxidant genes, is thought to regulate antioxidant defenses in several lung diseases. In addition, interaction between EETs, autophagy, and Nrf2 has been reported. The aim of this study was to explore the effect of 14,15-EET on cigarette smoke condensate (CSC)-induced inflammation in a human bronchial epithelial cell line (Beas-2B), and to determine whether the underlying mechanisms involved in the regulation of Nrf2 through inhibition of autophagy. Autophagy and expression of autophagy signaling pathway proteins (LC3B, p62, PI3K, Akt, p-Akt, and p-mTOR) and anti-inflammatory proteins (Nrf2 and HO-1) were assessed via Western blot analysis. Autophagosomes and autolysosomes were detected by adenoviral mRFP-GFP-LC3 transfection. Inflammatory factors (IL-6, IL-8, and MCP-1) were detected by ELISA. Lentiviral vectors carrying p62 short hairpin RNA were used to interfere with p62 expression to evaluate the effect of p62 on Nrf2 expression. Nrf2 expression was determined through immunocytochemistry. 14,15-EET treatment resulted in a significant reduction in IL-6, IL-8, and MCP-1 secretion, and increased accumulation of Nrf2 and expression of HO-1. In addition, 14,15-EET inhibited CSC-induced autophagy in Beas-2B cells. The mechanism of the anti-inflammatory effect of 14,15-EET involved inhibition of autophagy and an increase in p62 levels, followed by translocation of Nrf2 into the nucleus, which then upregulated expression of the antioxidant enzyme HO-1. 14,15-EET protects against CSC-induced lung inflammation by promoting accumulation of Nrf2 via inhibition of autophagy.

Background/Aims: Epoxyeicosatrienoic acids (EETs), a type of lipid mediators produced by cytochrome P450 epoxygenases, exert anti-inflammatory, angiogenic, anti-oxidative and anti-apoptotic effects. However, the role of EETs in cigarette smoke-induced lung injury and the underlying mechanisms are not fully known. The aim of this study was to explore the effects of CYP2J2-EETs on cigarette smoke extracts (CSE)-induced apoptosis in human bronchial epithelial cell line (Beas-2B) and the possible mechanisms involved. Methods: Cytochrome P450 epoxygenase 2J2 (CYP2J2) and its metabolites EETs were assessed by western blotting or LC-MS-MS. Cell viability and apoptosis were determined by MTT assay and AnnexinV-PI staining. Reactive oxygen species (ROS) were assessed by measuring H2DCFDA. Caspase-3, HO-1, MAPK and endoplasmic reticulum (ER) stress-related markers GRP78, p-elF2a, and CHOP were evaluated by western blotting. Results: CSE suppressed expression of both CYP2J2 and EET by Beas-2B cells. CSE also induced apoptosis, the generation of ROS and the ER stress in Beas-2B cells. These changes were abolished by pretreatment with exogenous 14,15-EET while pretreatment with 14,15-EEZE, a selective EET antagonist, abolished the protective effects of 14,15-EET. In addition, EETs increased the expression of antioxidant enzyme HO-1. Furthermore, 14,15-EET reduced CSE-induced activation of p38 and JNK. Conclusion: The data suggest that CYP2J2-derived EETs protect against CSE-induced lung injury possibly through attenuating ER stress.