14,15-Epoxyeicosatrienoic acid suppresses cigarette smoke condensate-induced inflammation in lung epithelial cells by inhibiting autophagy

Abstract

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.

β-Arrestin2 Inhibits Expression of Inflammatory Cytokines in BEAS-2B Lung Epithelial Cells Treated with Cigarette Smoke Condensate via Inhibition of Autophagy

“…These results indicated that cigarette smoke activated autophagy and induced the secretion of inflammatory cytokines in BEAS-2B cells and that the inhibition of autophagy inhibited the CSC-induced inflammatory response. In a previously published study by our group, we showed that in CSC-induced BEAS-2B cells, autophagy promoted secretion of the inflammatory cytokines, IL-6, IL-8, and MCP-1, by reducing p62 aggregation, which reduced translocation of Nrf2 into the nucleus, downregulating the expression of heme oxygenase-1 (HO-1), which can reduce the expression of inflammatory cytokines [29].…”

“…The procedure used to transfect and evaluate adenoviral mRFP-GFP-LC3 has been described previously [29]. Briefly, the day before transfection, good quality BEAS-2B cells were seeded into 24-well plates and cultured overnight.…”

“…We have also previously shown that 14, 15-epoxyeicosatrienoic acid treatment reduces the CSC-induced inflammatory response in bronchial epithelial cells in vitro by inhibiting autophagy through the PI3K/AKT/mTOR signaling pathway [29]. LC3 is a characteristic microtubule-associated protein light chain; when autophagy is activated, LC3-I is converted to LC3-II, and as a consequence, the LC3-II level would be expected to rise.…”

Characterisation of the relationship between Heme-oxygenase-1 and adiponectin

“…Indeed, these agents induce HO-1 expression, at least in part, by modulation of broad acting transcriptional repressors and transcription factors. More specifically, they involve post-translational downregulation of BACH1 and upregulation of NRF2 [275,291,297,298]. The latter is recognised as a key regulator of cellular defence against oxidative stress that is also strongly implicated in modulation of adipogenesis and adipose tissue remodelling [289,299,300].…”

“…It is well established that CoPP induces HO-1 by modulating the transcriptional repressors and transcription factors, the downregulation of BACH1 and upregulation of NRF2 [275,291,297,298].…”