The PI3K-Akt-HIF-1α Pathway Reducing Nasal Airway Inflammation and Remodeling in Nasal Polyposis

Fei He, Huaigang Liu, Wei Luo Ear, Nose & Throat Journal Aug 28, 2019 | OnlineFirst

Abstract Objective: Previous studies suggested that hypoxia-inducible factor-1α (HIF-1α) plays an important role in the progression of inflammation and remodeling of chronic rhinosinusitis with nasal polyposis. However, the molecule mechanisms of HIF-1α activation and regulation of cytokine expressions, such as interleukin (IL) 25 and IL-17RB, in nasal polyposis are not clear. Method: The IL-25 and IL-17RB levels in human nasal epithelial cells after stimulation by lipopolysaccharide (LPS) were detected by enzyme-linked immunosorbent assay method, and the proteins of HIF-1α and p-Akt were detected by Western blot method. Moreover, we evaluated the cytokine levels in the nasal mucosa of a murine model of nasal polyposis. Results: The levels of IL-25 and IL-17RB showed dose- and time-dependent release in response to LPS stimulation. The proteins of HIF-1α and p-Akt were both increased significantly after LPS stimulation. After inhibition of PI3K/Akt pathway by PI3K inhibitor LY294002, the levels of IL-25 and IL-17RB and HIF-1α were decreased by LPS stimulation. Conclusions: Inhibition of PI3K or HIF-1α pathway could significantly reduce growth factor production and decrease nasal inflammation. The HIF-1α pathway could be a novel therapeutic approach for reducing nasal airway inflammation and remodeling in nasal polyposis. Keywords chronic rhinosinusitis, nasal polyposis, hypoxia-inducible factor-1α, PI3K, signaling pathway Introduction There are 2 subtypes of the chronic rhinosinusitis (CRS), with polyps (CRSwNP) and without polyps (CRSsNP). The prevalence of CRSwNP ranged from 0.5% to 4.3%, which is less common than CRSsNP in national population surveys.1 However, there are about 38% to 69% of CRSwNP cases commonly leading to revision endoscopic sinus surgery therapy.2–4 Olfactory impairment is the common symptom of CRSwNP patients5 and is also likely to endorse nasal obstruction.6 Previous studies suggested that hypoxia-inducible factor 1α (HIF-1α) was overexpressed in the lung and nasal mucosa, which played an important role in airway inflammatory responses.7,8 The member of interleukin (IL) 17 cytokine family, IL-25, has been overexpressed in many inflammatory animal models, such as atopic dermatitis, asthma, and pulmonary fibrosis. It is said that eosinophils or TH2 cytokines would been producted in bronchoalveolar lavage fluid and lung tissue after IL-25 intraperitoneal or intranasal administration.9,10 Recently, IL-25 also reported to play a main role in promoting TH2-mediated inflammation.11 However, IL-25 roles in Asian patients with CRSwNP at present have not been illuminated. Chronic rhinosinusitis with polyps is a multifactorial disease, as a result of the persistent inflammation of nasal cavity and sinuses. The pathogenesis of CRSwNP remains unclear. In the present study, the mechanisms between the IL-25 production and HIF-1α and lipopolysaccharide (LPS) activation in vitro and in vivo were investigated. We had emphasized the role of PI3K/Akt/HIF-1α pathway in regulating IL-25 expression by LPS stimulation. Materials and Methods Cells Lines Human nasal epithelial cells (HNEpCs) were purchased from American Type Culture Collection and were grown in standard culture medium (RPMI-1640 containing 10% fetal bovine serum, 2 mM l-glutamine, 50 U/mL penicillin, and 50 μg/mL streptomycin). The cells were cultured in an incubator at 37°C in 5% CO2. Lipopolysaccharide Stimulation When HNEpCs reached 80% to 90% confluence, the cells were washed by phosphate-buffered saline (37°C, pH 7.4) and added fresh culture medium. The LPS (Sigma, St Louis, Missouri) was added to the cultures at the concentrations of 0, 0.1, 0.5, and 1 μg/mL and incubated at 37°C for 0 to 24 hours. Hypoxia-Inducible Factor 1α shRNA Transfection and Inhibitor Treatment Human nasal epithelial cells were transfected by HIF-1α short hairpin RNA (shRNA) or control shRNA 36 hours before LPS (1 μg/mL) stimulation. The used HIF-1α shRNA oligonucleotide primers were: forward 5′-AATTCHCCHHCCHCYHHAHACACAATCATATCTCGAGATATGATTGTGTCTCCACGGTTTTTTG-3′ and reverse 5′-GATCCAAAAAACCGCTGGAGACACAATCATATCTCGAGATATGATTGTGTCTCCAGCGGCCGGCG-3′. After 24-hour transfection, the cells were harvested. The LY294002 (Sigma), PI3K-specific inhibitor, was dissolved in dimethyl sulfoxide (DMSO). Serial concentrations of LY294002 (0, 5, 10, 20 μmol/L) and DMSO vehicle (