Additional file 1: Fig. S1. of Overexpression of programmed cell death 5 in a mouse model of ovalbumin-induced allergic asthma

Correlation between PDCD5 level in BALF and lung function. (TIF 40 kb

Effect of β-arrestin knockdown on CSE-stimulated MAPK activation.

<p>Western blot analysis of phospho-ERK1/2 (p-ERK1/2), total ERK1/2 (t-ERK1/2), phospho-p38MAPK (p- p38MAPK) and total p38MAPK (t-p38MAPK) in cells treated with Lipofectamine2000 (vehicle), nonspecific control siRNA (NC-siRNA, 100 nM) or β-arrestin2–targeted siRNA (A2-siRNA, 100 nM) before CSE stimulation as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097788#pone-0097788-g004" target="_blank">Figure 4</a>. (A, B) Representative western blot is from 3 independent experiments. (C, D) Data are the means ± SEM of 3 separate experiments. Cells were pretreated with ERK1/2 inhibitor PD98059 (50 µM) or p38MAPK inhibitor (5 µM) for 60 min before CSE treatment. (E) Quantitative RT-PCR analysis of MUC5AC mRNA expression. Data are means ± SEM (n = 3). NS, not significant.</p

β₂-Adrenoceptor Involved in Smoking-Induced Airway Mucus Hypersecretion through β-Arrestin-Dependent Signaling

<div><p>Progression of chronic obstructive pulmonary disease is associated with small airway obstruction by accumulation of inflammatory mucous exudates. However, the mechanism of mucin hypersecretion after exposure to cigarette smoke (CS) is still not clear. In this study, we explored the contribution of β₂-adrenoceptor (β₂-AR) signaling to CS extract (CSE)-induced mucus hypersecretion <i>in vitro</i> and examined the effect of a β-blocker on airway mucin hypersecretion <i>in vivo.</i> NCI-H292 epithelial cell line was used to determine the contribution of β₂-AR signaling to CSE-induced MUC5AC production by treatment with β₂-AR antagonists propranolol and ICI118551 and β₂-AR-targeted small interfering RNA. The effect of propranolol on airway mucus hypersecretion was examined in a rat model exposed to CS. MUC5AC expression was assayed by real-time PCR, immunohistochemistry and ELISA. β₂-AR and its downstream signaling were detected by western blot analysis. We found that pretreating NCI-H292 cells with propranolol, ICI118551 for 30 min or β₂AR–targeted siRNA for 48 h reduced MUC5AC mRNA and protein levels stimulated by CSE. However,inhibiting the classical β₂AR–cAMP-PKA pathway didn’t attenuate CSE-induced MUC5AC production, while silencing β-arretin2 expression significantly decreased ERK and p38MAPK phosphorylation, thus reduced the CSE-stimulated MUC5AC production. In vivo, we found that administration of propranolol (25 mg kg⁻¹d⁻¹) for 28 days significantly attenuated the airway goblet cell metaplasia, mucus hypersecretion and MUC5AC expression of rats exposed to CS. From the study, β₂-AR–β-arrestin2–ERK1/2 signaling was required for CS-induced airway MUC5AC expression. Chronic propranolol administration ameliorated airway mucus hypersecretion and MUC5AC expression in smoking rats. The exploration of these mechanisms may contribute to the optimization of β₂-AR target therapy in chronic obstructive pulmonary disease.</p></div

β₂-AR blockade attenuates cigarette smoke extract (CSE)-induced MUC5AC production from NCI-H292 cells.

<p>Cells were pre-incubated with 10⁻⁵ M propranolol or 10⁻⁶ M ICI118551 for 30 min before adding CSE (Pro+CSE, ICI+CSE) or were incubated with media (CON), CSE, 10⁻⁵ M propranolol (Pro), or 10⁻⁶ M ICI118551 (ICI) alone for 24 h. Quantitative RT-PCR and ELISA of MUC5AC mRNA (A) and protein level (B), respectively, and MUC5AC mRNA (C) and protein secretion (D) of cells transfected with Lipofectamine2000 (vehicle), a nonspecific control siRNA (NC-siRNA, 100 nM) or β₂-AR–targeted siRNA (β₂-siRNA, 100 nM) for 48 h before incubation with or without CSE for 24 h. Data are means ± SEM (n = 3).</p

Study protocol.

<p>32 rats were randomly divided into 3 groups for treatment: Group C (n = 12): exposure to air and administrated with distilled water as vehicle, Group S (n = 12): exposure to cigarette smoke and administrated with vehicle, Group S/P (n = 8) exposure to cigarette smoke and administrated with prapronolol. Rats in Group S and Group S/P were exposed to cigarette smoke by a whole-body inhalation instrument generated from commercial cigarettes at 20 cigarettes’ inhalation for 2 h followed by a 4-h recovery, repeated twice a day. Rats were exposed to CS 6 days per week. At 12 weeks later, 4 rats in Groups C and S were killed to evaluate lung function and histopathological alteration, respectively. From weeks 13 to 16, distilled water, 3–5 ml kg⁻¹d⁻¹, was intragastrically administered to Group C, Group S as vehicle, and propranolol, 25 mg kg⁻¹d⁻¹ (Kangpu Pharmaceutical Co., China) to Group S/P. The remaining rats were killed after 16 weeks of CS exposure.</p

Effect of cAMP–protein kinase A (PKA) inhibition on CSE-stimulated MUC5AC expression.

<p>Western blot analysis of phospho-PKA (p-PKA) and total PKA (t-PKA) levels in cells preincubated with (A) Rp-8-Br-cAMPs or (B) H89 for 30 min before stimulation with or without CSE for 15 min. (C, D) RT-PCR analysis of MUC5AC mRNA expression after incubation with or without CSE for 24 h. Data are means ± SEM from 3 separate experiments. ***<i>P</i><0.001 compared to CSE alone. NS, not significant.</p

Schematic of the β₂-AR signaling pathway involved in CS-induced upregulation of MUC5AC production in NCI-H292 cells, a mucin-expressin cell line from human airway epithelia.

<p>Wtih CSE stimulation, <i>β₂-AR</i> is activated, which increases cAMP and recruits many <i>β</i>-arrestins. Increased cAMP binds and activates PKA but does not stimulate MUC5AC transcription and production. <i>β</i>-arrestin2 phosphorylates ERK1/2 and p38MAPK, the key players, which activate transcription factors to stimulate MUC5AC transcription and production. β-AR antagonists, kinase inhibitors and molecular biological methods of interfering with signaling molecules are also shown.</p

The characteristic of asthma control among nasal diseases population: Results from a cross-sectional study

<div><p>Asthma affects a large number of patients in China, but comprehensive evaluation of risks associated with poor asthma control in asthmatic patients with nasal diseases was still limited. We conducted this study to provide a comprehensive estimate of asthma control in Chinese asthma patients with combined nasal diseases, to explore the effect of kinds of nasal diseases on the asthma control, and to identify risk factors associated with uncontrolled asthmatic patients with combined nasal diseases. 1756 asthma patients concomitant with nasal diseases aged ≥17 years and representing all regions of mainland China were surveyed. Multivariate logistic regression model with all related demographic characteristics and disease characteristics factors entered was used to identify risk factors associated with uncontrolled asthma. 56.4% of asthmatic patients with combined allergic rhinitis or rhinosinusitis or rhinopolyp remained uncontrolled. Concomitant without allergic rhinitis, younger age, better treatment adherence and higher education level might positively impact asthma control among asthmatic patients with combined nasal diseases. Perennial allergic rhinitis (OR = 1.5, P = 0.021), moderate-severe allergic rhinitis (OR = 2.2, P = 0.001) were all found to significantly increase the risk of uncontrolled asthma among asthma patients with combined allergic rhinitis. The high prevalence of uncontrolled asthma indicates that asthma management among adult Chinese asthmatic patients comorbid with nasal disease is still a challenge. Efforts should be made to raise the awareness of asthma management and to provide sufficient treatment will greatly contribute to improved quality of asthma management. It is possible to minimize the influence of allergic rhinitis on asthma control by improving nasal function, especially for more severe allergic rhinitis and perennial allergic rhinitis.</p></div

Effect of β-arrestin knockdown on CSE-stimulated MUC5AC expression.

<p>Cells were transfected with Lipofectamine2000 alone (vehicle), a nonspecific control siRNA (NC-siRNA, 100 nM), β-arrestin1–targeted siRNA (A1-siRNA, 100 nM) or β-arrestin2–targeted siRNA (A2-siRNA, 100 nM) for 48 h before incubation with or without CSE for 24 h. (A, B) Western blot analysis of β-arrestin1 and β-arrestin2 protein level to access the knockdown efficiency. (C, D) Quantitative RT-PCR analysis of MUC5AC mRNA expression. Data are means ± SEM (n = 3). NS, not significant.</p

Si-RNA sequences used in the experiments.

<p>Si-RNA sequences used in the experiments.</p