Azithromycin ameliorates airway remodeling via inhibiting airway epithelium apoptosis

Accepted Manuscript Azithromycin ameliorates airway remodeling via inhibiting airway epithelium apoptosis

Yuanqi Liu, Yue Pu, Diandian Li, Liming Zhou, Lihong Wan PII: DOI: Reference:

S0024-3205(16)30676-2 doi: 10.1016/j.lfs.2016.11.024 LFS 15090

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Life Sciences

Received date: Revised date: Accepted date:

6 October 2016 17 November 2016 25 November 2016

Please cite this article as: Yuanqi Liu, Yue Pu, Diandian Li, Liming Zhou, Lihong Wan , Azithromycin ameliorates airway remodeling via inhibiting airway epithelium apoptosis. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Lfs(2016), doi: 10.1016/j.lfs.2016.11.024

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Title page Azithromycin ameliorates airway remodeling via inhibiting airway epithelium apoptosis

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Department of Pharmacology, West China School of Preclinical and Forensic Medicine,

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Sichuan University, Chengdu, Sichuan 610041, PR China

Sichuan University “985 project -- Science and Technology Innovation Platform for

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Yuanqi Liu1,2, Yue Pu1,2, Diandian Li3, Liming Zhou1,2, Lihong Wan1,2*

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Department of Respiratory Medicine, West China Hospital, Sichuan University,

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Chengdu, Sichuan 610041, PR China *

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Novel Drug Development”, Sichuan University, Chengdu, Sichuan 610041, PR China

Correspondence should be addressed to

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Professor Lihong Wan, Department of Pharmacology, 3-17 Renmin South Road, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu,

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Sichuan 610041, P.R. China

E‑mail: [email protected]

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Tel: 86-28-85501278

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Abstract Aims: Azithromycin can benefit treating allergic airway inflammation and remodeling. In the present study, we hypothesized that azithromycin alleviated airway epithelium

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injury through inhibiting airway epithelium apoptosis via down regulation of caspase-3 and Bax/Bcl2 ratio in vivo and in vitro.

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Main methods: Ovalbumin induced rat asthma model and TGF-β1-induced BEAS-2B

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cell apoptosis model were established, respectively. In vivo experiments, airway

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epithelium was stained with hematoxylin and eosin (HE) and periodic acid–Schiff (PAS) to histologically evaluate the airway inflammation and remodeling. Airway epithelium index

(AI)

was

further

analyzed

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apoptotic

by

terminal

deoxynucleotidyl

transferase-mediated dUTP nick end labeling (TUNEL), while expression of apoptosis

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related gene (Bax, Bcl2, Caspase-3) in lungs were measured by qRT-PCR and western blotting, respectively. In vitro experiments, apoptosis were evaluated by Flow cytometry

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(FCM) and TUNEL. Above apoptosis related gene were also measured by qRT-PCR and western blotting.

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Key findings: Compared with the OVA group, azithromycin significantly reduced the inflammation score, peribronchial smooth muscle layer thickness, epithelial thickening and goblet cell metaplasia (P<0.05), and effectively suppressed AI of airway epithelium (P<0.05). Moreover, the increasing mRNA and protein expressions of Caspase-3 and Bax/Bcl-2 ratio in lung tissue were all significantly decreased in azithromycin-treated rats (P<0.05). In vitro, azithromycin significantly suppressed TGF-β1-induced

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BEAS-2B cells apoptosis (P< 0.05) and reversed TGF-β1 elevated Caspase-3 mRNA level and Bax/Bcl-2 ratio (P<0.05). Significance: Azithromycin is an attractive treatment option for reducing airway

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epithelial cell apoptosis by improving the imbalance of Bax/Bcl-2 ratio and inhibiting

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Caspase-3 level in airway epithelium.

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Key words: azithromycin, airway epithelium apoptosis, caspase-3, bax/bcl-2

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Text Introduction Airway epithelial cell is the first physical barrier in the airway, which is also an

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important target of inflammatory insults [1]. Epithelial damage is now considered as a novel pathogenic mechanism of asthma, contributing to airway inflammation and

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remodeling [2]. Following lung injury, epithelial cells produced a big amount of

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transforming growth factor-β1 (TGF-β1), inflammatory cells and fibroblasts to induce

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persistent and severe subepithelium fibrosis [3]. Additionally, TGF-β1 played a crucial role in cell differentiation, proliferation, and apoptosis process, including apoptosis of

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airway epithelial cells [4,5]. Thus, it is interesting to explore novel therapeutic agents to restore epithelium integrity via inhibiting epithelial cells apoptosis process.

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Long-term administration of azithromycine (AZM), the 15-member macrolide, at the dose of 250 mg daily could reduce the severity of bronchial hyperresponsiveness in

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patients with mild asthma [6] and in a murine model of chronic asthma, 75 mg/kg AZM ameliorates not only airway inflammation but also airway remodeling [7]. In our

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previous study, we also found azithromycin at the dose of 25 mg/kg possess significant protective effects against allergic airway inflammation, including significant reduction of the number of lymphocytes, eosinophils and neutrophils and inhibition of IL-2, IL-4, IL-5, IL-13, TNF-α levels in BAL fluid [8]. Importantly, azithromycin also possess significant protective effects against airway remodeling via suppressing TGF-β1 expression in rat lungs [8]. Moreover, Hodge et al found that azithromycin could

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notably improve the phagocytosis of apoptotic bronchial epithelial cells by alveolar macrophages (AMs) [9]. A recent report has also demonstrated that the proportion of apoptotic bronchial epithelial cells was markedly reduced in low-dose

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azithromycin-treated COPD patients [10]. Conversely, corticosteroid (CS) therapy actually induced airway epithelial apoptosis in mice model [22,23]. This might be the

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major reason that some patients may continue to have asthma symptoms and a

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progressive decline in lung function despite their use of CS therapy. Based on these

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literature evidences, we hypothesized that azithromycin was likely to be a competitive drug in long-term asthma treatment by inhibiting airway epithelium apoptosis. As an

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inducer of apoptosis in variety epithelial cells, TGF-β has been demonstrated highly

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Bcl-2 downregulated [33].

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enhanced apoptosis in human bronchial epithelial cells by caspase-3 activation [11] and

To investigate the potential mechanisms of azithromycin in airway epithelium apoptosis,

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OVA-induced rat model and TGF-β1-induced BEAS-2B cells apoptosis model were used in this study. The present study showed not only peribronchial smooth muscle

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layer thickness, epithelial thickening and goblet cell metaplasia but also AI of airway epithelium was significantly inhibited in azithromycin-treated OVA rat model. Consistently, AZM markedly suppressed TGF-β1-induced BEAS-2B cells apoptosis. Moreover, these effects appeared to be mediated through the inhibition of level of Caspase-3 and Bax/Bcl-2 ratio in vivo and in vitro.

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Materials and Methods Cell culture Transformed human bronchial epithelial cells, BEAS-2B cells donated by Dr. Xiaodong

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Sun (Chengdu, China) were cultured in Dulbecco's modified Eagle's medium (DMEM) (Harry Biotech, Chengdu, China) supplemented with 15% heat-inactivated FBS (Gibco,

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10099-141) and seeded on 6-well culture dishes at 1.5×105 cells/ml. When cells reached

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60%-70% confluence, the media was changed and cells received serum-free media for

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24 hours. Then, cells were subjected to stimulation with 3.3, 10 and 30 μg/ml azithromycin (AZM) and recombinant human TGF-β1 (30 ng/ml, PEPROTECH, USA,

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0715209) for an additional 72 hours. TGF-β1 was used as a positive control in this study.

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Apoptosis assay Flow cytometry (FCM)

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Apoptosis was measured by using an annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (Annexin V-FITC/PI, Vazyme BioTECH, A211-02). In brief,

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cells were plated at a density of 4×105 cells per well into 6-well plate and cultured in DMEM medium supplemented with 15% heat-inactivated fetal bovine serum and antibiotics at 37°C in 5%CO2 (v ) in a humidified incubator for 24 h and treated with different concentrations of azithromycin. Untreated cells were used as negative control. After 72 h, the cells were harvested by trypsinization (Solarbio, T1350-100), washed twice with cold PBS (2000 rpm, 5 min), and resuspended in 100 μL of binding buffer.

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Annexin V-FITC (5 μL) and propidium iodide (PI, 5 μL) were added to each sample, and the mixture was incubated in the dark for 10 min at room temperature. Cells were immediately subjected to FACS analysis (BD Accuri C6, USA) within 1 h. Ex=488 nm

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and Em=530 nm. Both PI and Annexin V negative cells were considered as normal, PI negative and Annexin V positive cells were considered as early apoptotic , cells that

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were both PI and Annexin V positive were considered as late necrotic, and cells that

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were PI positive and Annexin V negative were considered as mechanically injured

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during the experiment. All the experiments were conducted in triplicates. Apoptosis rate (%) = number of apoptotic cells/total number of nucleated cells×100%.

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TUNEL staining

TUNEL staining for apoptotic cells was carried out using the terminal deoxynucleotidyl

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transferase-mediated dUTP nick end labeling (TUNEL) assay according to the manufacturer’s instructions. Briefly, after 72 hours treatment with different

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concentrations of azithromycin, the cells were fixed by adding 4%paraformaldehyde (Kelong Chemical Reagent Factory, China) and incubating for 30 minutes. The fixed

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cells were washed in PBS, permeabilized with 0.1% Triton X-100 for 10 minutes on ice, washed in PBS twice, and then incubated with 50 μl of terminal deoxynucleotidyl transferase end-labeling solution for 60 minutes at 37˚C in a humidified chamber in dark. Cells containing green granules in the nucleus were regarded as positive for TUNEL. Nuclei were stained by DAPI. Fluorescence signals were detected with a fluorescence microscope system (IX73, OLYMPUS, Tokyo, Japan). DAPI and

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apoptosis positive cells were enumerated using the National Institutes of Health ImageJ software. Animals and ethics statement

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Forty male Sprague-Dawley (SD) rats, aged 6-7 weeks and weighing 190-210 g, were obtained from Da-Shuo Biological Technology Co., Ltd. (Chengdu, China), and used

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after 1 week of acclimation. All experimental procedures were conducted in accordance

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with the guidelines of the Experimental Research Institute of Sichuan University in

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agreement with the guidelines of the Canadian Council on Animal Care (Permit Number: 2003-149). Animal ethics approval has been obtained from our institution. The

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holding room was under standard conditions of ambient temperature (20±1°C), humidity (60±10%), and with a 12-h light/dark cycle (lights on at 8:00 AM) throughout

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the whole study. Experimental groups

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All rats were divided into four groups randomly (n=10): the control group (saline challenge), the OVA group (OVA challenge + saline treatment), the dexamethasone

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(DEX) group as a positive control (OVA challenge + 0.5 mg/kg DEX treatment), and the azithromycin (AZM) group (OVA challenge + 25 mg/kg AZM treatment, corresponding to human clinical equivalent dosage 250 mg/daily) [6,10]. OVA-induced rat asthma model and treatment A schematic diagram of the treatment schedule is shown in Fig 1. Rats were immunized by a subcutaneous injection of 1 mg of OVA (Sigma-Aldrich, USA) in 1 ml saline and

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200 mg aluminum hydroxide (Aldrich, USA) in 1 ml of saline on day 1 and 6. From day 7 to 13, after initial sensitization, rats were given aerosol challenges with 1%OVA for 30 min using an ultrasonic nebulizer (NE-U12; Omron Co., Tokyo, Japan) once daily.

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The control rats received aerosol challenges with saline in a similar manner. From day 7-13, OVA-sensitized rats were intragastrically with 0.2 ml of saline containing DEX

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(0.5 mg/kg) or AZM (25 mg/kg) 1 h prior to OVA administration, while the control

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group was treated in the same way with normal saline. On day 14, all rats were

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sacrificed by decapitation. The inferior lobes of right lungs from 10 animals in each group were rapidly removed, dissected and stored at -80°C.

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Lung tissue histopathology

The left lung lobes of each rat were immediately fixed in 10% (v/v) neutral-buffered

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formalin for 24 h at 4°C and embedded in paraffin. Then, the tissues were sectioned at 4 μm thickness, and stained with hematoxylin and eosin (HE) (Sigma, St. Louis, MO) and

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periodic acid–Schiff (PAS) solution (IMEB, San Marcos, CA) to assess the inflammation and epithelial injury, respectively. The airway wall thickness (Wat) and

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the bronchial smooth muscle thickness (Wam) were quantified by Image-Pro® Plus 6.0 software (Media Cybernetics, Bethesda, Md), and the inflammatory degree was scored by two independent observers blinded to the experiment. Briefly, grades of 0 to 4 were given: for no inflammation (grade 0), occasional cuffing with inflammatory cells (grade 1), and when most bronchi or vessels were surrounded by a thin layer (1-2 cells: grade 2), a moderate layer (3-5 cells: grade 3), and a thick layer (>5 cells deep: grade 4) of

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inflammatory cells. An increment of 0.5 was given if the inflammation scores were in between 2 grades, and the total inflammation score was calculated by the addition of both peribronchial and perivascular inflammation scores (n=10 airways from 3-5

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animals). The thickness of the epithelial layer and mucus accumulation in the epithelial layer were quantified using PAS-stained sections with Image-Pro® Plus 6.0 software by

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dividing the epithelial area (μm2) or the PAS+ area in the epithelium (μm2) by the length

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(μm) of the basement membrane (μm) around a bronchus [12]. The diameter of

Apoptotic index (AI) measurement

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bronchus was 200-400 μm.

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The number of apoptotic cells in the lungs was measured by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay using

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TACS® 2 TdT diaminobenzidine kit (Trevigen, Gaithersburg, Md) to label the DNA damaged cells following the manufacturer’s instructions. The AI was calculated in each

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lung of rats at 400× magnification according to the following equation: AI = number of apoptotic cells/total number of nucleated cells×100%.

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mRNA and protein preparation The total RNA and protein of BEAS-2B cells and lung were extracted by E.Z.N.A.® Total DNA/RNA/Protein Kit (Omega, USA). Protein concentrations were determined using the BCA protein assay kit (Pierce Biotechnology, Rockford, IL). Quantitative real-time polymerase chain reaction (qRT-PCR) Total RNA (1 μg) was reverse-transcribed with an ImProm-II Reverse Transcription

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System kit (Promega, USA). ABI PRISM 7500 Sequence Detection System (Applied Biosystems, USA) was applied for the mRNA amplification of apoptosis related gene using the following forward and reverse primers (Table 1). The amplification conditions

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were 30 seconds at 95°C, followed by 39 cycles of 5 seconds at 95°C, 30 seconds at 58°C, and 34 seconds at 72°C. The mRNA levels of caspase-3, bax and bcl-2 were

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normalized by the expression level of β-actin. Experiments were performed in triplicate.

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All the data was analyzed using the 2−∆∆CT method (ΔCt=CtTarget gene–Ctβ-actin, ∆∆Ct =

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∆Ct Exp-ΔCt Control). Western blotting

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Equal amounts of total protein were loaded onto 12% SDS PAGE gels at 80 V for 80 min, electro transferred to PVDF membranes by the wet transfer method (250 mA, 90

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min), and blocked in 5%BSA at 4°C overnight. Subsequently, the membranes were incubated with an anti-β-actin (TA-09, ZSGB-BIO, China) antibody (dilution 1:1000),

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anti-caspase3 (19677-1-AP, Proteintech, China) antibody (dilution 1:200), anti-bcl-2 (zs-783, ZSGB-BIO, China) antibody (dilution 1:100), and anti-bax (50599-2-Ig,

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Proteintech, China) antibody (dilution 1:500) at room temperature for 2 h. After washing with TBST, the membranes were incubated with secondary goat anti-mouse IgG (ZB-2305, ZSGB-BIO, China) antibody (dilution 1:1000) or goat anti-rabbit IgG (ZB-2301, ZSGB-BIO, China) antibody (dilution 1:1000) at room temperature for 1 h, which were conjugated with horseradish peroxidase. Equal loading of protein in each lane were verified by reblotting the membrane with an anti-β-actin antibody (Zhongshan,

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China). Then proteins were detected by chemiluminescence reagent (GE Healthcare). Protein band density was quantified using Bio-Rad Quantity One v4.62. Expression levels of the proteins were normalized by β-actin as an internal standard.

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Statistics Statistical analysis was performed by one-way analysis of variance (ANOVA) with

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Bonferroni correction (GraphPad Prism version 5). All values were expressed as mean ±

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SEM, and P＜0.05 was considered statistically significant.

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Results

Azithromycin attenuates airway injury in OVA-induced rat

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To determine the effect of azithromycin on airway injury, histopathological analysis was performed on HE and PAS stained lung sections, respectively. As shown in Fig 2A, the

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OVA challenge markedly induced peribronchial infiltration of inflammatory cells, compared with the control rats. In contrast, azithromycin (AZM) or dexamethasone

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(DEX) treatment significantly prevented the inflammatory changes around the bronchus, which was confirmed by the inflammation score. Compared with control rats

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(saline-challenged), OVA-challenged rats presented much higher inflammatory scores (P<0.05) (Fig 2D), which as expected, were notably lowered by AZM or DEX treatment (P<0.05) (Fig 2D). Consistent with the inflammatory changes, the thickness of the peribronchial smooth muscle layer (Fig 2C), epithelial thickening (Fig 2E) and goblet cell metaplasia (mucus production) (Fig 2B,F) were also increased in OVA-challenged rats (P<0.05). However,

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AZM or DEX treated OVA-challenged rats showed a significant reduction in thickness of the peribronchial smooth muscle layer (Fig 2C) and goblet cell metaplasia (mucus production) (Fig 2B,F) compared to OVA-challenged rats (P<0.05). Nevertheless, the

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epithelial thickening was not affected by treatment of DEX (P＞0.05) (Fig 2E). Effect of azithromycin on airway epithelium apoptosis in OVA-challenged rats and

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human BEAS-2B cells

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To further evaluate the effect of AZM on airway epithelial injury, we performed TUNEL

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assay in lung sections to determine the apoptotic index. In control rats, only a small number of TUNEL-positive (TUNEL+) cells were localized in the bronchial epithelium

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(Fig 3A). However, an elevated amount of TUNEL+ cells were seen in the bronchial epithelium of OVA-challenged rats, which was reduced markedly by the administration

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of AZM and DEX (Fig 3A). The protective effect of AZM on apoptosis was also quantitatively evaluated with the apoptosis index (AI) that calculated the amount of

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apoptotic bronchial epithelium (Fig 3B). The AI was significantly higher in the OVA-challenged rats than that in the saline-challenged rats (P<0.05). However, AZM

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notably inhibited the AI increase in bronchial epithelium (P<0.05). Accordant with epithelial thickening, the AI in the AZM group was significantly lower compared with that in DEX group (P<0.05), implying that AZM treatment did provide a more complete protection in preventing OVA-induced apoptosis in the rat bronchial epithelium than DEX. To confirm the effect of AZM on bronchial epithelium apoptosis, human bronchial

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epithelium cells, BEAS-2B were incubated with 3.3, 10 and 30 μg/ml AZM-containing media for 72 h and cellular viability was determined by CCK8 assay, and AZM didn’t show any cytotoxic activity on human BEAS-2B cells (data not shown). Next,

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recombinant human TGF-β1 was subjected to induce apoptosis in BEAS-2B cells. After 72 h of treatment with TGF-β1 (30 ng/ml), BEAS-2B cells exhibited a more elongated

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shape than their untreated counterparts (data not shown). To analyze the induction of

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apoptosis by TGF-β1 (30 ng/ml), we determined the number of Annexin V stained cells

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by flow cytometry. As shown in Fig 5A-B, treated with TGF-β1 (30 ng/ml) significantly induced cell apoptosis and the apoptotic rate was 47%, whereas the apoptotic rate of

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control cell was only 6.8% (P<0.05). Consistent with it, TUNEL staining clearly displayed apoptotic cells in BEAS-2B cells compared to untreated TGF-β1 cells (Fig

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4A). On contrast, different concentration of AZM (10 and 30 μg/ml) treatment markedly decreased the apoptotic rate compared with that in un-treatment group (P<0.05). This

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attenuated effect was more dramatic with the higher dose of AZM (Fig 4B). Effects of azithromycin on the expression of Caspase-3 and balance of Bax/Bcl2 in

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OVA-challenged rats and human BEAS-2B cells Caspase-3, a key executioner of apoptosis in programmed cell death, is the down-stream caspase act upon both cell nucleus and cytosol targets. To test the hypothesis that the effects of azithromycin on airway epithelium apoptosis coupled with a decreased level of Caspase-3, qRT-PCR and western blotting analysis for mRNA and protein expressions of Caspase-3 in rat model and cells line were performed, respectively. As

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shown in Fig 4A,D and E, the mRNA and protein levels of caspase-3 significantly elevated in OVA-challenged rats, compared with saline-challenged rats (P<0.05), while azithromycin and dexamethasone treatment notably reversed this tendency (P<0.05). In

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vitro, TGF-β1 significantly induced increased the mRNA level of Caspase-3 in BEAS-2B cells compared to untreated control cells (Fig 6A), but not protein level (Fig

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6D, E). Also, azithromycin treatment significantly down-regulated the Caspase-3

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mRNA level in BEAS-2B cells, compared with untreated TGF-β1 cells (Fig 6A).

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Accumulating studies have shown that the increase of proapoptotic protein Bax and decrease of antiapoptotic protein Bcl-2 promoted cytochrome c release in mitochondria,

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and therefore activated the cascades of apoptosis [13]. In our study, we found that mRNA and protein expressions of the Bcl-2 and Bax were changed dramatically by

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AZM and DEX treatment, compared with the OVA-challenged rats (P<0.05). Moreover, the Bax/Bcl-2 ratio, which was significantly up regulated by OVA challenge, also

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decreased notably after AZM (P<0.05) and DEX (P<0.05) treatment (Fig 5B-F). Similar results were found in BEAS-2B cells. Briefly, AZM treatment significantly reversed the

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TGF-β1 elevated Bax/Bcl-2 ratio in BEAS-2B cells, compared with untreated TGF-β1 cells (Fig 6B-F). Interestingly, in Fig. 6D, AZM increased the protein level of Bcl-2 after TGF-β1 stimulating, which is controversial to the Fig. 4D, AZM decreased the protein level of Bcl-2 in OVA-treated lung of rat. We speculated the difference between the studies might be due to different species.

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Discussion Excessive airway epithelial damage has been demonstrated contributable to bronchial asthma through secreting immunomodulatory cytokines and growth factors [14]. Thus,

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the recent therapeutic strategies for asthma mainly focused on alleviating airway epithelial injury. In our previous study, we found that azithromycin at the dose of 25

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mg/kg significantly reduced OVA-dependent airway inflammation and airway

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remodeling via decreasing TGF-β1 levels in lung tissue [8]. Interestingly, earlier studies

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also showed that azithromycin could maintain airway epithelial integrity during pseudomonas aeruginosa infection [15]. However, the exact mechanism of azithromycin

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on reducing airway epithelial injury during asthma is still unclear. Consistent with our previous findings, the present study showed that azithromycin significantly alleviated

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OVA-dependent airway epithelial injury, including the epithelial thickening and goblet cell metaplasia (mucus production). Nevertheless, the epithelial thickening was not

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affected by DEX treatment. Therefore, we speculated that azithromycin may be a novel therapeutic strategy in treating airway epithelial injury.

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Apoptosis is an important and beneficial regulatory mechanism in normal airway epithelial cells [16], while excessive apoptosis of airway epithelial cells is generally believed as the main mechanism of airway injury [17,18]. Abundant evidence has demonstrated that the epithelial apoptosis was significantly induced in human and animal model of asthma [19,20,21]. Therefore, suppression of airway epithelial apoptosis may be a novel therapeutic target for airway disorders. Recently, azithromycin

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has been reported to significantly reduce the proportion of apoptotic bronchial epithelium in COPD subjects [22], which supports our present result that systemic AZM administering (25 mg/kg) notably alleviated the OVA-induced apoptosis index increase

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in bronchial epithelium. However, the present study also indicated that dexamethasone therapy could decrease OVA-induced apoptosis index in bronchial epithelium, which is

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not consistent with previous investigations showing that dexamethasone therapy

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increased apoptosis in the airway epithelium of human asthmatics [23,24,25]. The

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conflicting results might be attributed to different species or cell lines. For example, dexamethasone was considered as a potent inhibitor of apoptosis in A549 lung epithelial

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cells [26]. Notably, consistent with epithelial thickening, DEX didn’t provide the same protective effect against the apoptosis of bronchial epithelium in OVA-induced rats as

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AZM did.

TGF-β1 is regarded as inducer of apoptosis in variety epithelial cells, including gingival,

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gastric, prostate and bronchial epithelial cells [27-30]. In addition, we previously reported that the OVA induced significant airway inflammation and airway remodeling

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via increasing TGF-β1 levels in lung tissue [8]. These findings and the results of the present study emphasized the important role of TGF-β1-induced apoptosis in airway inflammation and airway remodeling. In the present study, we established the epithelial cells apoptosis model through recombinant human TGF-β1 stimulating BEAS-2B cells. Our data indicated that recombinant human TGF-β1 induced apoptosis in BEAS-2B cells. Moreover, AZM treatment markedly suppressed TGF-β1-induced airway

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epithelium apoptosis in BEAS-2B cells. Therefore, AZM possessed a significant anti-apoptosis effect in vitro and in vivo. The Bcl-2 family is a key cell survival protein in a number of airway inflammatory diseases, including exposure to cigarette smoke or allergens [31,32]. Previous studies

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demonstrated that Bcl-2 was down-regulated after TGF-β1 stimulation [33]. Lower

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expression of Bcl-2 was found in airway epithelial cells in severe asthma patients [19].

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On the other hand, Bax, the pro-apoptotic members, also play crucial roles in airway

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epithelial remodeling [17]. It has also been suggested that a balance between pro-survival Bax and pro-apoptotic Bcl2 proteins determines cell life or cell death.

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Roscioli et al found that asthmatic epithelial cells demonstrated an increased Bax/Bcl2 transcript ratio [34]. To be specific, increased Bax/Bcl2 ratio promoted the cytochrome

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C release in mitochondria to activate the cascades of apoptosis [13]. Moreover, during airway dysfunction and inflammation, epithelial apoptosis was promoted accompanied

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by nuclear condensation or Caspase-3 activation [35,36], which is believed to be the terminal regulator of apoptosis. In this rat model of asthma, azithromycin treatment

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resulted in the reduction of Caspase-3 mRNA and protein levels, as well as the Bax/Bcl-2 ratio. A similar effect was also observed in DEX treated rats. Notably, in vivo study Bcl-2 protein level was decreased in OVA+AZM- and OVA+DEX- treated lung, with contrary Bcl-2 mRNA was elevated in OVA+AZM- and OVA+DEX- treated lung. The difference of mRNA and protein level of Bcl-2 may be controlled by post-transcriptional regulation mechanism. Moreover, as mentioned above, in

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TGF-β1-induced airway epithelium apoptosis BEAS-2B cells model, AZM treatment significantly reversed the TGF-β1 elevated Caspase-3 mRNA and Bax/Bcl-2 ratio, suggesting that AZM inhibited airway epithelium apoptosis contributes to ameliorating

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airway injury via down-regulating Caspase-3 and Bax/Bcl-2 ratio. However, we think down-regulating TGF-β1 expression is more important for attenuating OVA-induced

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airway remodeling, since airway epithelium apoptosis was induced by over-expression

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of TGF-β1.

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There is a major limitation in this experiment. There are no in vivo functional measurements showing that azithromycin benefited bronchial hyperresponsiveness in

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the OVA-induced mice. In the future study, we will complete this experiment to fill up this gap.

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Conclusion

In summary, the results of this study suggested that treatment of azithromycin could

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reduce airway epithelial apoptosis by down-regulating Bax/Bcl-2 ratio and Caspase-3 level, thus ameliorating airway epithelial injury.

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Acknowledgements

This study was supported by the National Natural Science Foundation of China (No. 81470224 to Lihong Wan). Abbreviations OVA, ovalbumin; PAS, periodic acid-Schiff; HE, hematoxylin and eosin; Wat, airway wall thickness; Wam, bronchial smooth muscle thickness; AZM, azithromycin; DEX,

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dexamethasone; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; TGF-β1, transforming growth factor-β1; AMs, alveolar macrophages; AI, apoptotic index; qRT-PCR, quantitative Real-time Polymerase Chain Reaction.

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2 Royce SG, Li X, Tortorella S, Goodings L, Chow BS, Giraud AS, et al. Mechanistic

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insights into the contribution of epithelial damage to airway remodeling: novel

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therapeutic targets for asthma. Am J Respir Cell Mol Biol. 2014; 50: 180-192. 3 Khalil N, O'Connor RN, Flanders KC, Unruh H. TGF-beta 1, but not TGF-beta 2 or

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12 Nabe T, Morishita T, Matsuya K, Ikedo A, Fujii M, Mizutani N, et al. Complete dependence on CD4+ cells in late asthmatic response, but limited contribution of the cells to airway remodeling in sensitized mice. J Pharmacol Sci. 2011; 116: 373-83. 13 Suen DF, Norris KL, Youle RJ. Mitochondrial dynamics and apoptosis. Genes Dev. 2008; 22: 1577-90. 14 Holgate ST. The sentinel role of the airway epithelium in asthma pathogenesis.

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epithelial cells. Cytokine. 2015; 75: 165-73. 30 Cheng G, Shao Z, Chaudhari B, Agrawal DK. Involvement of chloride channels in TGF-beta1-induced apoptosis of human bronchial epithelial cells. Am J Physiol Lung

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TGF-beta1-induced apoptosis of human bronchial epithelial cells. Am J Physiol Lung Cell Mol Physiol. 2007; 293: L1339-47.

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asthma-related inflammatory cytokines. Physiol Rep. 2013; 1: e00123. 35 Cho IH, Gong JH, Kang MK, Lee EJ, Park JH, Park SJ, et al. Astragalin inhibits airway eotaxin-1 induction and epithelial apoptosis through modulating oxidative stress-responsive MAPK signaling. BMC Pulm Med. 2014; 14: 122. 36 Rehman R, Bhat YA, Panda L, Mabalirajan U. TRPV1 inhibition attenuates IL-13 mediated asthma features in mice by reducing airway epithelial injury. Int

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Immunopharmacol. 2013; 15: 597-605.

Figure legend

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Figure 1 Rat model of airway inflammation and treatment with azithromycin

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Figure 2 Effects of azithromycin on OVA-induced lung inflammation and airway

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epithelium injury.

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Histological examination of lung tissue was performed 24 h after the final OVA challenge. Lung tissues were fixed, sectioned at 4 μm thickness, and stained with HE

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and periodic acid-Schiff (PAS). (A) Representative HE stained sections of lung of rats in the control group, OVA group, OVA+AZM group and OVA+DEX group

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(Magnification×100). (B) Representative lung sections showing periodic acid-Schiff (PAS) staining from control group, OVA group, OVA+AZM group and OVA+DEX

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group (Magnification×100). (C) Quantified results of airway wall thickness (Wat) and the bronchial smooth muscle thickness (Wam) analyzed by Image-Pro® Plus 6.0

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software. (D) Total inflammation score. (E) Degree of epithelial thickening was quantified using PAS-stained sections by dividing epithelial area (μm2) in the bronchus (diameter: 200-400 μm) by the length (μm) of basement membrane. (F) Degree of mucus accumulation was calculated by dividing PAS+ area (μm2) in the bronchus by the length (μm) of basement membrane. Values are presented as mean ± S.E.M. *P<0.05, versus control group (n=10). #P<0.05, versus OVA group (n=10).

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Figure 3 Effects of azithromycin on OVA-induced airway epithelium apoptosis. (A) Representative Terminal deoxynucleotidyl transferase–mediated dUTP nick end

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labeling (TUNEL) stained sections of lung of rats in the control group, OVA group, OVA+AZM group and OVA+DEX group (Left: Magnification×100;

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Right:Magnification×400)Arrows indicated the TUNEL positive cells. (B) AI was

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calculated as described in Materials and Methods. Values are presented as mean ±

P<0.05, versus OVA+DEX group (n=10).

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S.E.M. *P<0.05, versus control group (n=10). #P<0.05, versus OVA group (n=10).

Figure 4 Effects of azithromycin on Caspase-3, Bax and Bcl-2 in lungs.

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(A-B) The mRNA levels of Caspase-3, Bax and Bcl-2 were determined by quantitative real-time polymerase chain reaction (qRT-PCR). mRNA value=2-ΔΔCt, ΔΔCt=

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(CtTarget-CtActin) of the treated rat- (CtTarget-CtActin) of the control rat. (C) The ratio of Bax/Bcl-2mRNA was calculated. (D) The protein expressions of Caspase-3, Bax and

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Bcl-2 were analyzed by Western blotting. β-Actin was used as an internal standard. (E) The relative levels of Caspase-3, Bax and Bcl-2expressions in the lung. The results were expressed as the ratio of the investigated gene to β-actin. The band intensity was analyzed by Bio-Rad Quantity One v4.62 software. (F) The ratio of Bax/Bcl-2protein was calculated. Values are presented as mean ± S.E.M. *P<0.05, versus control group (n=10). #P<0.05, versus OVA group (n=10).

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Figure 5 Effects of azithromycin on TGF-β1-induced apoptosis in BEAS-2B cells. (A) BEAS-2B cells were exposed to TGF-β1 (30 ng/ml) and different concentration of

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AZM for 72 h. Apoptosis in BEAS-2B cells after AZM treatment was demonstrated by flow cytometry (left panel) and TUNEL staining (right panel: light micrographs, DAPI

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and green granules). (B) The graph shows the apoptotic rate. Values are presented as

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mean ± S.E.M. *P<0.05, versus control group (n=9). #P<0.05, versus TGF-β1 group

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(n=9).

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Figure 6 Effects of azithromycin on Caspase-3, Bax and Bcl-2 in BEAS-2B cells. (A-B) The mRNA levels of Caspase-3, Bax and Bcl-2 were determined by quantitative

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real-time polymerase chain reaction (qRT-PCR). mRNA value=2-ΔΔCt, ΔΔCt= (CtTarget-CtActin) of the treated rat- (CtTarget-CtActin) of the control rat. (C) The ratio of Bax/Bcl-2 mRNA was calculated. (D) The protein expressions of Caspase-3, Bax and

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Bcl-2 were analyzed by Western blotting. β-Actin was used as an internal standard. (E)

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The relative protein levels of Caspase-3, Bax and Bcl-2 expressions in the lung. The results were expressed as the ratio of the investigated gene to β-actin. The band intensity was analyzed by Bio-Rad Quantity One v4.62 software. (F) The ratio of Bax/Bcl-2protein was calculated. Values are presented as mean ± S.E.M. *P<0.05, versus control group (n=9). #P<0.05, versus TGF-β1 group (n=9).

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Table 1 PCR primers

caspase-3 F

5’-TGCATACTCCACAGCACCTGGTTA-3’

82bp

caspase-3 R

5’-CATGGCACAAAGCGACTGGATGAA-3’

bax R

5’-GTCCAGCCCATGATGGTTCT-3’

bcl-2 F

5’-TTCTTTGAGTTCGGTGGGGTC-3’

bcl-2 R

5’-TGCATATTTGTTTGGGGCAGG-3’

257bp

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5’-TCCACCAAGAAGCTGAGCGAG-3’

304bp

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bax F

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Length

5’-GATCATTGCTCCTCCTGAGC-3’

β-actin R

5’-ACTCCTGCTTGCTGATCCAC-3’

β-actin F

5’-CGTGCGTGACATTAAAGAG-3’

β-actin R

5’-TTGCCGATAGTGATGACCT-3’

101bp

132 bp

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β-actin F

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Rat

primers

caspase-3 F

5’-ACGACAGGGTGCTACGAT-3’

caspase-3 R

5’-CCTCTCCTTTCCTTACGC-3’

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Human

Gene

bax F

5’-GCCTTCTTTGAGTTCG-3’

bax R

5’-CAGCCTCCGTTATCC-3’

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Specices

bcl-2 F

5’-GCCTTCTTTGAGTTCG-3’

bcl-2 R

5’-CAGCCTCCGTTATCC-3’

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199 bp

140 bp

140 bp