Background
Chronic obstructive pulmonary disease (COPD) is a respiratory disease characterized by persistent and irreversible airflow restriction, which is related with abnormal airway inflammatory response to harmful gases/particles [
1]. Currently, COPD is a global chronic disease with high morbidity, mortality and disability rate, and has become an important public health problem in the world [
2]. Smoking is the major risk factor for COPD, and cigarette smoke (CS)-induced oxidative stress can lead to the damage of airway epithelium and compromise the barrier function, which is the initial step and pathological basis of COPD progression [
3‐
6].
Airway epithelium is the first line of defense against inhaling particles and pathogens due to its barrier property, which plays a crucial role in regulating innate barrier immunity and maintaining homeostasis [
7]. Airway epithelial barrier dysfunction not only impairs the physical barrier activity of airway epithelium, but further exposes sub-epithelial layers to exogenous substances, leading to a series of pathological processes such as airway inflammation, airway remodeling, airway hyperresponsiveness and increasing the rate of COPD exacerbation [
7,
8]. The barrier function of airway epithelium is maintained by apical junctional complexes consisting of tight junction (TJ) and adherent junction (AJ). The TJ proteins, such as zonula occludens (ZO), claudin, occludin, and the AJ proteins, such as E-cadherin, jointly participate in the formation of airway epithelial barrier [
5,
6,
9]. Studies have reported acute changes in airway epithelial permeability due to oxidative stress and inflammation, and junction proteins gradually decomposed after long-term CS exposure, leading to the structural destruction of airway epithelial barrier [
10‐
12]. Clinical data also confirmed that the expressions of ZO-1, occludin, and E-cadherin was significantly down-regulated in the bronchial epithelium and lung tissues in COPD patients compared to healthy controls [
5,
13,
14]. Accordingly, the structural and functional disorder of airway epithelial barrier may be a key link in the occurrence and development of COPD [
5,
15].
In recent years, great progress has been made in the treatment of COPD, while current therapies are mainly palliative. It is of great significance to find novel effective therapies targeting airway epithelial barrier. The Global Initiative for Chronic Obstructive Lung Disease [
16] recommended Azithromycin (AZI), a semi synthetic 15 membered macrolide antibiotic, as anti-inflammatory drug in stable COPD and for patients who still develop exacerbations on bronchodilators/inhaled glucocorticoid therapy. AZI can inhibit the synthesis of bacterial proteins by blocking the assembly of ribosomal units, and exert powerful antibacterial effects on a wide spectrum of gram-positive and gram-negative bacteria and atypical bacteria [
17]. Because of the good safety, tolerance and special pharmacokinetic characteristics, it is widely used in clinical practice for various respiratory diseases. Several randomized controlled studies have shown that long-term additional treatment with AZI significantly reduced the exacerbation rate of COPD [
18‐
20]. Besides antibacterial effect, AZI also showed antioxidant, anti-inflammatory and other pharmacological activities. For instance, AZI treatment ameliorated epithelial cell shedding after injury in addition to a dampened inflammatory response in a mouse model in a SO
2-induced mouse model [
21]. AZI up-regulated the expression of nuclear factor erythroid 2-related factor 2 (Nrf2), an anti-oxidant transcription factor, thereby suppressing inflammatory response induced by CS exposure [
22]. However, the effects of AZI and its mechanisms on airway epithelial barrier dysfunction caused by CS have not yet been elucidated.
In this study, we first investigated the protective effects of AZI targeting airway epithelial barrier using in vivo and in vitro cigarette smoking models. Then we employed a LC–MS-based metabolomics approach to obtain the metabolic profiles of in vivo experimental model to evaluate the underlying mechanisms. Our results demonstrated that GSH-related Nrf2 signaling pathway can be pharmacologically manipulated by AZI, which may provide new possibilities in the treatment of airway epithelium damage-related respiratory diseases induced by CS, such as COPD.
Methods
Materials
Macrolides were purchased from Selleck Chemicals (Houston, TX, USA). The antibodies used in present study were listed as follows: anti-Bax (#T40051F) and anti-Bcl-2 (#T40056) antibodies were purchased from Abmart biomedical Co., Ltd (Shanghai, China). Anti-GAPDH (#97166S), anti-Nrf2 (#12721S), anti-E-Cadherin (#14472), anti-ZO-1 (#13663S) and Alexa Fluor 594/488 conjugated anti-rabbit (#8889S)/mouse (#4408S) IgG (H + L) F(ab')2 Fragments were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-Keap1 (#ab19403) antibodies were purchased from Abcam Biotechnology (Cambridge, MA, USA). The HRP-labeled goat anti-rabbit (#65-6120)/mouse (#62-6520) IgG (H + L) secondary antibodies, ECL Plus Western Blotting Detection Kit (#32209) and BCA protein assay kit (#23227) were purchased from Thermo Fisher Scientific (San Jose, CA, USA). All other reagents were purchased from Beyotime Biotechnology (Shanghai, China) or Sangon Biotech Co., Ltd. (Shanghai, China) unless otherwise indicated.
Animal experiments and treatments
Male Sprague Dawley (SD) rats (8 weeks old, weighed 180–200 g) were purchased from SLAC Laboratory Animal Co., Ltd. (Shanghai, China). Nrf2 knockout mice (C57BL/6N background) were purchased from Cyagen Biosciences Inc (Suzhou, Jiangsu, China). The animals were housed at a constant temperature (25 ℃) under a 12 h light–dark cycle and had free access to food and water. The protocol has the approval of the Animal Experimental Ethical Committee of Fudan University (2019 Huashan Hospital JS-112) and all animal studies were performed in accordance with the guidelines for the care and use of laboratory animals set by Fudan University (Shanghai, China). The CS-induced COPD rat model was established as our previous report [
23]. 30 SD rats were randomly divided into 5 groups (6 rats/ group): control group, COPD model group, AZI low-dose (25 mg/kg) group, AZI middle-dose (50 mg/kg) group and AZI high-dose (100 mg/kg) group. AZI was administered intragastrically 1 h before the first exposure every day. The CS-exposed mice model was established according to a previously described protocol [
24]. Both wild type mice (n = 18) and Nrf2 (−/−) mice (n = 18) were randomly divided into 3 groups (6 mice/ group) respectively: control group, CS group and AZI-treated group (100 mg/kg). Control group was treated with saline. After the experiment, the rats or mice were anesthetized with intraperitoneal sodium pentobarbital (40 mg/kg) for sample collection and further analysis.
Bronchoalveolar lavage fluid (BALF) preparation
After anaesthesia, tracheotomy was performed to insert the cannula into the trachea. BALF was collected from the right lungs through three lavages of 1 ml saline for rats or two lavages of 0.5 ml saline for mice. Extracted BALF was immediately centrifuged at 1000 rpm for 5 min at 4 ℃ and used for further study.
Immunohistochemistry staining
Briefly, immediately after bronchoalveolar lavage, the right lungs were immersed in 4% paraformaldehyde for 24 h. After tissue fixation and paraffin embedding, 5 μm sections were incubated with anti-E-cadherin, ZO-1, Keap1 and Nrf2 antibodies (1:100 dilution) at 4 ℃ overnight followed by incubation with secondary antibody, at last stained with DAB and counterstained with hematoxylin.
Cell acquisition and culture
Primary bronchial epithelial cells (PBECs) were obtained from bronchial brushings in six healthy subjects (Additional file
1: Table S1). The Medical Ethics Committee of Huashan Hospital approved the study (KY2019-508), and all subjects gave their written informed consent. After protease digestion and centrifugation (1200 rpm, 5 min), PBECs were cultured in bronchial epithelial cell medium (#3211, ScienCell, San Diego, California, USA), supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin and 1 × bronchial epithelial cell growth supplement (#3262, ScienCell, San Diego, California, USA) using poly-L-lysine-coated flasks and employed for experiments at passage 2–4 without mycoplasma contamination (Additional file
2: Fig. S1). Human bronchial epithelial cells (HBECs, ZQ0001, ScienCell, San Diego, California, USA) were cultured in Keratinocyte Medium (#2111, ScienCell, San Diego, California, USA), supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin and 1 × keratinocyte growth supplement (#2162, ScienCell, San Diego, California, USA). Unless otherwise stated, cell culture reagents were purchased from Gibco (Carlsbad, CA, USA).
Cell treatment and cigarette smoke extract (CSE) preparation
Cells were pre-treated with or without AZI (0.5, 5, 50 μM), vitamin C (50 μM) or TBHQ (30 μM) for 1 h and subsequently exposed to vehicle (medium) or 3% CSE for 24 h. As our previous reports [
25], CSE was prepared by the combustion of one cigarette (12 mg tar/cigarette; Double Happiness, China), using a pump and passing the smoke through 10 mL of non-FBS culture medium at a rate of 5 min/cigarette. The resulting solution was adjusted to pH 7.4 with 1.0 M NaOH and strained through 0.22 μm gauge filters. The OD value of the obtained solution was 0.2 at 408 nm by a microplate reader, which represented 100% concentration and was diluted to the desired concentration with non-FBS culture medium. The fresh CSE was used within 30 min.
Measurement of transepithelial electrical resistance (TEER)
Airway epithelial permeability changes were evaluated by TEER measurement using a Millicell ERS-2 V-Ohmmeter (Millipore Co., Bedford, MA, USA) monitoring for 24 h at specified time points. In detail, PBEC/HBECs cells (105/well) were seeded in 12-well hanging inserts (0.4 μm, PET, Cat.No: MCHT12H48, Millipore, Darmstadt, Germany) with 500 μl apical and 1000 μl basolateral volumes of complete medium. HBECs were incubated for 48 h to yield a cell monolayer. Before TEER measurement, hanging inserts were equilibrated at room temperature for 10 min. After soaking in 70% ethanol and rinsing with medium, the electrode was inserted vertically into the chamber (below the liquid level without touching the bottom). TEER was calculated by the following equation: TEER (Ω/cm2) = (Rsample– Rblank) × effective membrane area (cm2). TEER values were corrected for background resistance of medium without cells.
Inflammatory cytokines and oxidative stress indexes
The levels of IL-6 and TNF-α in the BALF or culture supernatant were determined with ELISA kit (Jianglai industrial limited ByShare Ltd, Shanghai, China) according to the manufacturer's instructions. The contents of GSH and ROS, the activity of GST, GS and GCL in lung homogenate or cell lysate were detected using commercial assay kit (Sangon Biotech Co., Ltd., Shanghai, China) according to the manufacturer's instructions.
Western blot
Total protein was extracted by the RIPA (Beyotime, Shanghai, China), separated by 8–12% SDS-PAGE and electro transferred onto a PVDF membrane (Millipore, Bedford, MA, USA). The membrane was blocked with 5% skim milk for 1 h at room temperature and incubated with primary antibodies Bax, Bcl-2, Keap1, Nrf2, ZO-1, E-cadherin (1:1000 dilution) overnight at 4 ℃, followed by HRP-conjugated secondary antibodies for additional 1 h at 37 ℃. Proteins were visualized with BIO-RAD Molecular Imager (Version 6.0, USA) using enhanced chemiluminescence reagents.
Flow cytometry
The flow cytometry assays were performed according to manufacturer's instructions. Apoptosis was determined with the Annexin V-FITC/PI apoptosis detection kit (BD Bioscience, San Jose, CA, USA) and a flow cytometer (Beckman Coulter Inc., Brea, CA, USA).
Immunofluorescence staining
Briefly, cells were seeded on confocal dishes and treated as indicated for 24 h. The dishes were washed three times with PBS, fixed in 4% paraformaldehyde for 20 min at 37 ℃, and then permeabilized with 0.3% Triton X-100 for 10 min. Cells were then blocked with 3% BSA at room temperature for 1 h followed by incubation with a primary antibody E-cadherin, ZO-1, Keap1 and Nrf2 (1:100 dilution) at 4 ℃ overnight. Dishes were washed three times with PBS and incubated with Alexa Fluor 594/488 conjugated-goat anti-rabbit/mouse IgG for 1 h, and then labeled with hoechst for 15 min. Finally, Dishes were washed three times with PBS, visualized and photographed under confocal laser scanning microscope (Leica TCS SP8, Germany).
Short hairpin RNA (shRNA) interference
HBECs grown to 30–50% confluency were transfected by lentiviral-delivered shRNAs targeting Nrf2 (multiplicity of infection = 20, GENECHEM Incorporation, Shanghai, China), and simultaneously strengthened by HitransG P (1x). After 8 h transfection, cells were incubated with fresh medium for another 48 h, and subsequently screened with puromycin (2 μg/mL) for 72 h. Monoclonal cells were maintained for further experiment. The sequences of shRNA-Nrf2 are as follows: 5’-CCGGCATTTCACTAAACACAA-3’.
For lung tissue homogenate samples, 400 μl MeOH (containing 100 ng/mL Warfarin as Internal Standard) was added to the samples (100 μl), which were then vortexed for 60 s, followed by centrifugation at 14,000
g and 4 ℃ for 15 min. Finally, the supernatants were transferred to HPLC glass vials and stored at − 20 ℃ prior to LC–MS analysis. For data acquisition, the metabolomics data was acquired by Agilent 1290 UHPLC system coupled to a quadruple time-of-flight mass spectrometer (6530 Q/TOF–MS). Analytes in samples were separated with an ACQUITY BEH C18 (2.1 × 100 mm, 1.7 μm) (Waters Technologies, Milford, MA, USA); the column temperature was 30℃. The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B), and the flow rate was 0.3 mL/min. Gradient elution was carried out as follows: 5–5% B over 0–3 min; 5–10% B over 3–3.5 min; 10–40% B over 3.5–12 min; 40–60% B over 12–22 min; 60–80% B over 22–26 min; 100–100% B over 26.3–30 min. Re-equilibration was at 5% B for 3 min. A quality control sample was employed to monitor the system stability by injecting after each 10 injection. The MS condition was set according to previous study [
26]. For data processing, non-targeted LC–MS data from multiple runs were extracted and aligned using MS-DIAL software. The metabolites were identified according to MS-FINDER software based on MS1 and MS2 analysis. The raw MS data files were converted to the mzXML format using ProteoWizard, and processed using the MS-DIAL software for peak detection, alignment, and isotope annotation. After processing, a peak list containing the mass-to-charge ratio (m/z), retention time and peak intensity was generated. The peak list was imported to MetaboAnalyst 5.0 by sum, then features were calculated the
p values using
t-test and p < 0.05 were considered as differential metabolites, and were further identified based on the exact molecular weight and the MS/MS spectrum similarity using the MS-FINDER software according to METLIN and HMDB data. The metabolite set enrichment analysis (MSEA) of different metabolites was conducted based on online MetaboAnalyst 5.0 platform.
Data and statistical analysis
Data analysis was performed using GraphPad Prism software (Version 7.0, La Jolla, CA, USA), and expressed as means ± SD. The t-test was performed to measure the differences between the two groups and ANOVA followed by a Dunnett’s test was performed to compare the differences among three or more groups. A two-sided P value less than 0.05 were considered to be significantly different.
Discussion
In our present study, we found that AZI not only possessed anti-inflammatory and anti-oxidative properties, but also ameliorated airway epithelial barrier dysfunction by improving TEER and apical junctional complexes. Our study also revealed that AZI significantly up-regulated the expression of Nrf2 to promote GSH metabolism, highlighting the novel role of Nrf2/GCL/GSH signaling pathway in maintaining airway epithelial barrier function. These results demonstrated that AZI prevented CS-induced airway epithelial barrier dysfunction through Nrf2/GCL/GSH signaling pathway.
The bronchial epithelium is responsible for maintaining the airway homeostasis of respiratory system. Destruction of airway barrier integrity exposes sub-epithelial layer to inhaled particles, triggering airway inflammation and immune responses, indicating that airway epithelial barrier dysfunction is closely related with respiratory diseases [
5,
9]. It has been confirmed that smoking disrupted apical junctions of airway epithelium, and the reduction of apical junction genes has been observed in the lung tissues of COPD patients [
31]. In addition, TJ proteins were also significantly suppressed in lung tissue of patients with end-stage COPD and in air–liquid interface differentiated epithelial cells from these patients [
13]. Similarly, our experiments in vitro revealed that CSE exposure caused the degradations of TJ protein ZO-1 and AJ protein E-cadherin with subsequent TEER decline, which eventually leads to airway epithelial barrier dysfunction. It was well known that structural and subsequent functional destruction of epithelial barrier is a typical feature of chronic airway inflammation. Many innate and adaptive immune mediators that may be up-regulated after long-time cigarette smoking, including cytokines, chemokines and apoptosis factors, could regulate the airway epithelium barrier function [
5]. As found in our study, CS-increased secretions of pro-inflammatory cytokines and airway epithelium cell apoptosis in vitro and in vivo, which was consistent with above reports.
AZI is commonly indicated for the treatment of respiratory bacterial infection, and exerts immunomodulatory activities in chronic inflammatory disorders, such as COPD [
32‐
34]. Clinically, in patients with severe COPD, continuous therapy of AZI combined with nebulized colistin dramatically prevented the exacerbations of COPD [
35]. Preventive administration of AZI reduced the frequency of acute exacerbation and improved the quality of life in COPD patients [
20]. However, the underlying mechanism is not completely clear. Here, our study provided evidence that AZI treatment counteracted the CSE-induced TEER reduction and disruption of ZO-1 and E-cadherin, along with the inhibition of inflammatory response and apoptosis in vitro and in vivo. Consistently, a recent study revealed that AZI treatment substantially enhanced epidermal characteristics partially by up-regulation of tight junction proteins in bronchial epithelial cells [
36]. In addition, pretreatment of AZI inhibited the secretions of IL-6 and IL-8 in CS-exposed bronchial epithelial cells, suggesting that AZI may be beneficial for smoking-induced airway epithelial barrier dysfunction [
37].
Metabolomics is considered as a promising method to accurately determine all low molecular weight metabolites of an organism and reveal its biology and response to pathophysiological stimuli [
38]. The results of metabolomics usually contain a continuous stream of high-content information, which will essentially help to understand the differentiating metabolite profiles from a global perspective [
39]. Therefore, metabolomics has been widely applied in various fields, such as drug toxicity, disease diagnosis, and pharmacodynamic study [
40‐
42]. To elucidate the mechanisms though which AZI ameliorated CSE-induced airway epithelial barrier dysfunction, we performed metabolomics profiling. Interestingly, our experimental results showed that metabolite set enrichment analysis revealed pathways upregulated by AZI treatment, including GSH metabolism. GSH, the most abundant non-protein thiol compounds in mammalian tissues and cells, is known as the most important endogenous molecule to resist oxidative stress, detoxify xenobiotics and regulate cell proliferation, apoptosis, immune function, and fibrogenesis [
43]. Due to the central role of GSH in maintaining cellular redox homeostasis, it is absolutely necessary for a series of biochemical reactions to protect airway epithelial cells from CS-induced oxidative stress [
44]. It has been known that CS-induced oxidative stress weakened GSH levels in airway epithelium and disrupted tight junctions, epithelial barrier integrity, finally leading to the impairment of epithelial barrier function [
45,
46]. Herein, our study showed that the major molecules or enzymes involved in GSH synthesis and metabolism pathway, including GSH, GCL, GS, GST,
l-Glutamic acid and pyroglutamic acid, were all significantly decreased by CSE exposure, while these molecules or enzyme activity were prominently restored by AZI treatment. In fact, AZI has already been reported to suppress ROS release in epithelial cells and prevent oxidative damage in macrophages harvested from 8 transplant recipients [
47], indicating the antioxidant potentials of AZI. For the first time to our knowledge, our experimental results highlighted a role for AZI promoting glutathione metabolism in the lungs of CS exposure.
Oxidative stress is highly correlated with the impairment of glutathione metabolism in COPD pathogenesis [
48]. GSH synthesis and metabolism are mainly determined by GCL, GS, GST, which are directly regulated by Nrf2 transactivation [
49,
50]. Nrf2, a transcription factor significantly expressed in airway epithelial cells, is known to regulate antioxidant and cytoprotective genes through activating antioxidant response elements, showing protective effects on airway epithelium [
51]. Previous studies have confirmed that Nrf2 pathway regulates more than 500 genes, including genes that regulate oxidative stress (GCL), inflammation (NF-κB), apoptosis (Bcl-2 and Bax), and autophagy (p62) [
52]. It has been confirmed that Nrf2 and some of its target genes constituted a protective signaling pathway against oxidative stress and inflammation in COPD development [
53,
54]. And the deficiency of Nrf2 contributes to the pathogenesis of COPD, accompanied with dysregulation of GSH metabolism [
49,
51,
55,
56]. A recent observational longitudinal study revealed that GSH was significantly reduced in the blood samples of COPD patients, moreover the expression of Nrf2 in PBMCs were significantly down-regulated in COPD patients at follow-up compared with non-COPD patients [
57]. Also, it was reported that activating Nrf2/GCL/GSH antioxidant signaling pathway by quercetin could attenuate toosendanin-induced oxidative stress to prevent hepatotoxicity [
58], and Nrf2/GCL/GSH axis could protect mitochondria from methylglyoxal-induced cells [
59]. As expected, our results demonstrated that AZI treatment significantly prevented Nrf2 suppression which was induced by CS exposure in vitro and in vivo. Moreover, our experiments further verified the role of Nrf2 by using Nrf2-shRNA, Nrf2 agonists, antioxidant and Nrf2 knockout mice, confirming that the protective effects of AZI on CS-induced airway epithelial barrier dysfunction primarily depends on the activation of Nrf2/GCL/GSH signaling pathway. In addition, our results (in Additional file
4: Fig. S2) further confirmed that, compared with the baseline of control cells, AZI slightly increased the expression of Nrf2, E-cadherin and ZO-1, but the increased value was not statistically different. And AZI also did not affect the levels of IL-6, GCL and GSH in normal HBECs. These results are in accordance with previous reports [
60,
61], indicating that AZI would not adversely affect the function and stability of normal cells.
It is also noteworthy that not only AZI, but some other macrolides (e.g. erythromycin, clarithromycin) have also been reported to prevent COPD exacerbations and improve patient quality of life and symptoms [
62]. Thus, to further explore whether the protection of airway epithelial barrier is the common pharmacological activity of macrolides, we have repeated the in vitro experiments using erythromycin (EI, 14-membered ring macrolide) and spiramycin (SPI, 16-membered ring macrolide). Data in Fig.
8 revealed that EI could also protect the airway epithelial barrier against CSE and its molecular mechanism was similar to that of AZI, while SPI had no similar pharmacological activity and regulatory effect. In fact, the structure of 16-membered ring macrolide is different from that of 14- and 15-membered ring macrolide, and the bond mode (the hydrogen bond, the hydrophobic bond and the van der Waals force) to the target are also different, which lead to different activity. Pharmacological studies have confirmed that anti-inflammatory and immunomodulatory effects were mainly found in 14- and 15-membered ring macrolides, such as EI and AZI [
63], which may explain why SPI showed no obvious effects on CS-induced airway epithelial barrier dysfunction. There are also reports that macrolides with 14- and 15-membered ring, instead of 16-membered ring, have functions other than antibacterial effect: strengthen the epithelial cell barrier and strengthen the tight junction [
64]. However, the exact mechanisms of macrolides with 14- and 15-membered ring in protecting the airway epithelial barrier still need to be further studied.
Nevertheless, there are some limitations in this study: (1) We only investigated the preventive effect of AZI, but its clinical therapeutic effect on protecting airway epithelial barrier is still worthy of further verification; (2) We have already found that Nrf2 is the key mediator in the protective effect of AZI on airway epithelial barrier, but how AZI activates Nrf2 is still unknown, which is our next research objective; (3) Different pharmacological effects of macrolides on airway epithelial barrier may be related to the difference of structural characteristics, which requires molecular docking and structure modeling methods to further clarify the underlying mechanisms.
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