key: cord-1055304-2g03olcy
authors: Mao, Yajun; Feng, Hong
title: Vitamin D3 alleviates cigarette smoke extract-mediated epithelial-mesenchymal transition and fibrogenesis by upregulating CC16 expression in bronchial epithelial cells
date: 2022-03-29
journal: Exp Ther Med
DOI: 10.3892/etm.2022.11284
sha: d38a679ad44523355909cdf821b0db48377417ad
doc_id: 1055304
cord_uid: 2g03olcy

Vitamin D3 supplementation has been previously reported to inhibit the occurrence and development of chronic obstructive pulmonary disease (COPD). However, the underlying mechanism remains unclear. Epithelial-mesenchymal transition (EMT) and fibrogenesis have been associated with the development of COPD. The aim of the present study was to investigate the potential effects and mechanism of vitamin D3 in an in vitro model of cigarette smoke (CS)-induced EMT and fibrosis, with specific focus on the role of club cell protein 16 (CC16). CS extract (CSE) at different concentrations (5, 10 and 20%) was used to treat 16-HBE cells to induce EMT and fibrogenesis following which they were treated with vitamin D3. Subsequently, the 20% CSE group was selected for further experiments, where 16-HBE cells were divided into the following five groups: The control group; the CSE group; the low-dose vitamin D3 group (250 nM); the medium-dose vitamin D3 group (500 nM); and the high-dose vitamin D3 group (1,000 nM). Western blot analysis was used to detect the protein expression levels of the EMT-related proteins E-cadherin, N-cadherin, Slug and α-SMA, fibrogenesis-related proteins collagen Ⅳ and fibronectin 1, proteins involved in the TGF-β1/SMAD3 signaling pathway and CC16. Immunofluorescence was used to measure the protein expression levels of E-cadherin, N-cadherin and collagen Ⅳ. Specific CC16 knockdown was performed using short hairpin RNA transfection to investigate the role of CC16. The results of the present study found that vitamin D3 could increase the protein expression level of CC16 to inhibit the activation of the TGF-β1/SMAD3 signaling pathway; thereby reducing the 20% increase in CSE-induced EMT- and fibrogenesis-related protein expression levels. Following CC16 knockdown, the inhibitory effects of vitamin D3 on EMT- and fibrogenesis-related protein expression were partially reversed. To conclude, these results suggest that vitamin D3 can inhibit the protein expression levels of EMT- and fibrogenesis-related proteins induced by CSE, at least partially through the function of CC16. These findings are expected to provide novel theoretical foundations and ideas for the pathogenesis and treatment of COPD.

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation to and from the lung, which has become a growing global health issue (1, 2) . In addition, air pollution and genetic factors are important risk factors for COPD (3) (4) (5) (6) . However, currently available therapeutic options for COPD, including methylxanthines, β2-agonists, anticholinergics, corticosteroids and phosphodiesterase 4 inhibitors, were not able to completely halt the progression of COPD pathogenesis nor reduce the mortality rate (7) (8) (9) . Therefore, there remains to be an urgent demand to explore novel treatment strategies to prevent the development of COPD.

Although the pathogenesis of COPD has not been fully elucidated, it has been proposed that epithelial-mesenchymal transition (EMT) and fibrogenesis may be involved (10, 11) . During EMT, epithelial cells gradually lose their epithelial phenotype and acquire typical mesenchymal characteristics (12) (13) (14) (15) . These are characterized by enhanced mitotic ability and extracellular matrix production, accompanied by the upregulation of N-cadherin, slug and α-SMA expression and the downregulation of E-cadherin expression (12) (13) (14) (15) . The main pathological changes that occur during fibrosis are increases in fibrous connective tissue generation in the organ tissue, decreases in parenchymal cells (16) . Continuous EMT and fibrosis progression can lead to the destruction of organ structure and functional decline (17) . This serves to be one of the main mechanism of small airway narrowing and is now considered to be the most important mechanism of COPD progression (18) . Epithelial cell fibrogenesis Vitamin D3 alleviates cigarette smoke extract-mediated epithelial-mesenchymal transition and fibrogenesis by upregulating CC16 expression in bronchial epithelial cells process is frequently accompanied with upregulation of fibrogenesis-related protein expression, such as collagen IV and fibronectin 1 (FN1) (19) . Previous studies have provided evidence that EMT is active in the airway of smokers, particularly in patients with COPD who smoke, suggesting that smoking-induced EMT can contribute to the pathogenesis of COPD (15, 20) .

Patients with COPD and acute obstructive pulmonary disease were frequently found to have vitamin D deficiency (21) . Vitamin D (calcitonin) deficiency was reported to be positively associated with the severity of COPD (21) . In addition, patients with COPD or more severe disease were found to have lower serum vitamin D levels (22) . A previous study has suggested that treatment with vitamin D in patients with COPD and vitamin D deficiency can reduce the risk of moderate-to-severe disease (23) . Furthermore, a recent study on patients with COPD caused by COVID-19 showed that although vitamin D deficiency is not a determinant of disease severity, supplementation does confer a positive role in alleviation (24) . Cigarette smoke extract (CSE) can inhibit vitamin D-induced vitamin D receptor (VDR) translocation (25) . Mathyssen et al (26) previously documented that the use of vitamin D supplements may reduce CSE-mediated cellular inflammatory responses by upregulating cathelicidin expression in bronchial epithelial cells. However, the underlying molecular mechanism remain unknown (26) . In another previous study, although vitamin D deficiency exacerbated pulmonary fibrosis and EMT (27) , no significant associations between the two conditions could be found. Vitamin supplements have been found to delay the process of fibrosis (28) . However, the role of vitamins in CSE-induced fibrogenesis in bronchial epithelial cells remains unclear.

Club cell protein 16 (CC16) is the most abundant protein in the bronchoalveolar lavage fluid (29, 30) . It is encoded by the secretoglobin family 1A member 1 gene and serves an anti-inflammatory and antioxidant role in vivo (29, 30) . Previous studies have shown that CC16 mainly exerts anti-inflammatory effects in smoke-exposed lungs, such that COPD has been associated with CC16 deficiency (31, 32) . Smokers and patients with COPD were found to exhibit reduced airway CC16 immunostaining, which decreased further with increasing COPD severity (31) (32) (33) . Exposing mice to CSE was found to reduce the airway expression of CC16 (34) . Therefore, CC16 is becoming of interest as a target molecule for the treatment of COPD. CC16 has been associated with the occurrence and development of COPD, with the severity and prognosis of the disease (34) . The dynamic changes in CC16 expression can be used to predict changes in the condition of patients with COPD and to evaluate the clinical outcome. Combined with the comprehensive analysis of other common clinical indicators, the length of the stay in hospital can be shortened as analyzed by linear correlation analysis and multiple linear regression analysis (33, 35, 36) . Therefore, the present study hypothesized that CC16 is associated with lung fibrogenesis during COPD. However, the effects of vitamin D3 on the expression of CC16 after CSE exposure and its underlying molecular mechanism remain unclear.

In COPD, the bronchial epithelium is the first immune barrier triggered by cigarette smoke (37) . The present study aimed to investigate the effect of vitamin D3 supplementation on EMT and fibrogenesis in bronchial epithelial cells after CSE treatment. In addition, the role of CC16 in these processes and the expression of EMT and fibrogenesis-related markers were detected.

Bioinformatics Methods. The interaction between CC16 and FN1 was analyzed using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database (version 11.5; https://string-db.org/cgi/input.pl).

Bronchial epithelial cell culture. The 16-HBE cells were purchased from the American Type Culture Collection and were cultured in DMEM (Sigma-Aldrich; Merck KGaA) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin G sodium and 100 µg/ml streptomycin sulfate (Invitrogen; Thermo Fisher Scientific, Inc.). They were cultured at 37˚C in a humidified incubator with 5% CO 2 . The cells were cultured until they reached 70-80% confluence. Preparation of CSE. The smoke from 15 lit cigarettes (Sichuan China Tobacco Industry Co., Ltd.) was slowly inhaled into a 50 ml syringe, and then injected it into DMEM pre-heated in a 37˚C water bath (38) . The pH of DMEM was adjusted to 7.4 and sterilized using a 0.22-µm filter (EMD Millipore) and stored at -80˚C. Serum-free DMEM was used to dilute 100% CSE to the required CSE concentrations (5, 10 and 20%). Cells were treated with vitamin D3 (250, 500 or 1,000 nM; Sigma-Aldrich; Merck KGaA) or vehicle (0.1% ethanol) for 30 min prior to 24 h treatment at 37˚C with CSE and vitamin D3 or vehicle, after shRNA transfection (when it was required) (26) .

Cell Counting Kit (CCK)-8 assay. Cell viability was performed using CCK-8 assay. A total of 1x10 4 16-HBE cells/well were seeded into 96-well plates and pre-treated with CSE followed by vitamin D3. In total, 10 µl CCK-8 reagent (Abcam) was added into each well and the samples were incubated at 37˚C for 1-4 h. Subsequently, the absorbance in each well was measured at 460 nm using a microplate spectrophotometer (Bio-Rad Laboratories, Inc.).

Total RNA was extracted from the cells using TRIzol ® (Invitrogen; Thermo Fisher Scientific, Inc.). The concentration was assessed using a Nanodrop ® 2000 spectrophotometer (Thermo Fisher Scientific, Inc.) before being reverse transcribed into cDNA. cDNA was synthesized using the PrimeScript™ RT kit (cat. no. RR037A; Takara Bio, Inc.) under the following conditions: 15 min at 37˚C and 5 sec at 85˚C. The reactions were performed using SYBR Green Taq Mix (cat. no. RR096A; Takara Bio, Inc.) in a StepOnePlus™ PCR system (Thermo Fisher Scientific, Inc.) under the following conditions: 45˚C for 3 min, 95˚C for 10 sec, 40 cycles at 95˚C for 15 sec then 58˚C for 1 min. The primer sequences used were as follows: CC16 forward, 5'-CAG AGA CGG AAC CAG AGA CG-3' and reverse, 5'-CAG ATC TCT GCA GAA GCG GA-3' and GAPDH forward, 5'-GGA GCG AGA TCC CTC CAA AAT-3' and reverse, 5'-GGC TGT TGT CAT ACT TCT CAT GG-3'. Gene expression was evaluated using the 2 -ΔΔCq method (39) using GAPDH as a reference gene.

Western blot analysis. The total protein was extracted from the cells using RIPA lysate buffer (Beyotime Institute of Biotechnology) containing protease inhibitors, phosphatase inhibitors and phenylmethylsulfonyl fluoride (all from Beyotime Institute of Biotechnology). The protein concentration was determined using a BCA protein determination kit (Beyotime Institute of Biotechnology). A total of 30 µg protein per lane extract was separated using 6-12% SDS-PAGE and transferred onto PVDF membranes. Subsequently, the PVDF membranes were blocked with TBS containing 5% BSA (Sigma-Aldrich; Merck KGaA) for 2 h at room temperature, before being incubated overnight at 4˚C with the following primary antibodies: E-cadherin (1:1,000; cat. no. 14472; Statistical analysis. Statistical analysis was performed using GraphPad 8.0 (GraphPad Software, Inc.). Data were expressed as means ± SEM of three independent experiments. Comparisons were performed using one-way analysis of variance with Tukey-Kramer post hoc test. P<0.05 was considered to indicate a statistically significant difference.

After treating the 16-HBE cells with different concentrations of CSE (5, 10 and 20%) for 24 h, cell viability was decreased in a dose-dependent manner, comparing with that in the control group (Fig. 1A) . CSE at a concentration of 20% was used for subsequent experiments. Western blot analysis revealed that the protein expression level of E-cadherin was decreased, whilst the protein expression levels of N-cadherin, slug and α-SMA were increased, in a dose-dependent manner following CSE treatment ( Fig. 1B and C) . The results of immunofluorescence were consistent with those in the western blot analysis results, revealing a decrease in E-cadherin protein expression and an increase in N-cadherin protein expression compared with those in the control group (Fig. 1D) . These results suggest that CSE promoted the transformation of these 16-HBE epithelial cells into mesenchymal-like cells.

It was first found that CC16 interacted with FN1, which is associated with fibrogenesis (40), from analysis using the STRING database ( Fig. 2A) . Therefore, western blot analysis was used to measure the protein expression level of fibrogenesis-related proteins. Compared with that in the control group, the protein expression level of CC16 in 16-HBE cells were decreased following CSE treatment. Furthermore, the protein expression levels of the fibrogenesis-related proteins collagen IV and FN1, in addition to those in the TGF-β1/SMAD3 signaling pathway, were increased in the CSE treatment groups in a dose-dependent manner compared with that in the control group (Fig. 2B) . Immunofluorescence results also showed that the expression level of collagen IV was also increased in the CSE treatment group compared with that in the control group (Fig. 2C ). In summary, these results suggest that CSE induced fibrogenesis in the 16-HBE cell line.

Vitamin D3 treatment did not appear to cause toxicity to 16-HBE cells treated with 20% CSE, instead increasing cell viability (Fig. 3A) . Western blot analysis found that the protein expression level of E-cadherin was increased, whilst the protein expression levels of N-cadherin and slug were decreased following supplementation with vitamin D3, compared with those in the 20% CSE treatment alone group for 24 h (Fig. 3B and C) . The results from immunofluorescence were consistent with those in western blot analysis (Fig. 3C) , suggesting that vitamin D3 supplementation can reverse EMT induced by CSE.

Compared with that in the CSE alone group, the protein expression level of CC16 was increased following supplementation with vitamin D3.

Western blot analysis was used to also measure the protein expression level of fibrogenesis-related proteins. The results showed that compared with that in the CSE alone group, the protein expression level of fibrogenesis-related proteins collagen IV, FN1, TGF-β1 and SMAD phosphorylation were decreased following administration with vitamin D3 (Fig. 4A) . Immunofluorescence results showed that compared with that in the CSE alone group, the protein expression level of collagen IV was inhibited following supplementation with vitamin D3 (Fig. 4B) . These results suggest that vitamin D3 can inhibit the CSE-induced fibrogenesis of bronchial epithelial cells (Fig. 4B) .

To assess the role of CC16 in fibrogenesis and EMT, specific shRNAs were used to inhibit the expression level of CC16 in the 16-HBE cell line. Compared with that in the scrambled shRNA control group, both shRNA-CC16-1 and shRNA-CC16-2 significantly reduced CC16 expression in the 16-HBE cell line, with shRNA-CC16-1 being more potent (Fig. 5A) . According to western blot analysis, CC16 knockdown markedly reduced the protein expression level of E-cadherin whilst promoting the protein expression levels of N-cadherin, slug and α-SMA, compared with those in the vitamin D3 treatment group (Fig. 5B) . Subsequent western blot analysis revealed that knocking down CC16 expression led to significant increases in the protein expression levels of fibrogenesis-related proteins compared with those in the vitamin D3 treatment group (Fig. 5C) . These results suggest that after transfection with shRNA-CC16-1, the inhibitory effects of vitamin D3 on EMT and fibrogenesis were reversed in the 16-HBE cell line. In addition, these suppressive effects of vitamin D3 on CSE-induced EMT and fibrogenesis was at least partially mediated by CC16.

COPD is a type of chronic bronchitis and/or emphysema with the pathological characteristics of airflow obstruction, which can further develop into common chronic diseases such as pulmonary heart disease and respiratory failure (41) . COPD is associated with aberrant inflammatory reactions caused by harmful gases and harmful particles (42) . Among these harmful factors, cigarette smoke considered to be one of the most common harmful particles that can cause COPD (43) . Metal substances (such as Arsenic, cadmium and lead) and tobacco derivatives contained within cigarette smoke increases the risk of lung adenocarcinoma, such that ~6 million deaths are caused by tobacco use every year worldwide (44) (45) (46) . Previous studies have shown that smoking contributes to lung remodeling prior to COPD, where CSE induces bronchial epithelial damage by increasing the susceptibility to respiratory infections (47) (48) (49) . During this process, cells tend to undergo EMT and anchor-independent growth (50) . In the present study, 16-HBE cells exposed to CSE exhibited EMT characteristics in a dose-dependent manner, with the highest N-cadherin, slug and α-SMA protein expression levels and the lowest E-cadherin protein expression levels being observed following 20% CSE exposure. Previous studies have found signs of peribronchiolar fibrosis in the small airways of patients with COPD, which EMT appearing to be involved in this process (47, 51) . The present study observed that the expression of fibrogenesis-related proteins collagen IV and FN1 were significantly upregulated in 16-HBE cells after being treated with 20% CSE for 24 h. These results suggest that CSE can induce EMT and fibrogenesis in bronchial epithelial cells.

According to analysis using the STRING database, CC16 is associated with the fibrosis-related protein FN1. CC16 is a major protein that is secreted by club cells in the bronchial epithelium and is eliminated by the kidneys (30) . After observing this association between CC16 and fibrogenesis, the expression levels of CC16 was measured after CSE exposure. Previous study reported that CC16 can exert anti-inflammatory properties in smoke-exposed lungs of mice and proposed that COPD may be associated with CC16 deficiency (33) . In the present study, downregulation of CC16 expression in CSE treated 16-HBE cells was also observed.

Over the past decade, a number of studies have shown that vitamins serve an important role in COPD (52) (53) (54) . Vitamin A is essential for the preservation and integrity of the lung epithelium (55) . Compared with that in the healthy group, the levels and intake of vitamin A in patients with COPD was significantly reduced whilst serum vitamin A levels were also found to be positively associated with the severity of COPD (56) . In a previous clinical study that involved Korean patients with COPD, vitamin C was found to exert protective effects against COPD (57) . In addition, another previous study hypothesized that the protective effects of vitamin C against COPD was due to its antioxidant effects (52) . Vitamin B12, vitamin E and vitamin K were also found to serve key roles in inhibiting the occurrence and development of COPD (58-61). However, accumulating evidence has found that vitamin D, which is a class of fat-soluble vitamins that includes vitamin D3 and D2, can exert a role in COPD (62, 63) . Its reported physiological effects include regulation of calcium and phosphorus metabolism, cell proliferation and differentiation, immune regulation and promotion of bone growth (64) . Therefore, vitamin D has been of interest for the potential treatment and prevention of various diseases, such as chronic kidney disease and cystic fibrosis (65) (66) (67) . In a previous in vivo study, vitamin D3 supplementation was found to alleviate lung injury in rats with COPD whilst also reducing cell apoptosis in the lung tissues (68) . Clinical studies have also shown that vitamin D3 supplementation can alleviate moderate or severe COPD to reduce the incidence of upper respiratory tract infections . Furthermore, CSE has been reported to inhibit vitamin D-induced VDR translocation (25) , but the vitamin D3/VDR axis can inhibit emphysema in patients with COPD (70) . However, the specific mechanism of the protective effects of vitamin D3 against COPD remains unclear. Therefore, the present study investigated the effects of vitamin D3 on CSE-induced EMT and fibrogenesis in 16-HBE cells.

A previous study found that combining aerobic exercise with vitamin D3 supplementation can upregulate the levels of serum CC16 in patients with lung injury caused by smoking (71) . However, the specific mechanism was not elucidated (71) . Compared with the findings reported by Mathyssen et al (26) , this previous study found no clear association between vitamin D3 supplementation and the degree of EMT of bronchial epithelial cells (71) . In the present study, EMT and fibrogenesis was induced in 16-HBE cells following exposure to CSE, which were alleviated by vitamin D3 in a dose-dependent manner. In addition, to verify the association between Vitamin D3 and CC16 in this process, CC16 shRNA was used to knock down its expression. The results showed that after CC16 expression was reduced, the protective effects of vitamin D3 on 16-HBE cells were significantly reversed, with EMT and fibrogenesis restored. This suggests that vitamin D3 can mediate protective effects by increasing the protein expression of CC16. This provides a novel mechanism to further the understanding of the protective role of vitamin D3 against COPD. However, further investigation is required.

During EMT, the TGF-β1/SMAD signaling pathway serves an important role (72) . A previous study has shown that smoking can increase TGF-β1 production (73) . The levels of EMT and TGF-β in the airway epithelial cells of patients with COPD were associated with the severity of peribronchial fibrosis and airway obstruction (74) . In the present study, upregulation of TGF-β1 expression was observed in CSE-treated 16-HBE cells. A previous study also suggested that the TGF-β1/SMAD2/3 pathway can be a potential therapeutic target for EMT in malignant tumor cells derived from epithelial cells (75) . Compared with that in heathy individuals, the expression level of TGF-β1 and its downstream signal, SMAD2/3 was found to be significantly increased in the airways of patients with COPD (73) . The present study showed that SMAD3 was activated in 16-HBE cells exposed to CSE. After treating the cells with vitamin D3, the expression of TGF-β1 and the activation of SMAD3 was partially inhibited whilst the expression of CC16 was upregulated.

Bronchial epithelial cells were used for the present study. However, in vivo models must be applied for verification. In addition, it is of importance to investigate further the exact mechanistic relationship between vitamin D3 and CC16.

In conclusion, vitamin D3 supplementation could inhibit CSE-mediated EMT and fibrogenesis by increasing the protein expression levels of CC16. After CC16 expression was knocked down using shRNA, the inhibitory effects of vitamin D3 on EMT and fibrogenesis was reversed, suggesting that vitamin D3 protected the bronchial epithelial cells by inhibiting EMT and fibrogenesis. To the best of our knowledge, the present study was the first to suggest that vitamin D3 can serve a inhibitory role in EMT and fibrogenesis by regulating CC16. This may furthering the understanding of the mechanism underlying the effects exerted by vitamin D3 supplementation in preventing the development of COPD.

Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: A systematic analysis for the global burden of disease study

Burden and clinical features of chronic obstructive pulmonary disease (COPD)

Chronic exposure to high levels of particulate air pollution and small airway remodeling

High COPD prevalence at high altitude: Does household air pollution play a role?

Genetic risk factors for the development of pulmonary disease identified by genome-wide association

Microbial exposure to dairy farmers' dwellings and COPD occurrence

Nonantibiotic pharmacological treatment of severe chronic obstructive pulmonary disease exacerbations

Chronic bronchitis and chronic obstructive pulmonary disease

New treatments for COPD in the elderly

Heparin-binding epidermal growth factor (HB-EGF) drives EMT in patients with COPD: Implications for disease pathogenesis and novel therapies

Small airway fibrosis in COPD

EGF/EGFR axis contributes to the progression of cholangiocarcinoma through the induction of an epithelial-mesenchymal transition

Physcion inhibits the metastatic potential of human colorectal cancer SW620 cells in vitro by suppressing the transcription factor SOX2

Caveolin-1 promotes gastric cancer progression by up-regulating epithelial to mesenchymal transition by crosstalk of signalling mechanisms under hypoxic condition

Cigarette smoke induced lung barrier dysfunction, EMT, and tissue remodeling: A possible link between COPD and lung cancer

Histopathological approach to patterns of interstitial pneumonia in patient with connective tissue disorders

Mechanisms of fibrosis: Therapeutic translation for fibrotic disease

The contribution of small airway obstruction to the pathogenesis of chronic obstructive pulmonary disease

Sulforaphane attenuates pulmonary fibrosis by inhibiting the epithelial-mesenchymal transition

Clinical significance of epithelial mesenchymal transition (EMT) in chronic obstructive pulmonary disease (COPD): Potential target for prevention of airway fibrosis and lung cancer

Association of serum vitamin D (25OHD) level with acute exacerbation of chronic obstructive pulmonary disease

Correlation of severity of chronic obstructive pulmonary disease with serum vitamin-D level

Vitamin D3 supplementation in patients with chronic obstructive pulmonary disease (ViDiCO): A multicentre, double-blind, randomised controlled trial

Vitamin D supplementation to prevent COVID-19 in patients with COPD: A research perspective

Inhibition of vitamin d receptor translocation by cigarette smoking extracts

Vitamin D modulates the response of bronchial epithelial cells exposed to cigarette smoke extract

Vitamin D deficiency exacerbates bleomycin-induced pulmonary fibrosis partially through aggravating TGF-β/smad2/3-mediated epithelial-mesenchymal transition

Vitamin D prevents experimental lung fibrosis and predicts survival in patients with idiopathic pulmonary fibrosis

Club cell secreted protein cc16: Potential applications in prognosis and therapy for pulmonary diseases

Urinary club cell protein 16 (CC16): Utility of its assay during acute bronchiolitis

Recombinant club cell protein 16 (CC16) ameliorates cigarette smokeinduced lung inflammation in a murine disease model of COPD

Repression of CC16 by cigarette smoke (CS) exposure

Club cell protein 16 (CC16) augmentation: A potential disease-modifying approach for chronic obstructive pulmonary disease (COPD)

Protective role for club cell secretory protein-16 (CC16) in the development of COPD

Reduced serum concentration of CC16 is associated with severity of chronic obstructive pulmonary disease and contributes to the diagnosis and assessment of the disease

Multiple biomarkers predict disease severity, progression and mortality in COPD

Cigarette smoke affects dendritic cell populations, epithelial barrier function, and the immune response to viral infection with H1N1

Silymarin attenuates cigarette smoke extract-induced inflammation via simultaneous inhibition of autophagy and ERK/p38 MAPK pathway in human bronchial epithelial cells

Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method

The program of renal fibrogenesis is controlled by microRNAs regulating oxidative metabolism

Global burden of COPD

Epidemiology of COPD

Cigarette smoke-induced autophagy impairment accelerates lung aging, COPD-emphysema exacerbations and pathogenesis

Metals transfer from tobacco to cigarette smoke: Evidences in smokers' lung tissue

The impact of tobacco additives on cigarette smoke toxicity: A critical appraisal of tobacco industry studies

Current cigarette smoking, access, and purchases from retail outlets among students aged 13-15 years-global youth tobacco survey, 45 countries

Exosomal microRNA-21 derived from bronchial epithelial cells is involved in aberrant epithelium-fibroblast cross-talk in COPD induced by cigarette smoking

Aberrant epithelial differentiation by cigarette smoke dysregulates respiratory host defence

Biochemical and morphological effects of cigarette smoke condensate and its fractions on normal human bronchial epithelial cells in vitro

Chronic cigarette smoke-induced epigenomic changes precede sensitization of bronchial epithelial cells to single-step transformation by KRAS mutations

Epithelial to mesenchymal transition is increased in patients with COPD and induced by cigarette smoke

Efficacy of ascorbic acid (Vitamin C) and/N-Acetylcysteine (NAC) supplementation on nutritional and antioxidant status of male chronic obstructive pulmonary disease (COPD) patients

Vitamin K deficiency: The linking pin between COPD and cardiovascular diseases?

The association between vitamin D and COPD risk, severity, and exacerbation: An updated systematic review and meta-analysis

Importance of vitamin-A for lung function and development

Serum vitamin A and inflammatory markers in individuals with and without chronic obstructive pulmonary disease

Dietary vitamin C intake protects against COPD: The Korea national health and nutrition examination survey in 2012

The protective effects of vitamin E on lung injury caused by high temperature and PM2.5 in COPD rats

Vitamin E isoform gamma-tocotrienol protects against emphysema in cigarette smoke-induced COPD

Addition of vitamin B12 to exercise training improves cycle ergometer endurance in advanced COPD patients: A randomized and controlled study

Low vitamin K status is associated with increased elastin degradation in chronic obstructive pulmonary disease

Vitamin D to prevent exacerbations of COPD: systematic review and meta-analysis of individual participant data from randomised controlled trials

Vitamin D deficiency in COPD: Biomarker, treatable trait

Vitamin D: Classic and novel actions

Vitamin D in chronic kidney disease

Treatment of vitamin D deficiency in cystic fibrosis

Comparison of vitamin D2 and vitamin D3 supplementation in raising serum 25-hydroxyvitamin D status: A systematic review and meta-analysis

Effects of 1,25-Dihydroxyvitamin D3 on the prevention of chronic obstructive pulmonary disease (COPD) in rats exposed to air pollutant particles less than 2.5 micrometers in diameter

Clinical phenotypes, comorbidities, and exacerbations according to serum 25-OH vitamin D and plasma fibrinogen levels in chronic obstructive pulmonary disease

Vitamin D3-vitamin D receptor axis suppresses pulmona r y emphysema by maintaining alveolar macrophage homeostasis and function

The interaction effect of aerobic exercise and vitamin D supplementation on inflammatory factors, anti-inflammatory proteins, and lung function in male smokers: A randomized controlled trial

Transforming growth factor (TGF) β 1 and smad signalling pathways: A likely key to EMT-associated COPD pathogenesis

Hydrogen sulfide alleviates cigarette smoke-induced COPD through inhibition of the TGF-β1/smad pathway

Imprinting of the COPD airway epithelium for dedifferentiation and mesenchymal transition

Epithelial-mesenchymal transition in primary human bronchial epithelial cells is Smad-dependent and enhanced by fibronectin and TNF-alpha

Not applicable.

No funding was received.

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

HF guided the project, analyzed the data and wrote the manuscript. YM conceived the technical details and designed the experiments. HF and YM performed the experiments. YM and HF confirm the authenticity of all the raw data. All authors read and approved the final manuscript.

Not applicable.

Not applicable.

The authors declare that they have no competing interests.