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Abstract

Objectives: Erdosteine, an oral mucoactive anti-oxidant molecule, interferes with the pathological processes seen in respiratory disorders including thickened or increased mucus production, increased oxidative stress, and chronic inflammation; however, its efficacy as an asthma therapy remains to be fully clarified. Therefore, the aim of this study was to assess the effects of erdosteine on epithelial barrier function in asthma. Methods: To investigate the effects of erdosteine on cell barrier expression in a mouse model of asthma, BALB/c mice (n = 8 per group; total of 40 mice) were exposed to saline (Sham), ovalbumin (OVA), or OVA plus TiO₂ inhalation (200 μg/m³; OVA + TiO₂). The mice were then treated with erdosteine orally (OVA + TiO₂ + Erdos) or intraperitoneal dexamethasone (OVA + TiO₂ + Dex). Bronchoalveolar lavage and histology were performed. Enhanced pause was used as an indicator of pulmonary function, and samples were collected. The effect of erdosteine on cell barrier expression was assessed by immunoblotting and immunohistochemical analyses. Results: OVA + TiO₂ + erdosteine mice exhibited decreased inflammation, and mucous gland hyperplasia, and increased pulmonary function compared with OVA + TiO₂ mice. Levels of claudin (CLDN)-4 and nectin-4 protein were higher in lung tissue from OVA + TiO₂ mice than Sham and OVA mice, and were reduced by erdosteine treatment. In contrast, CLDN14 and CLDN18 protein levels were lower in lung tissue from OVA + TiO₂ mice than those from control mice, but were increased by treatment with erdosteine. Conclusion: Cell barriers are involved in airway inflammation in asthma patients, and erdosteine reduces airway inflammation by changing the cell barrier.

Keywords

Asthma cell barrier erdosteine particulate matter

Introduction

Erdosteine acts as a direct scavenger of reactive oxygen species (ROS), which is important for regulating redox status and affects signaling pathways involved in apoptosis, angiogenesis, cell growth and arrest, the inflammatory response, and redox-regulated gene expression. Erdosteine has an indirect antioxidant effect against toxic agents.¹

As a mucolytic agent, erdosteine is approved for the treatment of acute and chronic lung diseases and has antioxidant, anti-inflammatory, and antibacterial properties. It is a prodrug metabolized to the ring-opening compound metabolite M1, which has mucolytic properties1¹; thus, erdosteine is used as a mucolytic agent due to its ability to enhance mucus clearance and reduce bacterial adhesiveness.^2,3 Erdosteine is mainly used to treat chronic bronchitis and chronic obstructive pulmonary disease (COPD), and is commercially available in many countries worldwide.⁴

Tight junctions (TJs) act as a semipermeable barrier to the paracellular transport of ions, solutes, water, and cells, and divide the apical and basolateral domains of the plasma membrane.^5,6 Claudins (CLDNs) are structural molecules of TJs responsible for changes in the electrolyte and solute permeability of epithelial cell layers.^5–13

However, it is not obvious how the signaling mechanism for CLDN4 affects airway cell barriers, nor the impact of erdosteine on CLDN4 in patients with asthma. In this study, we investigated how CLDN4 contributes to asthma and whether CLDN4 expression is changed by erdosteine in a mouse model of asthma. In addition, the plasma CLDN4 levels of patients with asthma were examined.

Materials and methods

Experimental design

Mice were obtained from the Experimental Animal Centre of Orient Bio (Seongnam, Korea). BALB/c mice (n = 8 per group; total of 40 mice) were exposed to saline (Sham), ovalbumin (OVA) or OVA plus TiO₂ (inhalation of 200 μg/m³; OVA + TiO₂) and then treated with erdosteine (OVA + TiO₂ + Erdos) or dexamethasone (OVA + TiO₂ + Dex) (Figure 1). TiO₂ nanoparticles as one of air pollutants was used as one of exacerbation factor for asthma. The mice were raised in conventional surroundings, with a room temperature of 25–26°C, humidity of 50%, and 12-h day/night cycle. Water and food were freely available. The effects of erdosteine were estimated based on the levels of junctional proteins participating in signal transduction pathways. The use of animal models was approved by the Soonchunhyang University Institutional Animal Care and Use Committee (IRB No.: SCHBC-Animal-2020-06).

Figure 1.

(a) Experiment protocol of TiO₂ exposure treat model. (b) OVA + TiO₂ group had a significantly greater airway hyperresponsiveness compared to sham group, which were significantly reduced through dexamethasone (Dex) treatment, and erdosteine (Erdos) was the same. (c) The numbers of total inflammatory cells, macrophages, eosinophils, and neutrophils in BALF were significantly increased in OVA group compared to sham group (p < 0.05) and were significantly reduced by Erdos or Dex. (d) Evaluation of oxidative stress was assessed by ROMO 1 assay. *p < 0.05 compared to sham. #p < 0.05 compared to OVA + TiO₂.

Animals

On days 0 and 14, 6-week-old female BALB/c mice (20–25 g) were sensitized with intraperitoneal injections of 50 μg of grade V chicken egg OVA (Sigma-Aldrich, St Louis, MO, USA) emulsified in 10 mg of hydroxyl aluminum plus 100 μl of Dulbecco’s phosphate-buffered saline (D-PBS). From day 21–23, 150 μg of grade III OVA (Sigma-Aldrich) in 50 μl of D-PBS was used for intranasal challenge in all mice BALB/c mice were sensitized and challenged with OVA as previously described.⁶

Control mice were sensitized and challenged with saline. Mice in the TiO₂ nanoparticles group were administered 200 μg/m³ of nanoparticles (spherical shape with a diameter of approximately 70 nm; National Institute of Standards and Technology, Gaithersburg, MD, USA) by inhalation 1 h after OVA challenge, daily for 3 days. For TiO₂ nanoparticles exposure, whole-body inhalation was used. Mice were anesthetized with 2.5 mg/kg tiletamine and xylazine (Zoletil and lumpum; Bayer Korea Co., Seoul, Korea). Enhanced pause as an indicator of pulmonary function was assessed following a challenge with 0, 5, 20, or 100 mg/ml methacholine (Sigma-Aldrich) using a pulmonary instrument (OCP-3000; Gobiz-KOREA., Gyeonggi-do, Korea) 1 min after each dose, with 3-min intervals between doses. On the following day, bronchial lavage fluid (BALF) was collected to DPBS 1 ml syringe at 4 times, and it was push/pull, and was obtained and centrifuged, and the supernatant was stored at −20°C. The cell pellet was resuspended for cell counting, and cytospin slides were prepared for modified Diff-Quick staining. The differential blood cell count was obtained under a light microscope using standard morphological criteria; there were at least 500 cells on each slide. A portion of the lung was fixed in 4% phosphate-buffered paraformaldehyde, embedded in paraffin, sectioned (4-μm thickness), and stained [hematoxylin and eosin (H&E), Periodic acid-Schiff (PAS), and immunohistochemistry (IHC) staining] (Figures 2 and 3).

Figure 2.

(a) H&E-stained (x400) lung tissue contained more inflammatory cell infiltrations in OVA and OVA + TiO₂ group than sham group. (b) PAS staining (x400) airways contained increased goblet cells in the OVA group. Whereas H&E and PAS staining lung tissue had lower inflammatory and goblet cells in OVA + TiO₂ + Erdos and OVA + TiO₂ + Dex group. The scale bars present 200 μm. *p < 0.05 compared to sham. #p < 0.05 compared to OVA + TiO₂.

Figure 3.

(a) IHC staining (x400) demonstrated that the levels of Nectin4, CLDN4 were increased in the lung tissue of OVA and OVA + TiO₂ group compared with sham group. The expression of Nectin4 and CLDN4 decreased when recovered after treatment with Erdos or Dex. (b) CLDN14 and CLDN18 levels were similar or decreased in the lung tissue of OVA and OVA + TiO₂ group compared to sham group, and were increased further by Erdos or Dex. The scale bars present 200 μm. *p < 0.05 compared to sham. #p < 0.05 compared to OVA + TiO₂.

Erdosteine and dexamethasone treatments

Erdosteine (50 mg/kg) (Daewoong Pharmaceutical, Seoul, Korea) or dexamethasone (3 mg/kg) (Sigma-Aldrich) was administered by oral gavage or intraperitoneal 1 h before OVA challenge with OVA plus TiO₂ for three consecutive days (Figure 1(a)).

Western blotting

Protein extracts of mouse lung tissue were collected as described previously.⁶ Protein was separated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, Burlington, MA, USA). The membranes were blocked in Tris-buffered saline containing 5% skimmed milk and 0.1% Tween-20 for 1 h at room temperature before incubation with nectin-4 antibody (1:1000; Novus, Centennial, CO, USA), CLDN4 antibody (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA), CLDN14 antibody (1:1000, Santa Cruz, Dallas, USA), and CLDN18 antibody (1:1000; ThermoFisher Scientific, Waltham, MA, USA) overnight at 4°C. The membranes were then incubated with a horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Detection was performed on X-ray film using enhanced chemiluminescence plus a Western Blot Detection System (ATTO Corporation, Tokyo, Japan). The relative protein abundance normalized to β-actin was determined by quantitative densitometry (Sigma-Aldrich).

Enzyme-linked immunosorbent assays

Mouse reactive oxygen species modulator 1 (ROMO1) levels in plasma samples from patients with asthma were determined by enzyme-linked immunosorbent assay (ELISA; RD-ROMO1-Mu; Reddot Biotech, Kelowna, Canada). The plasma was incubated at room temperature on the ELISA plate, followed by the addition of a tracer antibody and a streptavidin-peroxidase-conjugated secondary antibody. TMB (3, 3′, 5, 5′-tetramethylbenzidine) solution and stop solution were added, and the optical absorbance at 450 nm was read on a microplate reader. The minimum detection limit was set to 28 pg/ml for calprotectin, according to the manufacturer’s recommendations.

Histology of the lung tissue

Sections of fixed embedded tissues were cut into 4-μm-thick slices using a rotary microtome (model 2165; Leica Microsystems Nussloch GmbH, Nussloch, Germany) and placed on glass slides. The tissues were processed, embedded in paraffin, sectioned, and stained with H&E and PAS. Lung sections stained with H&E were assigned a value for alveolar inflammation by calculating the means of the numerical scores. The numerical scores for each field of view were classified as follows: 0, normal; 1, alveolar walls normal, few macrophages in the alveoli; 2, mild thickening of the alveolar walls and increased alveolar macrophages and eosinophils; 3, marked thickening of the alveolar walls and alveolar multinucleated giant cells and eosinophils in 30–50% of the field; 4, same as 3 but in >50% of the field; 5, complete consolidation.

Periodic acid-Schiff assay

Lung tissue was cut into 4-μm sections and placed in periodic acid solution (Abcam, Cambridge, UK). After washing with distilled water, the specimens were immersed in Schiff’s fluid. The slides were washed with distilled water, placed in hematoxylin, and rinsed with tap water. Bluing reagent was then applied. Before observation, the slides were mounted with a cover slip using mounting media (Immunobioscience Corp, Mukilteo, WA, USA). The number of goblet cells per crypt-villus was determined in five randomly selected fields (three mice per group, and eight mice per treatment group) using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Immunohistochemical analysis

Mouse lung sections were deparaffinized and rehydrated in an ethanol series. The sections were treated with 1.4% H₂O₂ in methanol for 30 min to inhibit endogenous peroxidase. The specimens were then treated for non-specific binding with 1.5% horse serum and incubated with anti-nectin-4 (1:200; Novus), CLDN4 (1:100; Santa Cruz), CLDN14 (1:50; Santa Cruz), and CLDN18 antibody (1:100; ThermoFisher Scientific), as described in detail previously.⁶

Statistical analyses

The data were analyzed using SPSS statistical software (ver. 22.0; SPSS Inc., Chicago, IL, USA). All data are expressed as the mean ± standard deviation. Group differences were compared by the two-sample t test, Mann-Whitney test, and Pearson χ² test for normally distributed, skewed, and categorical data, respectively. One- and two-way ANOVA were also performed. A post-hoc analysis was used to determine differences among groups (post-hoc Tukey’s test). A p-value <0.05 was considered to indicate statistical significance.

Results

Effects of erdosteine on airway inflammation in asthma

The OVA group had significantly decrease in pulmonary function compared to the Sham group (Figure 1(b)). The OVA + TiO₂ group had significantly decrease in pulmonary function compared to the OVA group (Figure 1(b)), but this was increased significantly by treatment with erdosteine or dexamethasone. The numbers of total inflammatory cells, macrophages, eosinophils, and neutrophils in BALF were significantly increased in the OVA and OVA + TiO₂ groups compared to the Sham group (p < 0.05) (Figure 1(c)), but were reduced by erdosteine and dexamethasone. Mouse-ROMO one levels in BALF from the lungs was increased in the lungs of the OVA and OVA + TiO₂ groups compared with the Sham group, and was significantly reduced by treatment with erdosteine or dexamethasone compared with the OVA + TiO₂ group (Figure 1(d)).

Effects of erdosteine on nectin-4 and CLDN expression in lung tissue

H&E and PAS staining demonstrated that inflammatory cells and goblet cell hyperplasia were increased in the lungs of the OVA and OVA + TiO₂ groups compared with the Sham group, and decreased with dexamethasone and erdosteine treatments (Figure 2).

IHC staining demonstrated that the levels of nectin-4 and CLDN4 were increased in the lungs of the OVA and OVA + TiO₂ groups compared with the Sham group (Figure 3(a)). The expression of CLDN4 decreased with dexamethasone or erdosteine treatment, whereas reduced expression of nectin-4 was observed in the dexamethasone and erdosteine treatment groups compared with the OVA + TiO₂ group (Figure 3(a)). CLDN14 and CLDN18 protein levels were lower in lung tissue from the OVA + TiO₂ than Sham group, and were increased by treatment (Figure 3(b)). Additionally, Western blotting using protein lysates from the lungs of mice revealed increased CLDN4 and Nectin4 protein levels in the OVA and OVA + TiO₂ groups. CLDN4 and Nectin-4 expression decreased with dexamethasone and erdosteine treatment (Figure 4(a)). CLDN14 and CLDN18 protein levels were lower in the OVA + TiO₂ group compared with the Sham group and were higher with erdosteine treatment (Figure 4(b)).

Figure 4.

Effects of Erdos on Nectin4 and CLDNs by Western blotting. (a) Levels of Nectin4 and CLDN4 were increased in the lungs of OVA group and OVA + TiO₂ group compared with sham group. And were decreases when recovered after treatment with Erdos or Dex. (b) CLDN14 and CLDN18 were decreased in lung of OVA group and OVA + TiO₂ group compared with sham group. After were increased Erdos or Dex-treated group. Densitometry of three western blotting with normalization to β-actin. *p < 0.05 compared to sham. #p < 0.05 compared to OVA + TiO₂.

Discussion

The objective of this study was to establish the role of cell barriers, such as CLDNs and nectin-4, in airway epithelial barrier function in a mouse model of asthma on exposure to TiO₂ nanoparticles (an air pollutant). The primary findings of this study are that CLDN4 and nectin-4 levels increased, and CLDN14 and CLDN18 levels decreased, in asthmatic mice exposed to TiO₂ nanoparticulates. The ROMO1 level increased in asthmatic mice exposed to TiO₂ nanoparticles. Erdosteine treatment effectively reversed the response to TiO₂ nanoparticles exposure. These results suggest that TiO₂ nanoparticles increase cell barrier dysfunction and the amount of ROMO1, which leads to airway inflammation and increase in pulmonary function, and that the response can be reversed by erdosteine treatment.

Erdosteine is a thiol-based mucolytic agent characterized by the presence of sulfhydryl (–SH) groups in active sites¹⁴ that plays many roles in metabolism and homeostasis.^15,16 Thiol-based drugs have traditionally been considered as mucolytic drugs because they decrease the viscosity, and especially the elasticity, of bronchial secretions by reducing S–S bonds in proteins in mucous. In our previous study, we reported that TiO₂ contributes to airway epithelial barrier dysfunction and results in airway inflammation and responsiveness.⁶

In this study, erdosteine treatment increased pulmonary function and decreased inflammation in asthmatic mice exposed to TiO₂ nanoparticles, implying that erdosteine can be used for treating TiO₂ nanoparticle-related airway diseases.

ROS generation activates various signaling pathways, which leads to the expression of inflammatory proteins, mucus secretion, and bronchoconstriction.¹⁷ Erdosteine elicits anti-inflammatory effects that counteract this response.^14,18 Increased oxidative stress, as expected in current smokers with mild-to-moderate COPD, can affect β2-adrenoceptor function, as shown in a small study of 30 smokers suffering from non-reversible mild-to-moderate COPD who received erdosteine 300 mg, NAC 600 mg, or placebo twice daily for 10 days1¹⁹

In the current study, asthmatic mice exposed to TiO₂ nanoparticles exhibited an increase in ROMO1; erdosteine treatment reduced ROMO1 levels, in agreement with the findings of previous studies,^18,19 suggesting that NAC is an effective antioxidant against this type of pollution.

Fine particulate matter (<2.5 μm in diameter; PM2.5) exposure results in greater lung function decline and histopathological changes, as reflected by increased mucin (MUC) 5ac, MUC5b, collagen I, and collagen III levels, and the presence of profibrotic cytokine α-smooth muscle actin and transforming growth factor beta 1 in lung tissue.²⁰ PM2.5 exposure markedly increases oxidative stress, inflammation, proteases, the hypersecretion of mucus and collagen, and airflow obstruction in rats with preexisting COPD.^21,22

We further observed an incremental increase in mucus gland hyperplasia and decrement in NAC in asthmatic mice after TiO₂ nanoparticles exposure, possible resulting from excessive oxidants and inflammation.

Airway epithelium exposed to noxious gases and anthropogenic/natural particulates, plays an important role as a physical barrier and modulator of allergic and inflammatory responses.^22,23 Barrier dysfunction in the lung allows allergens to affect the epithelium and produce various cytokines that mediate airway inflammation.^24–26

CLDN4 reportedly functions as a paracellular sodium barrier and is one of the four major CLDNs expressed in lung alveolar epithelial cells.^27–30 The role of CLDN4 has been studied in lung injury,³¹ cancer,³² and fibrosis.^33,34

In our study, CLDN4 was associated with a change in the epithelium in an OVA-sensitized/OVA-challenged asthma mouse model. As a critical component of epithelial TJs, dysregulation of CLDN4 in the airway epithelium can lead to cytokine release; in turn, this contributes to inflammatory cell activation and airway obstruction. These effects can be reversed by steroid treatment. Thus, these findings suggest that the regulation of lung epithelial barrier proteins may constitute a therapeutic approach for asthma.¹¹

A growing body of evidence has focused on airway epithelial barrier dysfunction as a central feature of asthma. The potential contributions of increased epithelial permeability and loss of epithelial polarity to asthma include heightened exposure to air space antigens and altered cell signaling due to the loss of segregation of normally polarized receptors and ligands. CLDN18 appears to play a nonredundant role in epithelial permeability barrier, potentially through effects on TJ organization or a selective effect on paracellular permeability.¹³

This study had several limitations. First, it did not assess cell barrier functions, such as transepithelial electrical resistance, or include the data of asthmatic patients. Penh as a measure of pulmonary function was used. More accurate measure for pulmonary function like measuring airway resistance would be needed. Moreover, the absence of calculation and justification of the sample size (animals) is another limitation of this study which requires further assessment for implementation of this compound in experimental trial.

In this study, CLDN4 and nectin-4 levels were increased, and CLDN14 and CLDN18 levels were decreased, in asthmatic mice exposed to TiO₂ nanoparticles, suggesting that TiO₂ nanoparticles can damage cell barriers and cause airway inflammation. Further studies are needed to elucidate the role of cell barriers in asthmatic patients whose symptoms are exacerbated by air pollutants.

Conclusion

The results of our study suggest that cell barriers, such as CLDNs and nectin-4, play a role in airway inflammation related to air pollutants, and that erdosteine is an effective treatment modality. A better understanding of the regulation of cell barriers, including CLDN4 and CLDN18, in airway diseases may provide new insight leading to targeted therapeutics.

Footnotes

Acknowledgements

The authors would like to thank the participants of the Soonchunhyang University Bucheon Hospital birth cohort study (USBHS) for their commitment.

Author contributions

AS designed the study and wrote the manuscript. MH performed data collection, interpretation, and journal submission assistance for this manuscript. PH, SM, DY, SH, and AR contributed to the conduct of the study and data collection. All authors read and approved the final manuscript.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (NRF-2020R1A2C1006506) and Soonchunhyang University.

Ethics approval

Ethical approval for this study was obtained from the Institutional Animal Care and Use Committee in Soonchunhyang University Bucheon Hospital (approval No. SCHBC-Animal-2020-06). Animal welfare: All the animal experimentations were performed according to the Institutional Animal Ethical Committee of Soonchunhyang University Bucheon Hospital (approval No. SCHBC-Animal-2020-06) and all the procedures performed throughout the research have complied with ethical guidelines and were included in institutional ethical policies.

Data availability statement

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

ORCID iD

An-Soo Jang

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Erdosteine effects cell barriers causing decreased inflammation and increased pulmonary function in asthmatic mice exposed to nanoparticulate pollution

Abstract

Keywords

Introduction

Materials and methods

Experimental design

Animals

Erdosteine and dexamethasone treatments

Western blotting

Enzyme-linked immunosorbent assays

Histology of the lung tissue

Periodic acid-Schiff assay

Immunohistochemical analysis

Statistical analyses

Results

Effects of erdosteine on airway inflammation in asthma

Effects of erdosteine on nectin-4 and CLDN expression in lung tissue

Discussion

Conclusion

Footnotes

Acknowledgements

Author contributions

Declaration of conflicting interests

Funding

Ethics approval

Data availability statement

ORCID iD

References