Hypoxia disrupts human bronchial epithelial barrier integrity via tight junction protein remodeling and enhanced paracellular leakage

Abstract

Background

Hypoxia can impair cell and organ function, and cause apoptosis and various diseases. At present, there are many studies on pulmonary hypoxia but few studies on bronchial injury. The study aimed to research the impact of hypoxia on the barrier function of human bronchial epithelial cells and the expression level of tight junction proteins.

Methods

Primary human bronchial epithelial cells were allocated into four groups: (1) control group, (2) intermittent hypoxia group, (3) sustained hypoxia group, and (4) cigarette smoke exposure group. Apoptosis in each group was assessed by flow cytometric analysis. The expression levels of ZO-1, occludin, and claudin-1 were evaluated via Western blotting. Furthermore, trans-epithelial electrical resistance (TEER) was measured using an epithelial voltohmmeter to assess barrier function.

Results

(1) Compared with the control group, the intermittent hypoxia group exhibited no significant differences in apoptosis rate, TEER, or the expression of tight junction proteins ZO-1, occludin, and claudin-1 (P > 0.05). In contrast, both the sustained hypoxia and cigarette smoke groups demonstrated significantly elevated apoptosis rates (P < 0.05). Claudin-1 expression was significantly reduced in the sustained hypoxia group (P < 0.05), while the increase in ZO-1 expression was not statistically significant (P > 0.05). In the cigarette smoke group, expression levels of ZO-1, occludin, and claudin-1 were all markedly decreased (P < 0.05). (2) Compared with the control group, TEER values were significantly reduced in both the sustained hypoxia and cigarette smoke groups (P < 0.05). (3) A significant difference in ZO-1 expression was observed between the sustained hypoxia and cigarette smoke groups (P < 0.05).

Conclusions

Hypoxia modulates the expression of tight junction proteins in human bronchial epithelial cells, disrupts intercellular junctional integrity, increases epithelial permeability, and ultimately impairs barrier function.

Background

Oxygen is essential for all physiological functions in humans. Hypoxia, defined as a reduction in tissue oxygen concentration below physiological levels, can disrupt cellular metabolism. In severe cases, it may lead to organ dysfunction, cellular injury, apoptosis, and the onset of various diseases. Previous studies have demonstrated that intermittent hypoxia, characterized by repetitive cycles of hypoxia and reoxygenation, plays a pivotal role in the pathophysiology of obstructive sleep apnoea and contributes to cardiovascular morbidity and mortality [1, 2]. Moreover, hypoxia has been implicated in increasing the permeability of the blood–brain barrier, upregulating aquaporin expression, inducing vasogenic oedema, impairing brain function, and accelerating neurodegenerative processes [3]. In the gastrointestinal tract, hypoxia disrupts tight junction proteins, induces oxidative stress, and activates transcription factors, resulting in intestinal injury [4].

As the primary site of gas exchange, the lungs are continuously exposed to the external environment. Under hypoxic conditions, the respiratory barrier may be compromised, leading to impaired ion transport and reduced alveolar fluid clearance, ultimately contributing to alveolar oedema [5]. The bronchus, a critical component of the respiratory system, has been less extensively studied in this context, with most research focusing on pulmonary tissue. Berggren-Nylund, R. suggested that hypoxia may be a contributing factor in the pathogenesis of chronic lung diseases, and that bronchial and alveolar epithelial cells exhibit differential responses to hypoxic and fibrotic stimuli. Notably, the bronchial epithelium appears more susceptible to changes in oxygen concentration and airway remodelling than the alveolar epithelium [6]. However, the precise mechanisms through which hypoxia affects bronchial epithelial cells remain inadequately understood.

Numerous studies have explored the mechanisms of hypoxia-induced injury in various organs and cell types. Several years ago, Bouvry, D. et al. reported that hypoxia impairs alveolar epithelial function by promoting tight junction protein degradation and increasing cellular permeability [7]. Similarly, Luo, H. found that hypoxia downregulates the expression of tight junction-associated proteins, leading to junctional disruption and loss of barrier integrity in human urothelial cells [8]. Li, Y. also observed that hypoxia modulates intercellular tight junctions in the vascular barrier by altering occludin expression [9]. In a study by Meta Volcic et al., SARS-CoV-2 infection was shown to severely disrupt intestinal epithelial barrier function, depleting tight junction proteins, such as claudin-1, occludin, and ZO-1 [10].

Tight junctions are vital intercellular structures in epithelial and endothelial cells with complex molecular architecture. These junctions serve as selective barriers, restricting the free movement of solutes and water through paracellular spaces, thereby preserving intracellular homeostasis. In addition, they regulate ion and molecule transport between cells and maintain plasma membrane polarity. Tight junctions are composed of cytoplasmic scaffolding proteins—predominantly members of the zonula occludens (ZO) family—and transmembrane proteins, including occludin and claudins [11–13]. Among these, claudins play a key role in regulating paracellular permeability. Multiple claudin subtypes, including claudin− 1, − 3, − 4, − 5, and − 7, are expressed in human bronchi and bronchioles under physiological conditions. Their expression levels correlate with changes in epithelial solute and electrolyte permeability. Certain pathogens can exploit claudins as entry points, thereby undermining host defence mechanisms [14].

Human bronchial epithelial cells (HBEpicC) constitute the first line of defence in respiratory mucosal immunity, protecting against inhaled exogenous agents. Their structural and functional integrity depends on tight and adherens junctions formed between adjacent cells. However, the mechanisms underlying hypoxia-induced injury to HBEpicC remain poorly understood, particularly regarding potential alterations in barrier function and tight junction regulation. In contrast, the mechanisms of airway injury resulting from toxic gases, viral infections, and cigarette smoke exposure have been more thoroughly investigated. For instance, Eunsook Park et al., using a three-dimensional in vitro airway model, demonstrated that diesel exhaust particles impair ciliary clearance and disrupt epithelial barrier function in the upper airway. Western blot analysis showed decreased expression of ZO-1, occludin, and E-cadherin [15]. Similarly, Lei Hu et al. found that influenza A virus infection reduced occludin and ZO-1 expression in mice, leading to impaired epithelial barrier function, decreased transepithelial electrical resistance, and increased permeability [16]. Tatsuta, M. reported that cigarette smoke exposure damages airway mucosa by disrupting tight junctions and downregulating ZO-1 and occludin expression [17]. Furthermore, Shaikh, SB et al. observed reductions in both the protein and gene expression levels of ZO-1 and claudin-1 in the airways of smokers with chronic obstructive pulmonary disease [10].

Based on these findings, we hypothesise that the mechanism underlying hypoxic injury in HBEpicC may involve disruption of tight junction structures and alterations in the expression of tight junction proteins. Therefore, this study established an in vitro hypoxia model using HBEpicC, with a cigarette smoke-exposed HBEpicC model serving as the positive control. Through comparative analysis, this study aimed to investigate changes in barrier function and the expression of tight junction proteins in HBEpicC under hypoxic conditions, thereby providing a reference for clinical diagnosis and treatment.

Methods

This study was initiated on 01 April 2023 and concluded on 12 July 2024. It was approved by the Ethics Committee of Fujian Provincial Children's Hospital (Approval No. 2022ETKLD0111). Written informed consent was obtained from all participants.

Preparation of cigarette smoke extract (CSE)

Gellner et al [18] Two cigarettes (Jinsheng brand) were lit, and the air pump was initiated to intermittently supply air at a constant speed. The cigarettes burned out in about 2 min. The smoke was bubbled through 10 mL of culture medium (as shown in Fig. 1). Removed the suction device and rubber stopper, covered the centrifuge tube. After 1 h incubation, the medium was adjusted to pH7.4 and then passed through a 0.22 μm pore filter (Millipore). This solution was designated as 100% CSE and further diluted for cell culture applications.

Fig. 1.

Schematic diagram of the preparation device of cigarette smoke extract. The cigarette smoke was bubbled through culture medium, and the air pump was initiated to intermittently supply air at a constant speed

Identification and grouping of human bronchial epithelial cells

Human bronchial epithelial cells were cultured using the iCell Primary Epithelial Cell Culture System (PriMed-iCell-001, ICELL). Culture plates with adherent cells were washed three times with buffer, each wash lasting 3 min. Subsequently, the cells were fixed with 4% paraformaldehyde for 15 min and permeabilised with 0.5% Triton X-100 for 5 min. After blocking with 5% bovine serum albumin (BSA, Solarbio) at 37 °C for 30 min, the cells were incubated overnight at 4 °C with a cytokeratin 8 antibody (1:200). The following day, after washing, the cells were incubated with a Cy3-conjugated secondary antibody (1:200), counterstained with DAPI, and mounted for observation under a fluorescence microscope to confirm their identity as human bronchial epithelial cells. Once identified, the cells were divided into four experimental groups based on the treatment conditions: (1) control group: cells cultured under normoxic conditions for 6 h. (2) Intermittent hypoxia group: cells exposed to alternating cycles of 1.5 h in 5% O₂ followed by 1.5 h in 20% O₂, repeated twice (total duration of 6 h). (3) Sustained hypoxia group: cells continuously cultured in 5% O₂ for 6 h. Oxygen concentration was regulated using a tri-gas incubator. (4) Cigarette smoke group: Cells treated with cigarette smoke extract (CSE) at a final concentration of 5% and incubated continuously for 6 h.

Flow cytometric analysis

Following treatment, cells from each group were harvested. The cells were washed twice with phosphate-buffered saline (PBS) via centrifugation to remove the supernatant and resuspended in precooled 1 × Binding Buffer. The cell suspensions were transferred to flow cytometry tubes, to which Annexin V-FITC and propidium iodide (PI) solutions (MULTI SCIENCES) were added. The mixtures were gently shaken and incubated in the dark at room temperature for 10 min. Precooled 1 × Binding Buffer was subsequently added, and the samples were analysed using the NovoCyte™ flow cytometer (NovoCyte 2060R, ACEA Biosciences, Hangzhou).

Western blot analysis

Post-treatment, total protein was extracted from each group using RIPA lysis buffer (Beijing Applygen Technologies Inc.). After high-speed centrifugation, the supernatant was collected, and protein concentration was quantified using the BCA protein assay kit (Elabscience). Protein loading volumes were calculated based on the concentration, with approximately 15 µg of protein loaded per lane. Samples were denatured and subjected to SDS–PAGE (XiLong Scientific Co., Ltd.) for 1.5 h, followed by protein transfer onto PVDF membranes (Millipore) using a constant current method.

The membranes were blocked with 5% skimmed milk (Beijing Applygen Technologies Inc.) and incubated overnight at 4 °C with the following primary antibodies: mouse anti-β-actin (TransGen Biotech, 1:2000), rabbit anti-ZO-1 (Proteintech, 1:2000), rabbit anti-occludin (Proteintech, 1:5000), and rabbit anti-claudin-1 (affinity, 1:1000). On the following day, membranes were incubated at room temperature for 2 h with HRP-conjugated secondary antibodies: goat anti-mouse IgG (H + L) and goat anti-rabbit IgG (H + L) (Servicebio, 1:2000). The membranes were developed using enhanced chemiluminescence and imaged using an ultra-sensitive fully automated chemiluminescence imaging system (Tanon-5200, Shanghai Tanon Science & Technology Co., Ltd.).

Trans-epithelial electrical resistance (TEER) measurement

Cells were seeded in 24-well plates at approximately 4 × 10⁵ cells per well. The cell suspension (containing 5 × 10⁴ cells) was diluted according to experimental requirements. A volume of 0.3 mL of complete medium was added to the upper chamber and 0.5 mL to the lower chamber. The cells were incubated to allow the formation of a confluent monolayer. After 24 or 6 h of treatment, depending on the experimental group, TEER was measured using a RE1600 epithelial volt-ohm meter (Beijing Kinggong Hongtai Technology Co., Ltd.), and the data were analysed accordingly.

Statistical analysis

Data are presented as mean ± standard error of the mean (SEM). Comparisons between two groups were conducted using the Mann–Whitney U test. For multiple group comparisons, one-way or two-way analysis of variance (ANOVA) followed by Tamhane’s T2 post hoc test was employed. Statistical analyses were performed using Prism 9.5 software (GraphPad Software, San Diego, CA). A p value < 0.05 was considered statistically significant.

Results

Identification of HBEpicC

Immunofluorescence was employed to detect cytokeratin 8 expression in HBEpicC. As shown in Fig. 2, cell nuclei stained with DAPI exhibited blue fluorescence under UV excitation, while positive cytokeratin 8 expression was indicated by red fluorescence. The presence of red fluorescence confirmed the identity of the cultured cells as HBEpicC.

Fig. 2.

Immunofluorescence image of human bronchial epithelial cells (HBEpiC). Nuclei are stained blue, and positive DAPI staining is indicated in red. Scale bar: 100 μm. All results represent at least two independent experiments

Apoptosis induced by sustained hypoxia and cigarette smoke exposure

As illustrated in Figs. 3 and 4, there was no statistically significant difference in apoptosis rates between the intermittent hypoxia group and the control group (P > 0.05). The apoptosis rates were significantly elevated in both the sustained hypoxia and cigarette smoke groups compared to the control group, with the differences reaching statistical significance (P < 0.05). However, the apoptosis rates were similar between the sustained hypoxia group and the cigarette smoke group (P > 0.05).

Fig. 3.

Flow cytometry analysis of cell apoptosis. Apoptotic cells are identified by the binding of fluorescently labeled Annexin V-FITC, forming a green halo on the cell membrane. Cells that have lost membrane integrity exhibit a red-stained nucleus along with a green membrane halo. A Flow cytometry results of the control group; B intermittent hypoxia group; C prolonged hypoxia group; D cigarette smoke exposure group

Fig. 4.

Comparison of results among the four experimental groups. All results are representative of at least three independent experiments."***"indicates a significant difference compared with the control group (P < 0.001); and"****"indicates a highly significant difference compared with the control group (P < 0.0001)

Alterations in tight junction protein expression in HBEpiC under sustained hypoxia and cigarette smoke exposure

The results of Western blot analysis are presented in Fig. 5. Compared with the control group, the intermittent hypoxia group exhibited a slight reduction in the expression levels of occludin and claudin-1, although the differences were not statistically significant. In the sustained hypoxia group, both occludin and claudin-1 expression levels were reduced, with claudin-1 showing a statistically significant decrease (P < 0.05). In addition, ZO-1 expression levels increased in both the intermittent and sustained hypoxia groups; however, these changes were not statistically significant. In contrast, the cigarette smoke exposure group demonstrated a significant reduction in the expression of ZO-1, occludin, and claudin-1 (P < 0.05).

Fig. 5.

Western blot analysis of ZO-1, occludin, and claudin-1 expression levels in HBEpiC."*"indicates P < 0.05,"**"indicates P < 0.01 and"***"indicates P < 0.001 compared with the control group

When compared with the cigarette smoke group, the intermittent hypoxia group showed no statistically significant differences in apoptosis rate, expression levels of ZO-1, occludin, and claudin-1, or changes in transepithelial resistance (P > 0.05). In contrast, the sustained hypoxia group exhibited a significant difference in ZO-1 expression (P < 0.05), while the differences in apoptosis rate, occludin and claudin-1 expression levels, and transepithelial resistance were not statistically significant (P > 0.05).

Sustained hypoxia and cigarette smoke exposure reduce transepithelial resistance and compromise epithelial barrier integrity

As illustrated in Fig. 6, resistance measurements revealed no significant change in the intermittent hypoxia group compared with the control group. However, the sustained hypoxia and cigarette smoke groups exhibited a significant reduction in transepithelial resistance (P < 0.05), indicating impaired integrity of the epithelial cell barrier. However, there was no significant change in the resistance between the intermittent hypoxia group and the control group. Compared to the cigarette smoke group, the intermittent hypoxia group showed no significant change in the sustained hypoxia group (P > 0.05).

Fig. 6.

Measurement of transepithelial electrical resistance in each experimental group."*"indicates P < 0.05 and"**"indicates P < 0.01 compared with the control group

Discussion

Bronchial epithelial cells play a pivotal role in maintaining pulmonary airway homeostasis. Disruption of bronchial barrier integrity permits the translocation of inhaled particulates and harmful agents into the lung parenchyma, thereby increasing the risk of airway diseases. In this study, it was hypothesised that hypoxia impairs the tight junction structure between human bronchial epithelial cells (HBEpiC) and alters the expression of tight junction proteins. To investigate this, in vitro models of intermittent hypoxia, prolonged hypoxia, and cigarette smoke exposure were established, with the latter serving as a positive control. The deleterious effects of hypoxia on the bronchial epithelium were assessed and compared.

Previous studies have shown that hypoxic conditions can induce pulmonary disease [7], and structural components of the airway, such as the bronchial epithelium, may also be adversely affected. This was corroborated by the present findings, which demonstrated that sustained hypoxic exposure for 6 h increased apoptosis, reduced transepithelial resistance, and downregulated the expression of the tight junction protein claudin-1 in HBEpiC. These results are consistent with those of Hiranuma et al., who reported that RNA viruses compromise epithelial barrier integrity by downregulating the expression of claudin family members [19]. The data suggest that hypoxia disrupts tight junction structures by decreasing claudin-1 expression, thereby increasing intercellular permeability, impairing barrier function, and promoting apoptosis.

In contrast, intermittent hypoxia did not result in a significant increase in apoptosis, nor did it cause a notable decline in transepithelial resistance or substantial changes in permeability. Although minor reductions in tight junction protein expression were observed, these were not statistically significant. Consequently, intermittent hypoxia did not appear to cause significant damage to HBEpiC under the experimental conditions. Furthermore, no significant differences in ZO-1 expression were observed among the intermittent hypoxia, prolonged hypoxia, and control groups. This observation aligns with the findings of Luo et al., who reported that transient hypoxia (≤ 2 h) had no significant effect on the expression of tight junction proteins, such as ZO-1, claudin-1, and occludin, whereas prolonged hypoxia (up to 24 h) led to time-dependent downregulation of these proteins [8]. The absence of marked injury in the intermittent hypoxia group may be attributable to the specific parameters of the hypoxia-reoxygenation cycles employed. Due to experimental limitations, the severity and duration of hypoxia reoxygenation may have been insufficient to induce irreversible injury to tight junction proteins. It is also plausible that HBEpiC recovered partially under normoxic conditions between hypoxic episodes, thereby mitigating the extent of damage. The unaltered expression of ZO-1 may similarly be attributed to the short duration of hypoxic exposure. Future studies should incorporate more refined hypoxia paradigms to enable a comprehensive evaluation of these effects.

Cigarette smoke is a well-established risk factor for respiratory mucosal injury and airway disease. It is known to induce conditions, such as rhinitis, pharyngitis, and pneumonia, increase susceptibility to viral infections, and contribute to the development of malignancies, such as oropharyngeal, laryngeal, and lung cancers [20–23]. The deleterious effects of cigarette smoke on the airways predominantly stem from direct cytotoxicity to epithelial cells and disruption of epithelial barrier integrity [17, 21, 23]. The effects of cigarette smoke exposure were still accessible to the general public and were highly harmful. Both cigarette smoke and hypoxia represent common etiological factors inducing airway injury, which frequently coexist in clinical comorbidities, such as obstructive sleep apnea and so on. Therefore, we chose to use the cigarette smoke exposure HBEpiC model as a positive control in this study. In this study, a cigarette smoke-exposed HBEpiC model was employed as a positive control. Continuous exposure to 5% CSE for 6 h resulted in significant cellular injury, characterised by increased apoptosis, reduced transepithelial resistance, and enhanced cell permeability. Expression levels of tight junction proteins, including ZO-1, occludin, and claudin-1, were markedly diminished. These results are consistent with the findings of Miyoko Tatsuta, who reported that cigarette smoke exposure decreased TEER and suppressed the gene expression of claudin-1, claudin-3, and occludin, with a significant reduction in ZO-1 expression observed after 8 h of exposure [17]. Similar conclusions were drawn by Shaikh et al., who demonstrated that cigarette smoke disrupts tight junction protein expression and compromises epithelial barrier integrity [23].

In this study, a comparison was made between the prolonged hypoxia group and the cigarette smoke exposure group. Although the prolonged hypoxia group exhibited slightly higher apoptosis rates and greater reductions in epithelial resistance relative to the cigarette smoke exposure group, the decreases in occludin and claudin-1 expression were marginally less pronounced in the hypoxia group. However, these differences were minimal. ZO-1, a critical component of the tight junction complex, plays an essential role in regulating paracellular transport, maintaining epithelial cell polarity, facilitating tumor cell metastasis, and modulating gene transcription. Clinically, ZO-1 is frequently utilized as a key biomarker for evaluating intercellular barrier integrity [24]. Interestingly, in the prolonged hypoxia group, ZO-1 expression was slightly elevated compared with the control group, although the difference was not statistically significant. In contrast, a significant decrease in ZO-1 expression was observed in the cigarette smoke exposure group. This divergence may be related to the fact that the airway epithelium is a highly sensitive membrane structure, and in addition to the structural changes that affect the epithelial barrier, the cytotoxic effects of cigarette smoke are also important causative factors. Therefore, the impairment of protein expression is more significant [25, 26].

In conclusion, the present study demonstrates that hypoxic conditions alter the expression of tight junction proteins in HBEpiC, thereby compromising tight junction integrity and increasing intercellular permeability, ultimately impairing epithelial barrier function. Future research will aim to elucidate the extent of damage and the dynamic changes in tight junction protein expression under varying intensities and durations of intermittent hypoxia. In addition, using immunohistochemical staining and other biochemical assays to investigate the expression and localization of the apoptosis-related genes induced by hypoxia and animal models of hypoxia will be established to facilitate further investigation into the underlying mechanisms.

Author contributions

Jianmin Huang, Shan He, and Qiuyun Zhang conducted the experiments, collected data, and drafted the manuscript. Jianmin Huang, Shan He, Yongjing You, and Yunliang Liu performed the statistical analyses and contributed to the study design. All authors read and approved the final manuscript.

Funding

This study was supported by the Fujian Provincial Natural Science Foundation of China (grant number: 2022J011062) and the Startup Fund for Scientific Research, Fujian Medical University (grant number: 2022QH1214).

Data availability

All data generated or analyzed during this study are included in this article and its supplementary information files.

Declarations

Ethics approval and consent to participate

This study was conducted in accordance with the Declaration of Helsinki (2000) of the World Medical Association. Ethical approval was obtained from the Ethics Committee of Fujian Provincial Children's Hospital (approval number: 2022ETKLD0111), and written informed consent was obtained from all participants.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.