Introduction
With the worsening of air pollution, diseases such as asthma, acute respiratory infections and lung cancer are becoming prevalent. Asthma is a heterogeneous disease characterized by chronic airway inflammation and progressive airway remodeling, with >350 million patients worldwide (1). Long-term exposure to air pollutants can increase the frequency of acute asthma attacks and worsen disease progression (2,3). Benzo[a] pyrene (BaP), the most toxic among the polycyclic aromatic hydrocarbons (PAHs), is a widespread atmospheric organic pollutant. Major sources include car emissions, tobacco smoke, cooking and industrial burning (4,5). Epidemiological studies reveal that BaP exposure is markedly associated with more frequent acute asthma attacks, reduced lung function and aggravated clinical symptoms (5,6). Choi et al (7) reported that children exhibited a high risk of asthma diagnosis after exposure to high BaP level. In animal studies, BaP, with allergen ovalbumin (OVA) and dust mite extract, can increase allergen-specific immunoglobulin E (IgE) level, systemic T-helper 1/2 (Th1/Th2) responses and airway inflammation (8). Although evidence continues to grow regarding the harmful effect of BaP on respiratory health, the precise molecular mechanism involved in lung toxicity remains unknown.
The airway epithelium serves as a first barrier against external allergens, pollutants and pathogens (9). Toxic substances in polluted air can stimulate and damage this barrier, contributing to the onset and progression of asthma. Disruption of the airway epithelial barrier not only increases susceptibility to respiratory infections and exacerbation but also exposes the underlying tissues to external agents, initiating inflammation, remodeling and heightened airway reactivity (10,11). Epithelial cells maintain barrier through tight junctions, which seal the gaps between cells and regulate the selective permeability (12,13). Occludin and zonula occludens-1 (ZO-1) are key proteins involved in tight junction regulation (14). The discovery of occludin as the first transmembrane protein associated with tight junctions identified it as a crucial marker of barrier integrity (15). It plays a crucial role in stabilizing tight junctions and maintaining epithelial cohesion. Scaffolding protein-like function is performed by ZO-1, linking tight junction transmembrane proteins to the cytoskeleton and organizing the junction complex. Due to its central role, ZO-1 is widely used as a major marker for evaluating intercellular barrier function (16,17).
Elevated reactive oxygen species (ROS) production from air pollutant exposure is a key mechanism underlying airway epithelial barrier damage. Oxidative stress induced by ROS harms cell membranes and tight junction proteins, thereby reducing the integrity and functionality of the airway epithelium. As BaP is cytotoxic, mutagenic and highly lipophilic, it promotes oxidative damage by promoting ROS production (18). Increased ROS enhance epithelial permeability, while their reduction restores the expression of occludin and ZO-1 (19,20). However, the exact mechanism by which BaP-induced ROS production affects occludin and ZO-1 remains unclear. Reports indicate that the NOD-like receptor protein 3 (NLRP3) inflammasome disrupts the epithelial barrier and contributes to allergic airway inflammation, with ROS being essential for its activation (21). This suggests that BaP-induced ROS may activate the NLRP3 inflammasome and regulate occludin and ZO-1 expression in asthma. The present study used asthma mouse and epithelial cell models exposed to BaP to assess ROS level, NLRP3 activation and tight junction protein, aiming to clarify how BaP exacerbates asthma-related barrier damage via signaling pathways.
Materials and methods
Animal experimental design
A total of 48 specific-pathogen-free grade female BALB/c mice (6-8 weeks old, 18-22 g) were obtained from Beijing Sibeifu Biotechnology Co., Ltd. All procedures were approved by the Animal Ethics Committee of the Affiliated Hospital of Nanjing University of Traditional Chinese Medicine (approval no. 2022DW-48-02). All mice were housed with a standard temperature (20-24°C) and humidity (50-60%), with a 12 h light/dark cycle. The standard feed used in the present study was purchased from Nanjing Yinuojia Feed Co., Ltd. and was composed of corn, wheat, imported fish meal, chicken meal, soybean meal, soybean oil, amino acids, vitamins and minerals. The production batch number was 24100108, which met Chinese national hygienic standard for formula feed for laboratory animals. After sterilization by irradiation, it met the hygienic requirements of SPF grade laboratory mice.
As described previously (22,23), an aggravated asthma model was established using OVA sensitization and challenge combined with BaP exposure. The 24 mice were divided into three groups (n=8/group): control, OVA and OVA + BaP groups. On days 0 and 14, mice in OVA and OVA + BaP groups received intraperitoneal injection of 100 μg OVA (MilliporeSigma) with potassium aluminum sulfate, followed by an intranasal administration of 50 μl OVA on days 14, 25, 26 and 27. Group OVA + BaP also received 50 μl of BaP (MilliporeSigma) solution (4 μg/per mouse) on days 14, 16, 18, 20, 22, 24 and 26. Control mice received normal saline using the same manner. Mice were fasted overnight before blood sample collection on the final day. The mice were euthanized by filling with carbon dioxide at the rate of 30% CO2 tank volume/min.
Based on the initial results, a second batch of 24 mice were divided into 3 groups with 8 mice in each group. The temperature, light/dark cycle and humidity were the same as those of the first batch. The second batch of experiments was conducted using the same OVA + BaP and the control protocols. An additional group, OVA + BaP + NAC, received N-acetylcysteine (NAC) intragastrically at 250 mg/kg once daily from days 14 and 27, while other groups received 0.2 ml of normal saline.
Histological analysis
Left lung tissues were rinsed with phosphate-buffered saline (PBS), fixed in 4% formalin for 24 h at room temperature then dehydrated with graded alcohol, immersed the tissue in molten paraffin, forming wax blocks after cooling. Sections (4 μm thick) were dewaxed with xylene, rehydrated through gradient ethanol and stained with hematoxylin and eosin (5 min at room temperature). Tissue morphology was observed under a light microscope at ×200 magnification.
ELISA
Mouse serum IgE levels were measured using an Anti-OVA IgE ELISA kit (Cayman Chemical Company; cat. no. 500840). Standards and samples were added to duplicate wells and incubated for 2 h at room temperature. Detection working solution of IgE was added and shaken at room temperature for 1 h. Streptavidin-conjugated horse-radish peroxidase was then applied and incubated at room temperature for 30 min. After adding tetramethylbenzidine substrate solution, absorbance at 450 nm was measured using a microplate reader. The IgE level was determined using a standard curve.
Cell culture
Human bronchial epithelial (16HBE) cells, obtained from the Chinese Academy of Sciences (Shanghai, China), were cultured in Rosewell Park Memorial Institute (RPMI) 1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Biological Industries, Inc.) and 1% penicillin-streptomycin solution (Gibco; Thermo Fisher Scientific, Inc.). After the cells adhered, BaP was added at concentrations of 2.5, 5, 10 and 20 μM. Following BaP induction, NAC (10 mM; Beyotime Institute of Biotechnology) and MCC950 (10 μM; Selleck Chemicals) were administered for 48 h before sample collection.
ROS assay
Frozen sections of lung tissues were prepared and circled using a histochemical pen. A spontaneous fluorescence quenching agent was applied for 5 min, followed by a 10 min water rinse. A ROS dye solution was added and incubated for 30 min at 37°C in the dark. The nuclei were counterstained with DAPI, followed by three washings with an anti-fluorescence quenching agent and capturing images after sealing the slides. In 16HBE cells, dichloro-dihydro-fluorescein diacetate (DCFH-DA; Nanjing Jiancheng Bioengineering Institute) was used to assay ROS levels and was diluted 1:1,000 in RPMI 1640 medium. Cells were incubated at 37°C in 5% carbon dioxide for 30 min. After removing the medium and washing twice with PBS, images were captured under a fluorescence microscope.
Immunofluorescence staining
Paraffin-embedded lung tissue sections were deparaffinized and subjected to antigen retrieval in an ethylenediaminetetraacetic acid buffer. Immunol staining fix solution was used to fix 16HBE cells for 60 min at room temperature and permeabilized with Triton X-100 (Beyotime Institute of Biotechnology) in PBS. They were all blocked with 10% goat serum (Beyotime Institute of Biotechnology) for 60 min at room temperature. The lung tissues sections were incubated with anti-ZO-1 (1:200 dilution; cat. no. 21773-1-AP; Wuhan Sanying Biotechnology), anti-occludin (1:200 dilution; cat. no. 66378-1-Ig; Wuhan Sanying Biotechnology), anti-NLRP3, (1:200; cat. no. ab270449; Abcam), anti-apoptosis-associated speck-like protein containing a CARD (ASC; 1:200; cat. no. 67824T; CST) overnight at 4°C. The 16HBE cells with a density of 70-80% were incubated with anti-ZO-1 (1:400 dilution; cat. no. 21773-1-AP; Wuhan Sanying Biotechnology), anti-occludin (1:400 dilution; cat. no. 66378-1-Ig; Wuhan Sanying Biotechnology) anti-NLRP3 (1:100; cat. no. WL02635; Wanleibio Co., Ltd.) and anti-ASC (1:200; cat. no. sc-514414; Santa Cruz Biotechnology, Inc.) overnight at 4°C. On the following day, all samples were washed with PBS buffer and incubated with secondary antibodies (Wuhan Sanying Biotechnology) for 60 min at room temperature and stained with DAPI (Beyotime Institute of Biotechnology) for 5 min at room temperature. After applying anti-fluorescence quenching agents, images were acquired using a fluorescence microscope (Nikon Corporation).
Western blot analysis
Total proteins were extracted by radioimmunoprecipitation analysis lysis buffer. The protein concentration was determined with a bicinchoninic acid assay kit (Beyotime Institute of Biotechnology). Protein samples were mixed with the loading buffer at a 1:5 ratio and heated at 99°C for 10 min to denature proteins. 20 μg of protein were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with 10% separating and 5% stacking gel. After, proteins were transferred onto polyvinylidene difluoride membranes and blocked with 5% nonfat milk at room temperature for 1 h. Membranes were then incubated with primary antibodies [anti-ZO-1 (1:5,000; cat. no. 21773-1-AP; Wuhan Sanying Biotechnology), anti-occludin (1:5,000; cat. no. 66378-1-Ig; Wuhan Sanying Biotechnology), anti-NLRP3 (1:1,000; cat. no. WL02635; Wanleibio Co., Ltd.), anti-ASC (1:1,000; cat. no. 67824T; CST), anti-interleukin (IL)-18 (1:1,000; cat. no. A1115; ABclonal Biotech Co., Ltd.), anti-caspase1 (1:1,000; cat. no. WL03450; Wanleibio Co., Ltd.)] and anti-β-actin (1:5,000; cat. no. T0022; Affinity Biosciences) overnight at 4°C. The membranes were incubated with HRP Goat anti rabbit IgG (1:8,000; cat. no. BA1054; BOSTER Biological Technology Co., Ltd) or HRP Goat anti mouse IgG (1:5,000; cat. no. BA1050; BOSTER Biological Technology Co., Ltd.) for 1 h at room temperature the next day., then visualized and analyzed using an enhanced chemiluminescent detection system and Image Lab software (BL520; Bio-Rad Laboratories, Inc.).
Statistical analysis
All data are expressed as mean ± standard deviation and GraphPad Prism software (version 10.0; Dotmatics) was used to make statistical graphs. Comparisons between two groups were performed using a unpaired t-test, while multiple group comparisons were performed using one-way analysis of variance followed by Tukey's post hoc test to analyze significant differences between groups. P<0.05 was considered to indicate a statistically significant difference.
Results
BaP can aggravate OVA-induced epithelial tight junction disruption in asthmatic mice
To assess the effect of BaP, an air pollutant, on airway epithelial tight junction, BALB/c mice were used. Asthma was induced with OVA, while an enhanced model was established using OVA + BaP. As demonstrated in Fig. 1A, control mice exhibited smooth bronchial walls and orderly epithelium. The OVA group revealed disordered epithelium and inflammatory cell infiltration. Co-exposure to BaP and OVA results in an increased in inflammatory cell infiltration, more pronounced tracheal wall fold, more severe epithelium disorganization and some epithelial cell loss. Serum IgE levels were markedly elevated in the OVA group compared with control and further increased in OVA + BaP (Fig. 1B). Tight junction proteins ZO-1 and occludin indicated intercellular barrier function. In OVA-treated lung tissue, occludin and ZO-1 levels were reduced compared with control and further decreased in the BaP + OVA group (Fig. 1C). Immunofluorescence results (Fig. 1D) followed this trend, indicating BaP could disrupt tight junctions by downregulating ZO-1 and occludin.
BaP can induce disruption of the tight junction of airway epithelial cells
16HBE cells were treated with various BaP concentrations revealing reduced expression of tight junction markers. Western blot analysis (Fig. 2A) revealed reduced ZO-1 and occludin levels with increasing BaP dose. Immunofluorescence staining (Fig. 2B) revealed decreased ZO-1 (green) and occludin (red) fluorescence at 10 and 20 μM Bap, indicating reduced protein expression, while 2.5 and 5 μM revealed no significant change. These results suggested that BaP impaired tight junction integrity in a dose-dependent manner.
BaP induces NLRP3/caspase-1 pathway activation in lung tissue of asthmatic mice
Allergen-induced airway inflammation involves NLRP3 inflammasome activation. Previous studies report elevated NLRP3 in Ova-induced asthma (24,25), but the effects of BaP co-exposure remain unclear. Upon activation, NLRP3 converts procaspase-1 into caspase-1, leading to IL-1β and IL-18 maturation. Western blotting and immunofluorescence were used to detect inflammasome components. As revealed in Fig. 3A, NLRP3 and ASC expressions were higher in the OVA than control and further elevated in the OVA + BaP group. Additionally, fluorescence double staining revealed increased NLRP3 and ASC aggregation in the OVA + BaP group, indicating inflammasome activation. Exposure to BaP markedly increased NLRP3 and ASC expression, caspase-1, IL-18 and IL-1β levels compared with the control and OVA groups (Fig. 3B and C).
BaP activates the NLRP3/caspase-1 pathway in airway epithelial cells
As demonstrated in Fig. 4A, treatment with 10 and 20 μM BaP increased the fluorescence intensity of NLRP3 and ASC in 16HBE cells compared with the control, indicating elevated expression. Western blot analysis (Fig. 4B and C) further confirmed that BaP exposure upregulated NLRP3, ASC and caspase-1, leading to increased IL-18 and IL-1β levels, suggesting NLRP3 inflammasome activation.
BaP-induced ROS generation mediates NLRP3/caspase-1 signaling pathways in asthmatic mice
A crucial role is played by ROS in activating the NLRP3 inflammasome in bronchial epithelial cells, suggesting BaP may act by altering ROS production. Consequently, the present study investigated whether BaP can induce ROS generation. Elevated ROS level was demonstrated in the lung tissue of OVA-induced asthmatic mice after BaP exposure in Fig. 5A. To test this further, mice were treated with OVA + BaP, with or without NAC. The expression of NLRP3, ASC, caspase-1, IL-18 and IL-1β was increased by BaP compared with the control, whereas NAC reduced their expression (Fig. 5B-D). These findings demonstrated that BaP-induced ROS activated the NLRP3/caspase-1 signaling pathway, which can be suppressed by ROS inhibition.
BaP-induced ROS generation mediates NLRP3/caspase-1 signaling pathways in airway epithelial cells
To explore this at a cellular level, ROS levels in 16HBE cells were assessed using the DCFH-DA probe, with fluorescence intensity reflecting ROS levels. Compared with the control group, BaP-treated cells revealed increased ROS, especially at 20 μM (Fig. 6A). In addition, the selection of a 20 μM concentration was based on its ability to markedly reduce the levels of tight junction proteins ZO-1 and occludin. This concentration falls within the commonly used range of 1-50 μM for studies examining similar mechanisms (26,27). NLRP3 and ASC expressions were elevated by BaP compared with the control group, which was reduced by NAC (Fig. 6B and C). Similarly, caspase-1, IL-18 and IL-1β expression followed the same trend (Fig. 6D), confirming that BaP-induced ROS could mediate NLRP3/caspase-1 pathway activation in airway epithelial cells.
BaP induces asthma epithelial tight junction disruption by increasing ROS generation
Exposure to air pollutants may elevate ROS production, a key mechanism underlying airway epithelial barrier dysfunction. The cytotoxic, mutagenic and highly lipophilic properties of BaP help in inducing oxidative damage through ROS. Elevated ROS levels increase epithelium permeability, while blocking ROS restore tight junction proteins such as occludin and ZO-1. As demonstrated in Fig. 7A and B, ROS inhibitor NAC reduced ZO-1 and occludin in BaP-aggravated asthmatic mice. Meanwhile, NAC restored these proteins in BaP-treated 16HBE cells (Fig. 7C and D), suggesting that reducing ROS could alleviate BaP-induced airway barrier damage. This demonstrated that BaP disrupts the epithelial barrier in asthma by increasing ROS production.
BaP disrupts airway epithelial tight junctions through ROS-driven-NLRP3/caspase-1 signaling pathway
ROS-mediated NLRP3 signaling is a key molecular mechanism in tight and adherens junction disruption and airway epithelial barrier damage (28). Experimental results revealed that BaP markedly increases NLRP3 expression in 16HBE cells. The NLRP3 inhibitor MCC950 reversed the BaP-induced NLRP3 expression (Fig. 8A and B) and prevented the reduction of ZO-1 and occludin (Fig. 8C and D). Moreover, the ROS inhibitor NAC suppressed the BaP-induced NLRP3 expression. These results indicated that BaP disrupts airway epithelial barrier tight junctions through the ROS-driven NLRP3/caspase-1 signaling pathway.
Discussion
With advancing industrialization and urbanization, air pollution has become a growing concern and asthma incidence continues to rise yearly (29). Widespread environmental pollutants include PAHs, primarily originate from incomplete combustion of organic matter, such as forest fires, incineration and engine emission (30). Increased PAH exposure can lead to reduced lung function, potentially due to the production of ROS during PAH metabolism, which causes oxidative damage and disrupts respiratory barrier function. As one of the most toxic PAHs, BaP enhances airway hyperreactivity and lung inflammation in asthmatic mouse models co-exposed with the allergen Dermatophagoides farina 1, leading to elevated levels of epithelial cytokines such as thymic stromal lymphopoietin, IL-33 and IL-25 (31). Alone, BaP can induce oxidative stress, airway epithelial damage, lung toxicity and inflammation (32). It stimulates the airway by interfering with the forkhead box protein A2/IL-6/IL-6R/signal transducer and activator of the transcription 3 signaling pathway, promoting airway epithelial-mesenchymal transition (22). According to Ni et al (33), BaP enhances the expression and activity of the cytochrome P450 1A1, leading to toxic effects on human airway epithelial cells through DNA damage and oxidative stress. The present study established an air pollution-exacerbated asthma mouse model with co-exposure to BaP and OVA. The results demonstrated that BaP and OVA co-exposure markedly increased inflammatory cell infiltration in lung tissue, caused more disorganized airway epithelial cell arrangement with partial detachment and led to more pronounced tracheal wall folding. These findings indicated that BaP exposure can exacerbate OVA-induced asthma and aggravate lung tissue damage.
Tight junctions are essential components of the airway epithelial barrier located at the apical region of epithelial cell connections. They are crucial for maintaining epithelial barrier integrity and polarity, forming the structural basis for the airway resistance to harmful substances. Disruption of airway epithelial integrity is a significant marker in asthma pathogenesis, with substantial evidence revealing airway epithelial cell shedding in bronchial biopsies from patients with varying asthma severity (34). Expression levels of Claudin-18, Claudin-4, Claudin-1 and ZO-1 are reduced in the bronchial epithelial cells of asthmatic patients (35). He et al (36) found that exposure to air pollutants such as particulate matter ≤2.5 μm (PM2.5) decreases transcriptional and protein levels of tight junction markers E-cadherin, ZO-1 and occludin, exacerbating OVA-induced epithelial barrier damage. Diesel engine exhaust, a major urban pollutant, has been demonstrated both in vitro and in vivo to decrease transepithelial resistance and reduce ZO-1 and E-cadherin expression in airway epithelium (37,38). The present study investigated tight junction protein expression in lung tissues and airway epithelial cells of asthma mice induced by BaP, both in vitro and in vivo. Exposure to BaP suppressed ZO-1 and occludin expression in both asthmatic mice and 16HBE cells, leading to airway epithelial barrier disruption. This is the first study, to the best of the authors' knowledge, exploring the effect of BaP on the airway epithelial barrier in asthma. The findings further highlighted the toxic role of BaP in asthma pathogenesis, suggesting tight junctions as a promising target for mitigating barrier damage and its lasting impact on asthma. BaP is recognized as a ligand of aromatic hydrocarbon receptor (AHR) and is capable of activating AHR signaling, thereby influencing various cellular processes, including oxidative stress and inflammatory responses. Although the present study did not include experiments related to AHR, our previous research demonstrated the role of AHR in exacerbating asthma in mice exposed to BaP (23).
Research has demonstrated that NLRP3 inflammasome is vital for asthma development and progression, with two gain-of-function single-nucleotide polymorphisms in the NLRP3 gene linked to allergic asthma (39). NLRP3 inflammasome is an intracellular multiprotein complex consisting of NLRP3 receptor protein, the adaptor protein ASC and procaspase-1. Upon sensing external pathogens or danger signals, NLRP3 inflammasome cleaves procaspase-1 to activate caspase-1. Caspase-1 converts the pro IL-1β and IL-18 into active forms, facilitating their secretion (40,41). This cascade interferes with intercellular junctions, including tight and adherens junctions, compromising epithelial barrier integrity and function-processes closely tied to asthma onset and progression. In the present study, BaP-induced asthma mice and 16HBE cells exhibited activation of the NLRP3 inflammasome and downregulation of ZO-1 and occludin expression. Treatment with NLRP3 inhibitor MCC950 reversed the downregulation of ZO-1 and occludin, indicating that inhibiting NLRP3 activation can alleviate BaP-induced tight junction disruption. The NLRP3-IL-1β/IL-18 axis may regulate tight junction integrity through indirect signaling pathways.
Research indicates that ROS overproduction in the airways of individuals with asthma leads to oxidative stress and persistent inflammation (42). A crucial role is played by ROS in the activation of NLRP3 inflammasome in bronchial epithelial cells. Thioredoxin-interacting protein (TXNIP), a ligand for NLRP3, is reactive to ROS. Normally, the redox enzyme thioredoxin (TRX) binds to TXNIP and suppresses its activity (43). Elevated ROS levels cause dissociation of TRX-TXNIP, permitting TXNIP to connect with the leucine-rich repeat domain of NLRP3, thereby triggering inflammasome activation (44,45). Cytotoxicity, mutagenicity and high lipophilicity exhibited by BaP help it induce oxidative damage by generating ROS. The permeability of airway epithelium is increased by ROS and inhibiting ROS can restore the occludin and ZO-1 expression (46). It is hypothesized that BaP activates NLRP3 inflammasome by increasing ROS production, resulting in the downregulation of tight junction proteins and subsequent epithelial barrier disruption. In the present study, BaP exposure led to reduced expression of ZO-1 and occludin in asthma mice and 16HBE cells. Treatment with ROS inhibitor NAC not only restored ZO-1 and occludin levels but also inhibited the NLRP3 inflammasome activation. This indicates that inhibiting ROS can reduce airway barrier damage mediated by NLRP3-caspase-1.
In conclusion, the present study indicated that BaP impaired the airway epithelial barrier by downregulating the ZO-1 and occludin expression, with ROS playing a crucial role in BaP-induced airway epithelial barrier disruption. Furthermore, BaP appears to activate NLRP3 inflammasome through ROS generation. Mechanistically, the present study revealed a novel pathway in which ROS regulate NLRP3 inflammasome activation, leading to BaP-induced airway epithelial barrier disruption in asthma (Fig. 9).
However, the present study was subject to several limitations. The direct damage induced by cytokines or the indirect effects mediated by downstream inflammatory signals are not clear. Future research will aim to elucidate the regulation of tight junctions using IL-1 β/IL-18 neutralizing antibodies and receptor knockout models. It is also planned to employ conditional medium experiments to distinguish between direct and paracrine effects.
The OVA model is characterized by its strong immunogenicity and the immune response it induces closely parallels the immunopathological mechanisms observed in human asthma, with the added benefits of stability, reproducibility and low cost. However, although the OVA asthma model is mainly an acute model, it cannot accurately simulate the conditions of human asthma patients exposed to low concentrations of BaP over a long time. Future research should focus on developing a chronic exposure model to assess the generalizability of these findings.