Ozone Induces Oxidative Stress and Inflammation in Nasal Mucosa of Rats

Abstract

Background: The development of the global economy has led to changes in air pollution patterns. The haze phenomenon characterized by high concentrations of particulate matter 2.5 (PM2.5) has changed to complex pollution, and photochemical pollution characterized by ozone (O₃) has become increasingly prominent. Ozone pollution and its impact on human health has become an important topic that needs to be studied urgently. Objective: To investigate the effects of ozone on oxidative stress and inflammation in the nasal mucosa of a rat model. Methods: Thirty-two healthy female Sprague–Dawley rats, eight in each group, were divided into four groups using the randomized numeric table method: normal control group (NC group), normal rats with a low level of ozone inhalation exposure (NEL group, 0.5 ppm), medium ozone inhalation exposure (NEM group, 1 ppm), and high ozone inhalation exposure (NEH group, 2 ppm). The ozone inhalation exposure groups were placed in the ozone inhalation exposure system and exposed to different concentrations of ozone for 2 h each day for 6 weeks. Nasal secretion was measured, and nasal lavage and nasal mucosa were collected. Malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) activities were measured by colorimetric assay, and the nasal mucosa was analyzed by Western blot. Western blot (WB) was used to detect the expression of NF-κB p65 nuclear protein in nasal mucosa. The mRNA expression of NF-κB target genes IL-6 and IL-8 and tumor necrosis factor-α (TNF-α) was detected by real-time quantitative PCR (qRT-PCR), and the protein content of pro-inflammatory factors IL-6, IL-8, and TNF-α was detected by ELISA in serum and nasal lavage fluid. The nasal mucosa of rats was stained with hematoxylin-eosin (HE) to observe the pathological changes in the nasal mucosa. The data were analyzed by SPSS 20.0 software. Results: The amount of nasal secretion increased significantly in all groups after ozone exposure compared with that in the NC group. The MDA content of the nasal mucosa was significantly increased in the ozone-exposed group compared with the NC group, and the activity levels of SOD and GSH-Px in the nasal mucosa were lower in the ozone-exposed group than in the NC group. The mRNA expression of IL-6, IL-8, and TNF-α in the nasal mucosa of the ozone-exposed group was elevated, and the protein content of TNF-α, IL-6, and IL-8 in the nasal lavage fluid was elevated, and the content increased with the increase in ozone concentration. The expression of NF-κB p65 intracellular protein in the nasal mucosa of each ozone-exposed group was higher than that of the normal group, and the content increased with the increase in ozone concentration. Conclusions: Ozone inhalation exposure promotes oxidative stress and the release of inflammatory factors TNF-α, IL-6, and IL-8, leading to pathological damage of the nasal mucosa, the degree of which increases with increasing concentration. This pathological process may be related to the activation of the transcription factor NF-κB by ozone in the nasal mucosa of rats, which increases the expression of its target genes.

1. Introduction

In recent years, air quality has improved with the management of PM 2.5, yet the symptoms of allergic respiratory illness have not decreased significantly. The development of the global economy has led to an increasing number of automobile and industrial emissions, which leads to a change in air pollution patterns [1]. The haze phenomenon characterized by high concentrations of fine particulate matter (particulate matter 2.5, PM2.5) has shifted to complex pollution, and photochemical pollution characterized by ozone (O₃) has become increasingly prominent [2]. It is clear that ozone pollution and its impact on human health has become an important topic that needs to be studied urgently.

About 40–60% of ozone inhaled through the nose is absorbed in the nasal cavity, with the rest going downward into the trachea and alveoli of the lower respiratory tract [3]. As early as 1957, Stokinger et al. demonstrated in animal studies that ozone inhalation exposure causes lung damage [4]. It was found that long-term repeated low-dose ozone inhalation exposure (1~1.5 ppm for 62 weeks) in rats and other animals caused bronchitis, bronchiolitis, and other respiratory illnesses. Bates et al. found that humans can develop olfactory perception in 0.02 ppm ozone, 0.05 ppm, and higher concentrations cause dryness and irritation in the upper airway mucous membranes; 1 ppm ozone inhalation for 30 min can lead to headache, 0.75 ppm ozone inhalation for 2 h can lead to lung function decline, and mild exercise will aggravate these reactions [5,6].

Ozone is a highly reactive and powerful oxidant, and inhalation of ozone produces oxidation products in the lower respiratory tract, causing oxidative stress and inflammatory responses. Oxidative stress and inflammation are also key components of the effects of air pollutants such as PM2.5 on the respiratory tract [7,8]. However, the mechanism of ozone on the respiratory system remains unclear, and there is not much evidence of ozone-induced oxidative stress and inflammatory responses in the nasal mucosa of rats. Therefore, this study was designed to explore the effects of ozone inhalation exposure on the nasal mucosa and related inflammatory factors in rats through the observation of the effects of ozone inhalation exposure on inflammatory factors in rats by exposing healthy rats to ozone inhalation at different concentrations. These findings may provide some clues to understanding the mechanisms underlying ozone-induced mammalian nasal toxicity.

2. Materials and Methods

2.1. Model Preparation and Apparatus

2.1.1. Animal Grouping and Model Preparation

Female Sprague–Dawley (SD) rats of 6–7 weeks of age, with a body mass of about 180–200 g, were selected and provided by Shanghai Sipul-Bicai Laboratory Animal Co., Ltd. (Shanghai, China) and kept in the Department of Laboratory Animal Science, Fudan University, which has been approved by the Ethical Review Committee of the Department of Laboratory Animal Science, Fudan University (approval number 202011018Z). Thirty-two rats were randomly divided into four groups of eight rats each: normal rats in the low ozone inhalation exposure group (Group NEL, 0.5 ppm), medium ozone inhalation exposure group (Group NEM, 1 ppm), high ozone inhalation exposure group (Group NEH, 2 ppm), and normal control group (Group NC) using the randomized numerical table (RNT) method.

2.1.2. Ozone Inhalation Exposure Device

Animal ozone inhalation exposure was performed by an independently designed ozone inhalation exposure system, the animal exposure chamber produced by Shanghai Zhongjing Environmental Protection Science and Technology Co (ZJC-T01; ZJ Environmental Protection, Shanghai, China), which consisted of four parts. The ozone real-time detection and feedback system can continuously detect the ozone concentration in the exposure chamber in real time and automatically adjust the ozone generator’s work. They cooperate with each other to ensure that the ozone concentration in the exposure chamber is relatively constant and in the range of concentration required by the experiment. An aerodynamic hybrid system installed HEPA filters particles in the air, ensuring that the air entering the exposure chamber is clean, the air in and out of the chamber continues to circulate, and that the ozone in the chamber space has a relatively uniform distribution.

2.2. Methods and Observations

2.2.1. Ozone Inhalation Exposure

Inhalation exposure was conducted at a fixed time period every day when the feeding box containing rats was put into the exposure chamber. The ozone concentration was set at 0, 0.5, 1.0, and 2.0 ppm for 2 h/d for 6 weeks. The exposure chamber was placed at a fixed time period every day, and the ozone concentration range in the chamber during ozone exposure did not fluctuate by more than 10%. Groups were exposed to different concentrations of ozone for 2 h for 42 d. The ozone inhalation exposure was completed in 42 d.

2.2.2. Nasal Symptoms of Rats

The effects of ozone exposure on the nasal symptoms of rats were assessed within 15 min after the end of the last experiment. The filter paper method was used to determine the amount of nasal secretion. A thin strip of filter paper of appropriate width was trimmed and weighed. Then, one end was placed into the nasal cavity of one side of the rats for 5 min and then taken out and weighed. The difference in the mass of the two strips was calculated, which was used as an indicator of the amount of nasal secretion [7].

2.2.3. Collection of Experimental Specimens

After 24 h from the last inhalation exposure, 10% chloral hydrate was injected intraperitoneally to anesthetize the rats. The abdominal cavity was cut open, and the abdominal aorta was exposed. Blood was collected from the abdominal aorta, whole blood was centrifuged at 4 °C for 10 min at 1000× g, and the supernatant, i.e., serum, was stored at −80 °C to be detected by ELISA. The head of the rat was excised, the nasal septum and the lateral wall of the nasal cavity were exposed, and the nasal mucosa tissue of the nasal septum and the lateral wall of the nasal cavity was rapidly removed, and a part of the tissue was stored at −80 °C. The other part was fixed with 4% paraformaldehyde for pathologic examination.

2.2.4. Measures of Biomarkers of Oxidative Stress in Nasal Mucosa

Malondialdehyde (MDA) levels and the activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the nasal mucosa tissue homogenates were measured using their corresponding substrates (both from Nanjing Jiancheng Bioengineering Institute, Nanjing, China) through a spectrophotometry-based method.

2.2.5. Detection of NF-κB Protein Expression in Nasal Mucosa by Western Blotting

The cytoplasmic and cytosolic proteins of nasal mucosa tissues of each group were extracted, and the concentration of cytoplasmic and cytosolic proteins extracted by the BAC method was detected. Separation and concentration gels were prepared according to the SDS-PAGE preparation kit, and the samples were electrophoresed and transferred to the PVDF membrane by the “sandwich” semi-dry membrane transfer method. The membrane was submerged in the blocking solution and incubated at room temperature on a shaker for 1 h. For antibody incubation, the primary antibody was diluted with the blocking solution (dilution ratio: NF-κB p65 1:4000, Histone H3 1:5000) and incubated at room temperature for 15 min and overnight at 4 °C on a shaker. For the secondary antibody, the secondary antibody was diluted with 5% skimmed milk powder-TBST, HRP labeled secondary antibody that can recognize the corresponding primary antibody was added, and Goat-Anti-Rabbit IgG HRP, 1:10,000 was used for dilution. The membrane was washed and incubated at room temperature for 40 min with gentle shaking, and the color was developed by mixing equal volumes of ECL Luminescent Solution A and Solution B. The color was then detected by Quantity One software. Quantity One software (v4.6.6, Bio-Rad, Hercules, CA, USA) was used to analyze the grayscale values and compare the protein expression of each group semi-quantitatively.

2.2.6. Real-Time Quantitative PCR Was Used to Detect the mRNA Levels of IL-6, IL-8, and TNF-α in Nasal Mucosa

The total RNA of nasal mucosa tissues was extracted by the Trizol method, and cDNA was synthesized by reverse transcription of mRNA according to the kit, then amplified by SYBR Green PCR Master Mix Reagent (Qiagen, Stockach, Germany). The relative abundance of mRNA was normalized to β-actin. The PCR primers were designed by Primer Express 3.0.1 software and synthesized by Shanghai Sangong Biological Engineering Co (Shanghai, China). The primer names and sequences are presented in

Table 1. Sequences of primers used in qRT-PCR.

Genes	Primers (5′ to 3′)
IL-6	Forward: 5′-CCGGAGAGGAGACTTCACAG-3′ Reverse: 5′-ACAGTGCATCATCGCTGTTC-3′
IL-8	Forward: 5′-ACACTGCGCCAACACAGAAATTA-3′ Reverse: 5′-TTTGCTTGAAGTTTCACTGGCATC-3′
TNF-α	Forward: 5′-ACTCCCAGAAAAGCAAGCAA-3′ Reverse: 5′-CGAGCAGGAATGAGAAGAGG-3′
β-actin	Forward: 5′-AGCCATGTACGTAGCCATCC-3′ Reverse: 5′-CTCTCAGCTGTGGTGGTGAA-3′

. Relative quantitation values were expressed as the fold change over controls.

2.2.7. Pathological Observation of Nasal Mucosa

Thirty-two nasal mucosa of rats were fixed with 4% paraformaldehyde for 24 h, embedded in paraffin, and sectioned (slice thickness of 4 μm). The hematoxylin-eosinstaining (HE) method was used to observe the pathological changes in the nasal mucosa in various groups under a light microscope (BX-43, Olympus, Tokyo, Japan).

2.3. Statistical Processing

All data were analyzed using the statistical software SPSS 20.0 and expressed as the means ± standard deviation (SD). Differences among multiple groups were determined using one-way of variance ANOVA, followed by Tukey’s post hoc test. Comparisons between two groups were performed using an unpaired Student’s t-test. A p-value < 0.05 was considered to be statistically significant. The test level was taken as α = 0.05.

3. Results

3.1. Effect of Ozone on Nasal Secretion in Rats

The effect of ozone exposure on the amount of nasal secretion was concentration-dependent. The amount of nasal secretion was higher in all concentration groups than in the NC group. The amount of nasal secretion in the NEL, NEM, and NEH groups was statistically different from that in the NC group (p < 0.01) (Figure 1). Thus, inhalation of ozone exacerbated inflammatory symptoms in rats.

3.2. Effects of Ozone on Oxidative Stress in Rat Nasal Mucosa

MDA content in nasal mucosa was significantly increased in the ozone-exposed group compared with the normal group (p < 0.01), and the activity levels of SOD and GSH-Px in nasal mucosa were lower in the ozone-exposed group than in the normal group (p < 0.05), with statistically significant differences in the results and a concentration-dependent effect (Figure 2).

3.3. Effect of Ozone on the Levels of IL-6, IL-8, and TNF-α Protein in Nasal Lavage Fluid

The protein levels of IL-6, IL-8, and TNF-α in the nasal lavage fluid of rats exposed to different concentrations of ozone were significantly increased in the NC group (p < 0.05) and the highest in the NEH group (Figure 3).

3.4. Effects of Ozone Exposure on the Histopathology of Nasal Mucosa

In this study, the effect of ozone exposure on the amount of nasal secretion was a concentration-dependent effect. The results of the HE staining method to observe the effect of different concentrations of ozone on the histopathology of the nasal mucosa of rats are shown in Figure 4. The epithelial cilia of the nasal mucosa of the normal rats were neatly arranged, and the tissue level was clear, with intact cell structure. In the NEL group, after ozone exposure, nasal mucosal epithelial cilia were disorganized with shedding, the lamina propria was swollen and thickened, and the blood vessels were congested with inflammatory cells. In the NEM group, the nasal mucosal epithelial cells were partially necrotic, the cilia were disorganized and shedding, the lamina propria was swollen and thickened, and the blood vessels were congested with inflammatory cell infiltration. In the NEH group, the cilia of the nasal mucosa were obviously missing with the squamous epithelial hyperplasia, and a large number of inflammatory cells, such as neutrophils and lymphocytes, were seen. A large number of inflammatory cells, such as neutrophils and lymphocytes, were seen infiltrating the nasal mucosa and hypertrophied glands in the submucosal lamina propria. It can be seen that the pathological damage to the nasal mucosa of rats exposed to various concentrations of ozone was more severe than that of the non-exposed group, and the higher the concentration, the more severe the damage.

3.5. Effect of Ozone on the Expression of NF-κB p65 Protein and Its Inflammatory Factor Target Group mRNAs

The protein changes in NF-κB p65 in the cytoplasm and nucleus of the nasal mucosa of each group are shown in Figure 5. After ozone exposure in rats, the nuclear expression of NF-κB p65 in the nasal mucosa was higher in the NEL group than in the NC group (p < 0.05), and the amount of protein in the nucleus of NF-κB p65 in the NEM and NEH groups was further elevated, higher than that of the NC group (p < 0.05). The nuclear expression of each group showed a dose-dependent effect. The cytoplasmic content of NF-κB p65 gradually decreased in the NEL, NEM, and NEH groups compared with the NC group (p < 0.05), and the expression of IL-6, IL-8, and TNF-α mRNA increased with the ozone concentration (Figure 6). The trend of the changes in the expression of IL-6, IL-8, and TNF-α mRNA was the same as the changes in the nuclear expression of NF-κB p65 in the NEL, NEM, and NEH groups.

4. Discussion

The respiratory hazards of ozone pollution have received increasing attention in recent years [9,10]. Excessive atmospheric ozone concentrations have been associated with increased morbidity and mortality from disease, decreased lung function, and the development of asthma and allergic rhinitis [11]. It is predicted that near-surface ozone levels may increase and cause more associated health effects in the future if outdoor temperatures increase from year to year [12]. Currently, other than PM2.5, ozone is the only air pollutant included in the Global Burden of Disease (GBD) evaluation with independent effects in the American Cancer Society’s Respiratory Disease Cohort Study [13]. Atmospheric ozone pollution can induce oxidative stress and inflammation [14].

Other experimental studies of ozone inhalation exposure in animals are mostly conducted in rodents such as rats and mice, and the three inhalation exposure modes used are inhalation via respiratory mask, inhalation via nasal cannula, and inhalation via whole-body placement in an exposure chamber. In this experiment, we designed an animal ozone inhalation exposure system that more realistically simulates ozone inhalation via the respiratory tract in the natural state than inhalation via mask and nasal cannula [13,15].

In this study, a minute volume of 200 mL was assumed for 300 g rats [16], and inhalation of ozone at 0.5, 1, and 2 ppm concentrations for 6 consecutive weeks provided 0.27 mg, 0.54 mg, and 1.08 mg of total ozone, respectively, and, therefore, the ozone exposure levels in this study were higher than the World Health Organization’s Air Quality Guidelines for atmospheric ozone days in Shanghai, China. In 2022, the maximum 8 h average 90th percentile concentration was 165 µg/m³, which is significantly higher than the WHO standard [17].

Ozone exposure aggravated oxidative stress symptoms and nasal mucosal damage in rats [7]. In this experiment, MDA levels in the nasal mucosa of rats exposed to various concentrations of ozone increased with the increase in concentration, while SOD and GPX levels gradually decreased. Excessive MDA can be found in oxidative stress and damage to the structure of lipids, proteins, and DNA, leading to disruption of cellular structure and metabolism, which further leads to cellular damage and even apoptosis and decreases the antioxidant capacity of the nasal mucosa locally. Kheirouri et al. found that ozone caused oxidative stress in rats. Ozone was the most important factor in oxidative stress [18], and the expression levels of Caspases-3 and -8 mRNAs increased at 0.6 ppm ozone concentration. The nasal mucosa is more sensitive to ozone due to the characteristics of the nasal mucosa, in which about 40% of the ozone accumulates.

In the present study, it was found that ozone inhalation at medium and high concentrations could activate the NF-κB pathway in the nasal mucosa, which resulted in a significant enhancement of the expression of NF-κB p65 in the nucleus and a significant increase in the transcription of the downstream inflammatory factors IL-6, IL-8, and TNF-α mRNA and a significant increase in the levels of IL-6, IL-8, and TNF-α proteins in the nasal lavage fluid, which promoted the activation of inflammatory cells recruited to the nasal cavity. The activated inflammatory cells produced ROS, thus promoting the activation of inflammatory cells and the activation of inflammatory cells. These cells produce ROS, which promotes the cascade amplification of oxidative stress, further leading to oxidative stress injury and inflammatory response outbreak. Thus, ozone may promote the expression and secretion of inflammatory factors by activating the NF-κB pathway.

NF-κB is a transcription factor widely expressed in cells and associated with inflammatory responses. Nuclear translocation of NF-κB p65 is a hallmark of NF-κB pathway activation, which can rapidly activate the gene expression of pro-inflammatory factors with NF-κB-binding sequences, such as IL-6, IL-8, and TNF-α. Studies on human respiratory epithelial cells exposed to ozone have demonstrated that ozone-induced inflammatory responses in the respiratory tract are associated with the activation of the classical pathway of NF-κB [19]. Wu et al. exposed human bronchial epithelial cells to 0.4 ppm ozone for 4 h and demonstrated that ozone and its induced production of ROS resulted in the activation of NF-κB and mediated the secretion of IL-8 [20]. J. A. Kwon, M et al. exposed human nasal mucosa epithelial cells to ozone for 4 h and demonstrated that ozone and its induced ROS production resulted in the activation of IL-8 [21]. Nichols et al. exposed human nasal mucosa epithelial cells to ozone at 0.24 and 0.5 ppm for 3 h in an in vitro experiment and, for the first time, directly demonstrated that ozone activates NF-κB by inducing ROS production, leading to the release of TNF-α and thus triggering an inflammatory response [22]. It is evident that NF-κB and inflammatory factors play a role in the progression of inflammation. This result indicates that the ozone-induced inflammatory response may be related to the role of ozone in promoting the release of inflammatory factors through NF-κB entry into the nucleus. The sample size of this study is small, which is the limitation of this study. In the future, a larger-scale study will be carried out.

5. Conclusions

There are limitations in our study. Ozone-induced inflammation in rat nasal mucosa is a complex pathological process, and only three cytokines representing inflammatory response were selected for detection in this study, which represent only a part of the inflammatory response. In the future, the scope of detection needs to be expanded, or the omics method should be used to determine more comprehensively. In summary, the present study demonstrates that inhalation exposure to three different concentrations of ozone induced alterations to the main parameters of oxidative stress and inflammation, especially at high concentrations (2 ppm). It is suggested that exposure to ozone has the potential to induce toxicity, as well as oxidative damage, in the inflammatory response in the nasal mucosa of rats. This study may provide some clues to understanding the mechanisms underlying ozone-induced mammalian nasal toxicity.