Contributed equally
Allergic rhinitis (AR) is a common chronic inflammatory condition. It has been previously indicated that oxidative stress may contribute to allergic inflammation, including AR. Although molecular hydrogen (H2), an antioxidative agent, has been effective in treatment of numerous oxidative stress-associated diseases, the effect of inhalation of a high concentration of H2 on AR remains unknown. In the current study, female BALB/c mice were sensitized with ovalbumin (OVA) followed by intranasal OVA challenge to establish an animal model of AR. Mice were subjected to exposure to H2 and the inert gas helium at different frequencies and durations. The frequencies of sneezing/scratching and the body weights of mice were recorded. Histological analysis and multiplex cytokine assays were performed to evaluate the effects of H2 on AR. Challenge with OVA induced significant nasal mucosa inflammation. H2 inhalation reduced the infiltration of inflammatory cells into mucosa and lowered the levels of interleukin (IL)-5, IL-13 and monocyte chemoattractant protein-1 in serum. H2 inhalation slightly increased the level of interferon-γ, however the difference was not statistically significant. Treatment with H2 limited the weight increase in healthy mice and reversed the weight loss in mice with AR. Furthermore, H2 inhalation induced a therapeutic effect on AR in a dose-dependent manner. The current results demonstrate that H2 may demonstrate a therapeutic value for allergic diseases.
Allergic rhinitis (AR) is a common chronic inflammatory condition of the upper airways that affects 10–40% of the global population (
An increasing number of studies have indicated that molecular hydrogen (H2) induces beneficial effects in numerous oxidative stress-associated diseases (
AR is a chronic inflammatory condition associated with T-helper cell (Th)1/Th2 imbalance. In the present study, the beneficial effects of small molecule gases, H2 and helium (He), were compared in a murine model of AR. The expression levels of Th1/Th2 cytokines in the serum were also studied using a multiplex cytokine assay. The current study demonstrated that inhalation of H2 alleviated nasal allergic inflammation and reversed the weight loss caused by AR in mice.
A total of 106 female BALB/c mice (age, 6–8 weeks; weight, 19–22 g) were purchased from Shanghai Laboratory Animals Center (Shanghai, China) and housed in a temperature-controlled room (23±2°C) with 55±10% humidity under a 12-h light/dark cycle. The mice had
The murine model of AR was established as described previously with minor modifications (
Hydrogen gas was produced using the AMS-H-01 hydrogen oxygen nebulizer (Asclepius Meditec Co., Ltd., Shanghai, China), which simultaneously produced oxygen by electrolyzing water (vol/vol: 66.7% H2 and 33.3% O2). Mice were housed in a standard cage with food and water available
A total of 60 female BALB/c mice were randomly divided into six groups of 10 mice each, as follows: i) Control group, mice were exposed to ambient atmosphere; ii) control + H2 group, mice inhaled the H2 mixture for 4 h daily for 7 consecutive days before each intranasal NS challenge; iii) control + He group, mice inhaled the He mixture for 4 h daily for 7 consecutive days before each intranasal NS challenge; iv) AR group, AR mice were exposed to ambient atmosphere; v) AR + H2 group, AR mice inhaled the H2 mixture for 4 h daily for 7 consecutive days before each intranasal OVA challenge; and vi) AR + He group, AR mice inhaled the He mixture for 4 h daily for 7 consecutive days before each intranasal OVA challenge (
In dose-effect experiments, 4 control mice were exposed to air and 42 AR mice (7 groups; 6 AR mice in each group) were exposed to the H2 mixture for 0, 0.5, 1 or 2 h each time, once or twice a day, for 7 consecutive days.
The severity of nasal allergic symptoms was determined by measuring the frequencies of sneezing and nose-scratching for 10 min after the last intranasal OVA/NS challenge. In order to eliminate bias, mice were subjected to an observation in a single-blinded manner by examiners.
Mice were euthanized with an intraperitoneal overdose of sodium pentobarbital (100 mg/kg bodyweight) 24 h after the last intranasal challenge. Blood was collected from the retro-orbital sinus and centrifuged at 1,000 × g for 10 min at 4°C. Serum was collected and stored at −80°C for cytokine assays. Quantitative assays for multiple cytokines were performed using the commercially available Mouse Cytokine 23-Plex Immunoassay (Bio-Rad Laboratories, Inc., Hercules, CA, USA) according to the manufacturer's protocol. The 23-Plex Immunoassay mainly detects Th1 and Th2 cytokines.
Decapitated mouse heads were fixed in 4% paraformaldehyde for 48 h at room temperature, decalcified in 10% EDTA for 4–5 weeks, and embedded in paraffin. The embedded nasal cavities were cut into 4-µm-thin sections in the coronal direction. The sections were stained in hematoxylin solution for 5 min and stained in eosin-phloxine solution for 1 min at room temperature. Images were captured under optical microscopy at ×400 magnification.
Statistical analyses were performed using SPSS software (version 19.0; IBM Corp., Armonk, NY, USA). All data are presented as the mean ± standard error of the mean. Statistical analyses of data were performed using one-way analysis of variance followed by Least Significant Difference post hoc test for comparisons among groups. P<0.05 was considered to indicate a statistically significant difference.
The murine model of AR established via sensitization and challenge with OVA in the present study, has been previously used to investigate the pathological and physiological mechanisms of AR (
Sensitization and challenge with OVA can induce significant allergic inflammation in mouse nasal mucosa (
Th2 cytokines, interleukin (IL)-4, −5 and −13, serve roles in airway allergic inflammation, including AR and asthma (
To investigate the dose-effect association between H2 inhalation and allergic symptoms, different inhalation frequencies and durations were used in the murine models of AR. The frequency of sneezing decreased following administration of H2 in a dose-dependent manner (
Our earlier work indicated that AR could limit the increase in mice body weight (data not published). In the present study, the baseline weight in all groups was comparable. During the modeling phase, there was no significant difference in weight gain between mice after OVA-sensation or OVA-challenge alone, compared with the control (
The weight gain in the control + H2 group (−0.46±0.15 g) was significantly lower compared with the control group (0.57±0.17 g; P=0.001). Mice in the control + He group exhibited a 0.18±0.14 g weight gain which was not significantly different compared with the weight gain in the control group (P=0.161;
H2 is a medical gas with antioxidative, anti-inflammatory and antiapoptotic properties (
Oxidant/antioxidant imbalance serves an important role in airway inflammation (
The immune dysfunction resulting from the Th1/Th2 response imbalance is a major cause of chronic allergic inflammation, including AR (
Previous results concerning the effects of allergic inflammation on body weight are inconsistent. Loerbroks
There are certain limitations in the current study. The study only presented results for certain cytokines which expression levels between groups were statistically significantly different. Oxidative stress serves an important role in the pathogenesis of allergic diseases and H2 can selectively reduce the levels of ROS (
In conclusion, H2 inhalation at a high concentration may improve nasal allergic symptoms, reduce inflammatory cell infiltration in nasal mucosa, and regulate Th1/Th2 responses. The inhibitory effect of H2 inhalation on nasal allergic symptoms over a long period is more effective than that over a short period. At an equal inspiratory volume, multiple inhalation events result in greater improvements compared with single inhalation events. In addition, treatment with H2 exhibited a beneficial effect on mouse health through selective weight control. The present study indicated that H2 may have a therapeutic value for allergic diseases.
Not applicable.
The present study was supported by National Natural Science Foundation of China (cat. no. 81300809) and Shanghai Pudong New Area Science & Technology Development Fund (cat. nos. PKJ2016-Y02 and PW2016D-11).
The datasets of the present study are available from the corresponding authors.
SF, XL, XW, YZ, and ZM were responsible for performance of experiment, and data analysis. YW and WW were responsible for manuscript writing and revision, and experimental design.
The experiment protocols were approved by the Ethics Committee of Laboratory Animal Center of Tongji Universtiy (Shanghai, China; approval no: TJLAC-017-028).
Not applicable.
The authors declare that they have no competing interests.
allergic rhinitis
immunoglobulin E
interleukin-5
interleukin-13
interferon-γ
monocyte chemoattractant protein-1
normal saline
ovalbumin
reactive oxygen species
The protocol for the gas inhalation experiments in the murine model of allergic rhinitis. OVA, ovalbumin; alum, aluminum hydroxide; i.p., intraperitoneal injection; i.n., intranasal injection.
Frequencies of sneezing and nose scratching within 10 min after the last ovalbumin challenge in mice. Data are presented as the mean ± standard error of the mean. *P<0.001 vs. the control group, vs. the control + H2 group, and vs the control + He group; #P<0.05 vs. the AR group. H2, hydrogen gas; He, helium gas; AR, allergic rhinitis.
Effect of hydrogen inhalation on nasal mucosa inflammation. Representative sections of the nasal tissues (original magnification, ×400) after hematoxylin and eosin staining. (A) The control group. (B) The AR group. (C) The control + H2 group. (D) The AR + H2 group. (E) The control + He group. (F) The AR + He group. Yellow arrows indicate eosinophils; black arrows indicate lymphocytes. H2, hydrogen gas; He, helium gas; AR, allergic rhinitis.
Effect of hydrogen inhalation on serum expression of cytokines and chemokines. The expression of (A) IL-5, (B) IL-13, (C) MCP-1 and (D) IFN-γ. Data are presented as the mean ± standard error of the mean. *P<0.05 vs. the control group; §P<0.05 and §§P<0.01 vs. the AR group. H2, hydrogen gas; He, helium gas; AR, allergic rhinitis; IL, interleukin; MCP-1, monocyte chemoattractant protein-1; IFN-γ, interferon-γ.
Effect of different H2 inhalation frequencies and durations on nasal allergic symptoms. Mice were exposed to the H2 mixture for 0.5, 1 or 2 h each time, once or twice a day. The effects on (A) sneezing and (B) scratching. Data are presented as the mean ± standard error of the mean. *P<0.05 vs. the AR group. H2, hydrogen gas; He, helium gas; AR, allergic rhinitis.
Effect of ovalbumin-sensitization on the bodyweight of mice.
Group | Weight gain (g) | P-value |
---|---|---|
Non-sensitized | 0.58±0.12 | 0.078 |
Sensitized | 0.93±0.15 |
Weight gain=bodyweight after sensitization-initial bodyweight.
Effect of ovalbumin-challenge on the bodyweight of mice.
Group | Weight gain (g) | P-value |
---|---|---|
Control | 0.57±0.17 | 0.264 |
AR | 0.23±0.24 | |
Control + H2 | −0.46±0.15 | 0.644 |
AR + H2 | 0.60±0.21 | |
Control + He | 0.18±0.14 | 0.102 |
AR + He | −0.73±0.32 |
Weight gain=bodyweight after challenge-bodyweight before challenge. H2, hydrogen gas; He, helium gas; AR, allergic rhinitis.
Effect of AR on the bodyweight of mice.
Group | Weight gain (g) | P-value |
---|---|---|
Control | 1.57±0.41 | 0.081 |
AR | 0.95±0.20 | |
Control + H2 | −0.10±0.30 | 0.293 |
AR + H2 | 0.36±0.12 | |
Control + He | 0.57±0.17 | 0.560 |
AR + He | −0.38±0.14 |
Weight gain=bodyweight after challenge-initial bodyweight. H2, hydrogen gas; He, helium gas; AR, allergic rhinitis.
Effect of H2 inhalation on the bodyweight of mice.
Group | Weight gain (g) | P-value |
---|---|---|
Control | 0.57±0.17 | |
Control + H2 | −0.46±0.15 | 0.001 |
Control + He | 0.18±0.14 | 0.161 |
AR | 0.23±0.24 | |
AR + H2 | 0.60±0.21 | 0.008 |
AR + He | −0.73±0.32 | 0.002 |
Weight gain=bodyweight after gas inhalation-bodyweight before gas inhalation.
vs. Control group
vs. AR group. H2, hydrogen gas; He, helium gas; AR, allergic rhinitis.