Attenuation of cigarette smoke-induced airway mucus production by hydrogen-rich saline in rats

Abstract

Background: Over-production of mucus is an important pathophysiological feature in chronic airway disease such as chronic obstructive pulmonary disease (COPD) and asthma. Cigarette smoking (CS) is the leading cause of COPD. Oxidative stress plays a key role in CS-induced airway abnormal mucus production. Hydrogen protected cells and tissues against oxidative damage by scavenging hydroxyl radicals. In the present study we investigated the effect of hydrogen on CS-induced mucus production in rats.

Methods: Male Sprague-Dawley rats were divided into four groups: sham control, CS group, hydrogen-rich saline pretreatment group and hydrogen-rich saline control group. Lung morphology and tissue biochemical changes were determined by immunohistochemistry, Alcian Blue/periodic acid-Schiff staining, TUNEL, western blot and realtime RT-PCR.

Results: Hydrogen-rich saline pretreatment attenuated CS-induced mucus accumulation in the bronchiolar lumen, goblet cell hyperplasia, muc5ac over-expression and abnormal cell apoptosis in the airway epithelium as well as malondialdehyde increase in the BALF. The phosphorylation of EGFR at Tyr1068 and Nrf2 up-regulation expression in the rat lungs challenged by CS exposure were also abrogated by hydrogen-rich saline.

Conclusion: Hydrogen-rich saline pretreatment ameliorated CS-induced airway mucus production and airway epithelium damage in rats. The protective role of hydrogen on CS-exposed rat lungs was achieved at least partly by its free radical scavenging ability. This is the first report to demonstrate that intraperitoneal administration of hydrogen-rich saline protected rat airways against CS damage and it could be promising in treating abnormal airway mucus production in COPD.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Effect of hydrogen on lung histopathology of rats exposure to CS.

A. Representative H&E staining of lung sections. Con, sham control group; CS, cigarette smoke group; CS+H, hydrogen-rich saline pretreatment group; H, hydrogen-rich saline control group. (Scale bars  =  100 µm; lower-left insert: Scale bars  =  50 µm). B. Inflammation of rat lungs were scored. Hydrogen-rich saline significantly abrogated CS-induced lung inflammation. The results are presented as mean± SD (n = 10 rats per group) *p

Figure 2. Effect of hydrogen on goblet cell hyperplasia of the bronchial epithelium detected by AB/PAS-staining.

(A) Representative goblet cell staining determined by AB/PAS. Con, sham control group; CS, cigarette smoke group; CS+H, hydrogen-rich saline pretreatment group; H, hydrogen-rich saline control group. (Scale bars = 100 µm; lower-left insert: Scale bars = 50 µm); (B) Quantification of AB/PAS-positive area in the airway epithelium. AB/PAS-staining area and total area of corresponding bronchiolar epithelial were measured. AB/PAS-positive rates were presented as the ratio of AB/PAS-positive area to the total area. The values were expressed as mean ± SD from all the rats in each group.*p

Figure 3. Effect of hydrogen on CS-induced muc5ac expression in rat lung tissues.

(a) Effect of hydrogen on CS-induced muc5ac transcription analyzed by realtime RT-PCR. The levels of muc5ac mRNA were normalized to β-actin. Data are mean ±SD. * p<0.05, ** p<0.01 vs. the control; ## p<0.01 vs. CS group. (b) The upper panel was representative western blot analysis for muc5ac and GAPDH proteins in homogenized rat lung tissues. The bar graph (lower panel) showed muc5ac protein levels after normalized to the corresponding abundance of GAPDH. Data were presented as mean ± SD. *p<0.05, **p<0.01 vs. the control; # p<0.05 vs. CS group. (c) Representative immunohistochemistry for muc5ac in rat lung sections as indicated. Con, sham control group; CS, cigarette smoke group; CS+H, hydrogen-rich saline pretreatment group; H, hydrogen-rich saline control group. (Scale bars = 50 µm). Positive immunoreactivity for muc5ac was characterized by brown staining. (D) Percentage of Muc5ac-positive staining of the airway epithelium. Muc5ac-positive area and total area of corresponding bronchial epithelium were measured. Data were presented as the ratio of muc5ac-positive area to the total area. Hydrogen-rich saline pretreatment significantly decreased CS-induced muc5ac-positive area in the airway epithelium. The values were expressed as mean± SD (n = 10 per group). *p<0.05, **p<0.01 vs. the control, # p<0.05 vs. the CS group.

Figure 4. Effect of hydrogen on CS-induced airway epithelial cell apoptosis in rats.

Representative TUNEL staining in small airways. Con, sham control group; CS, cigarette smoke group; CS+H, hydrogen-rich saline pretreatment group; H, hydrogen-rich saline control group. (Scale bars = 50 µm). (B) The bar graph showed the apoptosis rate of airway epithelial cells in each group as indicated. Data were presented as mean ± SD of the apoptosis rate from all the rats in each group as indicated. *p

Figure 5. Effect of hydrogen on CS-induced MDA production in the BALF of rats.

MDA contents in the BALF were determined using a chemical reaction kit. Data were expressed as mean ± SD, n = 10 for each group.*p

Figure 6. Effect of hydrogen on CS-induced phosphorylation of EGFR and expression of Nrf2.

(a) Representative photographs of western blot for EGFR, p-EGFR (Tyr1068) proteins from homogenized rat lungs were shown in the upper panel. EGFR and p-EGFR(Tyr1068) protein levels were normalized to the corresponding GAPDH as shown in the lower bar graph. (b) The upper panel was representative western blot analysis for Nrf2 protein from homogenized rat lung tissues as indicated. The level of Nrf2 protein was normalized to the corresponding GAPDH as shown in the lower panel. Data were presented as mean ± SD from all rats in each group as indicated. *p