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<0.05, **p<0.01 vs. the control group; #p<0.05 vs. the CS group.
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<0.05, **p<0.01 vs. Control, # p<0.05 vs. CS group.
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<0.05, **p<0.01 vs. the control; #p<0.05 vs. CS group.
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<0.05, **p<0.01 vs. Control. #p<0.05 vs. the CS group.
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<0.05, **p<0.01 vs. Control; #p<0.05 vs. CS group.