Protective Effects of Hydrogen-Rich Saline Against Lipopolysaccharide-Induced Alveolar Epithelial-to-Mesenchymal Transition and Pulmonary Fibrosis

Abstract

BACKGROUND Fibrotic change is one of the important reasons for the poor prognosis of patients with acute respiratory distress syndrome (ARDS). The present study investigated the effects of hydrogen-rich saline, a selective hydroxyl radical scavenger, on lipopolysaccharide (LPS)-induced pulmonary fibrosis. MATERIAL AND METHODS Male ICR mice were divided randomly into 5 groups: Control, LPS-treated plus vehicle treatment, and LPS-treated plus hydrogen-rich saline (2.5, 5, or 10 ml/kg) treatment. Twenty-eight days later, fibrosis was assessed by determination of collagen deposition, hydroxyproline, and type I collagen levels. Development of epithelial-to-mesenchymal transition (EMT) was identified by examining protein expressions of E-cadherin and α-smooth muscle actin (α-SMA). Transforming growth factor (TGF)-β1 content, total antioxidant capacity (T-AOC), malondialdehyde (MDA) content, catalase (CAT), and superoxide dismutase (SOD) activity were determined. RESULTS Mice exhibited increases in collagen deposition, hydroxyproline, type I collagen contents, and TGF-β1 production in lung tissues after LPS treatment. LPS-induced lung fibrosis was associated with increased expression of α-SMA, as well as decreased expression of E-cadherin. In addition, LPS treatment increased MDA levels but decreased T-AOC, CAT, and SOD activities in lung tissues, indicating that LPS induced pulmonary oxidative stress. Hydrogen-rich saline treatment at doses of 2.5, 5, or 10 ml/kg significantly attenuated LPS-induced pulmonary fibrosis. LPS-induced loss of E-cadherin in lung tissues was largely reversed, whereas the acquisition of α-SMA was dramatically decreased by hydrogen-rich saline treatment. In addition, hydrogen-rich saline treatment significantly attenuated LPS-induced oxidative stress. CONCLUSIONS Hydrogen-rich saline may protect against LPS-induced EMT and pulmonary fibrosis through suppressing oxidative stress.

Figure 1

Hydrogen-rich saline treatment attenuates LPS-induced pulmonary fibrosis. Mice were instilled intratracheally with LPS (5 mg/kg). Hydrogen-rich saline was administered at a dose of 2.5, 5. or 10 ml/kg/d i.p. Lungs were harvested at the end of 28-day hydrogen-rich saline treatment therapy. (A–E) Collagen deposition was assessed with Masson’s trichrome staining on paraffin-fixed lung sections. Original magnification, ×400. Scale bars correspond to 20 μm. Fibrosis was also quantified by determination of hydroxyproline (F) and type I collagen (G, H) levels in lung tissues as described in “Material and Methods”. Data are expressed as means ±SEM (n=7 per each group). * p<0.05, ** p<0.01 vs. control group. ^# p<0.05, ^## p<0.01 vs. LPS group. H₂ represents hydrogen-rich saline.

Figure 2

Hydrogen-rich saline treatment attenuates LPS-induced EMT. Mice were instilled intratracheally with LPS (5 mg/kg). Hydrogen-rich saline was administered at a dose of 2.5, 5, or 10 ml/kg/d i.p. Lungs were harvested at the end of 28-day hydrogen-rich saline treatment therapy. (A–J) Lung sections were subjected to immunohistochemical analysis using antibodies against E-cadherin (A–E) or α-SMA (F–J). Original magnification, ×400. Scale bars correspond to 20 μm. (K, L) Protein expression of E-cadherin (K) or α-SMA (L) in lung homogenates was determined by Western blot analysis. Data are expressed as means ±SEM (n=7 per each group). ** p<0.01 vs. control group. ^## p<0.01 vs. LPS group. H₂ represents hydrogen-rich saline.

Figure 3

(A, B) Hydrogen-rich saline treatment suppresses LPS-induced TGFβ production. Mice were instilled intratracheally with LPS (5 mg/kg). Hydrogen-rich saline was administered at a dose of 2.5, 5, or 10 ml/kg/d i.p. Lungs were harvested at the end of 28-day hydrogen-rich saline treatment therapy. TGFβ-1 content and TGFβ protein expression in lung tissues were analyzed by ELISA and Western blot analysis, respectively. Data are expressed as means ±SEM (n=7 per each group). ** p<0.01 vs. control group. ^## p<0.01 vs. LPS group. H₂ represents hydrogen-rich saline.

Figure 4

Hydrogen-rich saline treatment attenuates LPS-induced lung oxidative stress. Mice were instilled intratracheally with LPS (5 mg/kg). Hydrogen-rich saline was administered at a dose of 2.5, 5, or 10 ml/kg/d i.p. Lungs were harvested at the end of 28-day hydrogen-rich saline therapy. MDA level (A), T-AOC level (B), CAT activity (C), and SOD activity (D) were determined as described in “Material and Methods”. Data are expressed as means ±SEM (n=7 per each group). * p<0.05, ** p<0.01 vs. control group. ^# p<0.05, ^## p<0.01 vs. LPS group. H₂ represents hydrogen-rich saline.