The Effect And Mechanism Of Antioxidants In Ameliorating Pulmonary Fibrosis In ARDs

Objectives: Firstly, The present study detected breath hydrogen of respiratory distress syndrome(ARDS) patients induced by sepsis in a clinical observation, suggesting that hydrogen may play an important role in lung injury and repair; Then we built a endotoxin-induced lung fibrosis mice model, aimed to determine Whether exogenous hydrogen had a protective effect in lipopolysaccharide(LPS)-induced lung fibrosis; in addition, we aimed to determine whether resveratrol(RSV), a natural antioxidant polyphenol, had anti-fibrotic effects in the murine model of LPS-induced pulmonary fibrosis.Methods: Part1: We measured breath hydrogen in sepsis-induced ARDS patients and volunteers by using a gas chromatograph method. Part2: Anesthetized male ICR mice were randomly divided into control group, model group and hydrogen-rich saline treatment group. Mice were administered a single intratracheal dose of purified LPS at 5 mg/kg. The pathological process of lung tissues was observed 7d, 14 d, 21 d, 28 d in the model group. The fibrotic areas in lung sections were detected by using Masson’s trichrome staining. The hydroxyproline content was determined by alkaline hydrolysis. Type I collagen levels was determined by commercially ELISA kits in lung tissues. The loss of E-cadherin accompanying by the acquisition of α-smooth muscle actin(α-SMA) was identified by using Western blot and immunohistochemical analysis, which indicating the development of epithelial mesenchymal transition(EMT). Transforming growth factor(TGF)-β1 content was measured using a commercially available ELISA kit. Levels of phosphorylated Smad2/Smad3 and Smad4 were detected by using Western blot analysis. Malondialdehyde(MDA) content, total antioxidant capacity(T-AOC), catalase(CAT) activity, and superoxide dismutase(SOD) activity in lung tissues were determined, which reflected the oxidative status in the lung. The hydrogen-rich saline treated mice were randomly divided into three groups and injected intraperitoneally with hydrogen-rich saline at 2.5、5、10 ml/kg once daily after LPS injection, hydrogen-rich saline was continued for 28 d. The control group received an identical dose of normal saline. Lungs were harvested at the time points indicated. The latter two groups received the same detection as the model group. Part3: Anesthetized male ICR mice were randomly divided into control group, model group and RSV treatment group. The RSV treated mice were injected intraperitoneally with RSV at 0.3 mg/kg once daily after LPS injection, RSV was continued for 28 d. The control and model group were treated the same as part2. Lungs were harvested at the time points indicated. All groups received the same detection as part2.Main Results: Part1: We found that breath hydrogen in ARDS patients was significantly decreased. Part2: LPS increased collagen deposition, hydroxyproline and type I collagen contents, and meanwhile induced EMT process, stimulated TGF-β1 production and Smad activation in lung tissues on day 21 to day 28 after LPS administration. In addition, LPS treatment resulted in a rapid induction of oxidative stress as evidenced by increase of MDA and decreases of T-AOC, CAT and SOD activities as early as 7 days after LPS treatment, which was persistent for at least 4 weeks. In contrast, hydrogen-rich saline treatment attenuated LPS induced EMT and pulmonary fibrosis, it suppressed LPS-induced oxidative stress, TGF-β1 production. Part3: Resveratrol had a protective effect in LPS induced EMT and pulmonary fibrosis, meanwhile it suppressed LPS-induced oxidative stress, TGF-β1 production and activation of Smad signaling pathway.Conclusions: Breath hydrogen in ARDS patients was decreased, which indicated that hydrogen may play an important role in ARDS lung injury and repair. Both hydrogen-rich saline and resveratrol may ameliorate LPS-induced EMT and pulmonary fibrosis through suppression of oxidative stress and TGF-β1/Smad signaling pathway. Application of antioxidants may represent a useful adjuvant pharmacologic approach to reduce ARDS-associated pulmonary fibrosis.