Effect of inhalation of hydrogen gas on Rho/ROCK pathway in lung tissues of septic mice with acute lung injury

Abstract

Objective To evaluate the effect of inhalation of hydrogen gas (H2) on Rho/Rho kinase (ROCK) pathway in lung tissues of septic mice with acute lung injury. Methods Forty male C57BL/6 mice, weighing 20-25 g, aged 6 weeks, were randomly divided into 4 groups (n=10 each) using a random number table: sham operation group (group Sh), sham operation+ inhalation of H2 group (group H2), sepsis group (group S), and sepsis+ inhalation of H2 group (group S+ H2). Sepsis was produced by cecal ligation and puncture (CLP). Both H2 and S+ H2 groups inhaled 2% H2 for 1 h starting from 1 and 6 h after CLP.The mice in each group were sacrificed at 24 h after CLP.The bronchoalveolar lavage fluid (BALF) was collected for determination of protein concentrations, polymorphonuclear neutrophil (PMN) count, and concentrations of tumor necrosis factor-alpha (TNF-α) and interleukin-1beta (IL-1β) (by enzyme-linked immunosorbent assay). The lung tissues were obtained for examination of the pathological changes which were scored and for determination of the wet/dry weight ratio (W/D ratio), activities of myeloperoxidase (MPO) and superoxide dismutase (SOD), malonaldehyde (MDA) level, the expression of Rho, ROCK1, ROCK2 and activated caspase-3, and phosphorylation of myosin phosphatase target protein 1 (MYPT-1) (by Western blot). Results Compared with group Sh, the concentrations of protein, PMN count, and concentrations of TNF-α and IL-1β in BALF, lung injury score, W/D ratio, and levels of MPO and MDA in lung tissues were significantly increased, the activity of SOD in lung tissues was significantly decreased, the expression of Rho, ROCK1, ROCK2 and activated caspase-3 was significantly up-regulated, and the phosphorylation of MYPT-1 in lung tissues was significantly increased in S and S+ H2 groups (P 0.05). Compared with group S, the concentrations of protein, PMN count, and concentrations of TNF-α and IL-1β in BALF, lung injury score, W/D ratio, and levels of MPO and MDA in lung tissues were significantly decreased, the activity of SOD in lung tissues was significantly increased, the expression of Rho, ROCK1, ROCK2 and activated caspase-3 in lung tissues was significantly down-regulated, and the phosphorylation of MYPT-1 in lung tissues was significantly decreased in group S+ H2 (P<0.05). Conclusion The mechanism by which inhalation of H2 attenuates acute lung injury is related to inhibition of Rho/ROCK pathway activation in lung tissues of septic mice. Key words: Hydrogen; Sepsis; Respiratory distress syndrome, adult; Rho factor; Rho-associated kinases