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Sepsis-associated acute lung injury (ALI), which carries a high morbidity and mortality in patients, has no effective therapeutic strategies to date. Our group has already reported that hydrogen gas (H2) exerts a protective effect against sepsis in mice. However, the molecular mechanisms underlying H2 treatment are not fully understood. This study investigated the effects of H2 on lung injuries in septic mice through the isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomic analysis. Male ICR mice used in this study were subjected to cecal ligation and puncture (CLP) or sham operation. And 2% H2 was inhaled for 1 h beginning at 1 and 6 h after sham or CLP operation. The iTRAQ-based liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was preformed to investigate lung proteomics. Sepsis-challenged animals had decreased survival rate, as well as had increased bacterial burden in blood, peritoneal lavage, and lung sample, which were significantly ameliorated by H2 treatment. Moreover, a total of 4,472 proteins were quantified, and 192 differentially expressed proteins were related to the protective mechanism of H2 against sepsis. Functional enrichment analysis showed that H2-related differential proteins could be related to muscle contraction, oxygen transport, protein synthesis, collagen barrier membranes, cell adhesion, and coagulation function. These proteins were significantly enriched in four signaling pathways, and two of which are associated with coagulation. In addition, H2 alleviates ALI in septic mice through downregulating the expression of Sema 7A, OTULIN, and MAP3K1 as well as upregulating the expression of Transferrin. Thus, our findings provide an insight into the mechanism of H2 treatment in sepsis by proteomic approach, which may be helpful to the clinic application of H2 in patients with sepsis.

Hydrogen gas (H2) has antioxidative, anti-inflammatory, and antiapoptotic effects and may have beneficial effects in severe sepsis. The purpose of this study was to investigate the mechanisms underlying these protective effects. Male Institute for Cancer Research mice were randomized into 6 groups: sham; sham + H2; severe sepsis; severe sepsis + H2; severe sepsis + zinc protoporphyrin IX (ZnPPIX), a heme oxygenase-1 (HO-1) inhibitor; and severe sepsis + H2 + ZnPPIX. Cecal ligation and puncture (CLP) was used to induce sepsis. Mice in the H2 groups received inhaled 2% H2 for 1 h at 1 h and 6 h after CLP or sham operation. Mice in the ZnPPIX groups received 40-mg/kg ZnPPIX by intraperitoneal injection 1 h before CLP. Tin protoporphyrin IX (TinPPIX), another HO-1 inhibitor, was also used in part for this study. Mice in the TinPPIX groups received 50-mg/kg TinPPIX through subcutaneous injection 6 h before CLP. The levels of biochemical markers, oxidative products, inflammatory mediator, the number of intestinal apoptotic cells, and the colony-forming unit numbers in the peritoneal lavage fluid were much higher in the severe sepsis group compared with the sham group. Intestinal injury in animals with severe sepsis was worse than that in animals in the sham group. H2 therapy in the animals with severe sepsis was associated with reduced intestinal injury, decreased numbers of colony-forming unit and apoptotic cells, reduced levels of biochemical markers, oxidative products, and high-mobility group box 1 protein. The protective effects of H2 were reversed by ZnPPIX and TinPPIX. Protein and messenger RNA expressions of HO-1 and nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) in the intestine were increased in the severe sepsis group compared to the sham group, and H2 further increased their expressions in the severe septic mice. Zinc protoporphyrin IX and TinPPIX inhibited the expression of HO-1 protein. Hydrogen has the capacity to protect mice from organ injury in severe sepsis through a mechanism involving HO-1.

Sepsis is the most common cause of death in intensive care units. Some studies have found that hyperoxia may be beneficial to sepsis. However, the clinical use of hyperoxia is hindered by concerns that it could exacerbate organ injury by increasing free radical formation. Recently, it has been suggested that molecular hydrogen (H2) at low concentration can exert a therapeutic antioxidant activity and effectively protect against sepsis by reducing oxidative stress. Therefore, we hypothesized that combination therapy with H2 and hyperoxia might afford more potent therapeutic strategies for sepsis. In the present study, we found that inhalation of H2 (2%) or hyperoxia (98%) alone improved the 14-day survival rate of septic mice with moderate cecal ligation and puncture (CLP) from 40% to 80% or 70%, respectively. However, combination therapy with H2 and hyperoxia could increase the 14-day survival rate of moderate CLP mice to 100% and improve the 7-day survival rate of severe CLP mice from 0% to 70%. Moreover, moderate CLP mice showed significant organ damage characterized by the increases in lung myeloperoxidase activity, lung wet-to-dry weight ratio, protein concentration in bronchoalveolar lavage, serum biochemical parameters (alanine aminotransferase, aspartate aminotransferase, creatinine, and blood urea nitrogen), and organ histopathological scores (lung, liver, and kidney), as well as the decrease in PaO2/FIO2 ratio at 24 h, which was attenuated by either H2 or hyperoxia alone. However, combination therapy with H2 and hyperoxia had a more beneficial effect against lung, liver, and kidney damage of moderate or severe CLP mice. Furthermore, we found that the beneficial effect of this combination therapy was associated with the decreased levels of oxidative product (8-iso-prostaglandin F2α), increased activities of antioxidant enzymes (superoxide dismutase and catalase) and anti-inflammatory cytokine (interleukin 10), and reduced levels of proinflammatory cytokines (high-mobility group box 1 and tumor necrosis factor α) in serum and tissues. Therefore, combination therapy with H2 and hyperoxia provides enhanced therapeutic efficacy via both antioxidant and anti-inflammatory mechanisms and might be potentially a clinically feasible approach for sepsis.

Acute lung injury (ALI) is still a leading cause of morbidity and mortality in critically ill patients. Recently, our and other studies have found that hydrogen gas (H₂) treatment can ameliorate the lung injury induced by sepsis, ventilator, hyperoxia, and ischemia-reperfusion. However, the molecular mechanisms by which H₂ ameliorates lung injury remain unclear. In the current study, we investigated whether H₂ or hydrogen-rich saline (HS) could exert protective effects in a mouse model of ALI induced by intratracheal administration of lipopolysaccharide (LPS) via inhibiting the nuclear factor κB (NF-κB) signaling pathway-mediated inflammation and apoptosis. Two percent of H₂ was inhaled for 1 h beginning at 1 and 6 h after LPS administration, respectively. We found that LPS-challenged mice exhibited significant lung injury characterized by the deterioration of histopathology and histologic scores, wet-to-dry weight ratio, and oxygenation index (PaO₂/FIO₂), as well as total protein in the bronchoalveolar lavage fluid (BALF), which was attenuated by H₂ treatment. Hydrogen gas treatment inhibited LPS-induced pulmonary early and late NF-κB activation. Moreover, H₂ treatment dramatically prevented the LPS-induced pulmonary cell apoptosis in LPS-challenged mice, as reflected by the decrease in TUNEL (deoxynucleotidyl transferase dUTP nick end labeling) staining-positive cells and caspase 3 activity. Furthermore, H₂ treatment markedly attenuated LPS-induced lung neutrophil recruitment and inflammation, as evidenced by downregulation of lung myeloperoxidase activity, total cells, and polymorphonuclear neutrophils in BALF, as well as proinflammatory cytokines (tumor necrosis factor α, interleukin 1β, interleukin 6, and high-mobility group box 1) and chemokines (keratinocyte-derived chemokine, macrophage inflammatory protein [MIP] 1α, MIP-2, and monocyte chemoattractant protein 1) in BALF. In addition, i.p. injection of 10 mL/kg hydrogen-rich saline also significantly attenuated the LPS-induced ALI. Collectively, these results demonstrate that molecular hydrogen treatment ameliorates LPS-induced ALI through reducing lung inflammation and apoptosis, which may be associated with the decreased NF-κB activity. Hydrogen gas may be useful as a novel therapy to treat ALI. munosorbent assay; H₂-hydrogen gas; HMGB1-high-mobility group box 1; HS-hydrogen-rich saline; i.t.-intratracheal; KC-keratinocyte-derived chemokine; LPS-lipopolysaccharide; MCP-1-monocyte chemoattractant protein 1; MIP-1α-macrophage inflammatory protein 1α; MIP-2-macrophage inflammatory protein 2; MPO-myeloperoxidase; PBS-phosphate-buffered saline; PMNs-polymorphonuclear neutrophils; TUNEL-deoxynucleotidyl transferase dUTP nick end labeling; W/D-wet-to-dry.