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.
Hydrogen gas (H(2)) as a new medical gas exerts organ-protective effects through regulating oxidative stress, inflammation and apoptosis. In contrast to H(2), hydrogen-rich saline (HS) may be more suitable for clinical application. The present study was designed to investigate whether HS can offer a neuroprotective effect in a rat model of permanent focal cerebral ischemia and what mechanism(s) underlies the effect. Sprague-Dawley rats were subjected to permanent focal cerebral ischemia induced by permanent middle cerebral artery occlusion (pMCAO). Different doses of HS or normal saline were intraperitoneally administered at 5min after pMCAO or sham operation followed by injections at 6h, 12h and 24h. Here, we found that HS treatment significantly reduced infarct volume and improved neurobehavioral outcomes at 24h, 48h and 72h after pMCAO operation in a dose-dependent manner (P<0.05). Moreover, we found that HS treatment dose-dependently increased the activities of endogenous antioxidant enzymes (SOD and CAT) as well as decreased the levels of oxidative products (8-iso-PGF2α and MDA) and inflammatory cytokines (TNF-α and HMGB1) in injured ipsilateral brain tissues at 6h, 12h and 24h after pMCAO operation (P<0.05). Thus, hydrogen-rich saline dose-dependently exerts a neuroprotective effect against permanent focal cerebral ischemia, and its beneficial effect is at least partially mediated by reducing oxidative stress and inflammation. Molecular hydrogen may be an effective therapeutic strategy for stroke patients.
Sepsis is the leading cause of death in critically ill patients. In recent years, molecular hydrogen, as an effective free radical scavenger, has been shown a selective antioxidant and anti-inflammatory effect, and it is beneficial in the treatment of sepsis. Rho-associated coiled-coil protein kinase (ROCK) participates in junction between normal cells, and regulates vascular endothelial permeability. In this study, we used lipopolysaccharide to stimulate vascular endothelial cells and explored the effects of hydrogen-rich medium on the regulation of adhesion of monocytes to endothelial cells and vascular endothelial permeability. We found that hydrogen-rich medium could inhibit adhesion of monocytes to endothelial cells and decrease levels of adhesion molecules, whereas the levels of transepithelial/endothelial electrical resistance values and the expression of vascular endothelial cadherin were increased after hydrogen-rich medium treatment. Moreover, hydrogen-rich medium could lessen the expression of ROCK, as a similar effect of its inhibitor Y-27632. In addition, hydrogen-rich medium could also inhibit adhesion of polymorphonuclear neutrophils to endothelial cells. In conclusion, hydrogen-rich medium could regulate adhesion of monocytes/polymorphonuclear neutrophils to endothelial cells and vascular endothelial permeability, and this effect might be related to the decreased expression of ROCK protein.
Endothelial injury is a primary cause of sepsis and sepsis-induced organ damage. Heme oxygenase-1 (HO-1) plays an essential role in endothelial cellular defenses against inflammation by activating nuclear factor E2-related factor-2 (Nrf2). We found that molecular hydrogen (H2) exerts an anti-inflammatory effect. Here, we hypothesized that H2 attenuates endothelial injury and inflammation via an Nrf2-mediated HO-1 pathway during sepsis. First, we detected the effects of H2 on cell viability and cell apoptosis in human umbilical vein endothelial cells (HUVECs) stimulated by LPS. Then, we measured cell adhesion molecules and inflammatory factors in HUVECs stimulated by LPS and in a cecal ligation and puncture (CLP)-induced sepsis mouse model. Next, the role of Nrf2/HO-1 was investigated in activated HUVECs, as well as in wild-type and Nrf(-/-) mice with sepsis. We found that both 0.3 mmol/L and 0.6 mmol/L (i.e., saturated) H2-rich media improved cell viability and cell apoptosis in LPS-activated HUVECs and that 0.6mmol/L (i.e., saturated) H2-rich medium exerted an optimal effect. H2 could suppress the release of cell adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1) and intercellular cell adhesion molecule-1 (ICAM-1), and pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β and high-mobility group box 1 protein (HMGB1). Furthermore, H2 could elevate anti-inflammatory cytokine IL-10 levels in LPS-stimulated HUVECs and in lung tissue from CLP mice. H2 enhanced HO-1 expression and activity in vitro and in vivo. HO-1 inhibition reversed the regulatory effects of H2 on cell adhesion molecules and inflammatory factors. H2 regulated endothelial injury and the inflammatory response via Nrf2-mediated HO-1 levels. These results suggest that H2 could suppress excessive inflammatory responses and endothelial injury via an Nrf2/HO-1 pathway.