Protective Effects And Mechanisms Of Molecular Hydrogen In Sepsis

Sepsis, a systemic inflammatory response syndrome induced by infection, is the most common cause of death in intensive care units [1,2]. However, to date, there is no effective treatment for sepsis. A large number of studies have found that both inflammatory response and oxidative stress play an important role in the pathogenesis of sepsis. In2007, Ohsawa et al reported that hydrogen gas (H2,<4%) could exert a therapeutic antioxidant activity by selectively reducing hydroxyl radicals (the most cytotoxic ROS) and effectively protect against transient cerebral ischemia-reperfusion damage [3,4]. Furthermore, recently, increasing researchers have shown that H2is a new therapeutic gas [4,6]. This study, using the different animal models, was designed to investigate the protective effects of H2in sepsis and sepsis-associated organ damage and molecular mechanisms.PART1:Protective Effects and Mechanisms of H2Inhalation in Moderate and Severe SepsisObjective:Oxidative stress plays an important role in the pathophysiological process of sepsis. H2has selective antioxidant properties. Therefore, the present study was designed to investigate the possible therapeutic effects and mechanisms of H2inhalation in a murine model of moderate and severe sepsis.Methods:Moderate and severe sepsis was induced by cecal ligation and puncture (CLP). H2treatment was given by inhalation of H2at different concentration (0%,1%,2%,4%) and different treatment time (0min,30min,60min,90min) beginning at1h and6h after CLP or sham operation, respectively. The arterial blood gas analysis was conducted during H2treatment, and survival rates of the animals were observed at different time points after sham or CLP operation. The organ (lung, liver, kidney) damage was measured. Furthermore, the changes of pro-inflammatory cytokine (HMGB1), anti-oxidant enzymes (SOD, CAT) and oxidative product (8-iso-PGF2a) in serum and organ tissue (lung, live, kidney) were detected. Finally, the role of Nrf2in this protection of H2in severe sepsis was investigated by measuring Nrf2expression in organ tissue and Nrf2null mice.Results:1. H2inhalation could significantly improve the survival rate of mice with moderate or severe sepsis in a concentration-and time-dependent manner. Treatment with2%H2for60min beginning at1h and6h after CLP operation was better.2. Moderate or severe septic mice showed significant multiple organ damage characterized by the increases of lung myeloperoxidase activity, wet-to-dry weight ratio, protein concentration in bronchoalveolar lavage, as well as failure of hepatic and renal function, which was significantly attenuated by2%H2treatment.3. The beneficial effects of H2treatment on sepsis and sepsis-associated damage were associated with the decreased levels of oxidative product (8-iso-PGF2a), increased activities of antioxidant enzymes [superoxide dismutase (SOD) and catalase (CAT)], and reduced levels of the late pro-inflammatory cytokine [high-mobility group box1(HMGB1)] in serum and tissue.4. H2inhalation could increase the expression of Nrf2organ tissue in severe septic mice. And H2treatment could not increase the survival rate of Nrf2null mice with severe sepsis.Conclusion:H2inhalation improves the survival rate and organ injury of moderate and severe sepsis in mice. Moreover, H2treatment decreases the levels of inflammatory cytokines and oxidative stress in serum and tissue via activation of Nrf2signaling pathway.PART2:Protective Effects and Mechanisms of Combination Therapy with H2and Hyperoxia in a Murine Model of Moderate and Severe SepsisObjective:Some studies have found that hyperoxia may be beneficial to sepsis. However, it could exacerbate organ injury or weaken the therapeutic effect by increasing free radical formation. Recently, it has been suggested that H2at low concentration could exert a therapeutic antioxidant activity. This study was designed to investigate whether combination therapy with H2and hyperoxia could afford more potent therapeutic effects in a murine model of moderate or severe sepsis. If yes, the relevant mechanisms would be studied.Methods:Moderate and severe sepsis was induced by CLP. The animals were exposed to98%O2or/and2%H2for3h starting at1and6h after CLP operation, respectively. The survival rate and organ damage in all groups were observed at different time points. In addition, the pro-inflammatory cytokines (high-mobility group box1[HMGB1] and tumor necrosis factor α [TNF-α]), anti-inflammatory cytokine (interleukin10[IL-10]), antioxidant enzymes (superoxide dismutase [SOD] and catalase [CAT]), and oxidative product (8-iso-prostaglandin F2α [8-iso-PGF2α) in serum and tissues (lung, liver and kidney) were measured.Results:1. Either98%O2or2%H2exposure for3h starting at1and6h after operation could improve the survival rate of moderate and severe CLP mice. However, combined treatment with H2and hyperoxia increase the survival rate of septic mice more significantly.2. Either98%O2or2%H2treatment alone can attenuate sepsis-associated organ injury (lung, liver and kidney) in moderate or severe CLP mice, including the improvement of histopathological changes and scores, lung MPO activity, lung W/D ratio, protein concentration in BAL, PaO2/FiO2ratio as well as liver and renal function (ALT, AST, BUN and Cr). Combination therapy with H2and hyperoxia showed a better protective effect in a synergistic way.3. The synergistic effects of combined therapy with H2and hyperoxia in sepsis might be associated with the decreased levels of oxidative product (8-iso-PGF2α) and pro-inflammatory cytokines (HMGB1and TNF-α), as well as the increased levels of anti-inflammatory cytokine (IL-10) and antioxidant enzymes activity (SOD, CAT) in serum and tissues.Conclusion:Combination therapy with H2and hyperoxia provides enhanced therapeutic efficacy via both antioxidant and anti-inflammatory mechanisms and might be potentially a clinically feasible approach for treating sepsis.PART3:Protective Effects and Mechanisms of H2Treatment on LPS-induced Acute Lung Injury in Mice Objective:Acute lung injury (ALI) is a common complication of critically ill patients. We have found that H2inhalation at low concentration exerts protective effects on the sepsis-induced lung injury, but the molecular mechanisms by which H2ameliorates lung injury remain unclear. In the current study, we aim to investigate the protective effects and relevant mechanisms of H2in a mouse model of ALI. Furthermore, the protective effects of hydrogen-rich saline against ALI were studied.Methods:ALI model was induced by intratracheal administration of lipopolysaccharide (LPS). Two percent of H2was inhaled for1h beginning at1h and6h after LPS administration, respectively. The indicators of lung injury and apoptosis were observed. Moreover, the changes in pro-inflammatory cytokines (TNF-a, IL-1β, IL-6, HMGB1) and chemokines (KC, MIP-1α, MIP-2, MCP1) levels in the bronchoalveolar lavage fluid (BALF), as well as the activity of NF-κB in the lung tissue were detected at different time points. In addition, the protection of hydrogen-rich saline (HS) against ALI was observed.Results:1. ALI induced by LPS was attenuated by H2treatment, including the improvement of histopathology and histologic scores, lung myeloperoxidase activity, wet-to-dry weight ratio, and oxygenation index (PaO2/FiO2), total protein in the bronchoalveolar lavage fluid (BALF).2. H2treatment dramatically prevented the LPS-induced pulmonary cell apoptosis, as reflected by the decrease in TUNEL (deoxynucleotidyl transferase dUTP nick end labeling) staining-positive cells and caspase-3activity.3. H2treatment markedly attenuated LPS-induced lung inflammation, as evidenced by down regulation of polymorphonuclear neutrophils and total cells in BALF, as well as pro-inflammatory cytokines (tumor necrosis factor a, interleukin1β, interleukin6, and high-mobility group box1) and chemokines (keratinocyte-derived chemokine, macrophage inflammatory protein [MIP]1a, MIP-2, and monocyte chemoattractant protein1) in BALF.4. H2treatment inhibited LPS-induced pulmonary NF-κB activation, which may be associated with the lung inflammation.5. Intraperitoneal injection of HS also played a protective role in LPS-induced ALI.Conclusion:Hydrogen gas and hydrogen-rich saline can dramatically alleviate ALI induced by LPS, which might be associated with down-regulation of inflammation and apoptosis by inhibiting pulmonary NF-κB activation.PART4:Protective Effects and Mechanisms of Hydrogen-rich Saline in a Rat Model of Septic ShockObjective:Hydrogen-rich saline (HS) was produced by dissolving H2to normal saline at a supersaturated level. Septic shock is the severe sepsis with hypotension and circulatory failure. This study was designed to investigate the effects and mechanisms of HS in a rat model of septic shock.Methods:Septic shock was induced by CLP in rats. The different doses of HS (5mL/kg,10mL/kg) were administrated intravenously at1h after CLP operation. Then, the blood gas, hemodynamic, organ (heart, lung, liver, kidney) function and survival rate of animals in all groups were detected at different time points, on this basis, the levels of inflammatory factors (IL-1β, IL-6, HMGB1, NO) in serum, as well as myeloperoxidase (MPO) activity and inducible nitric oxide synthase (iNOS) expression in organ tissues (heart, lung, liver, kidney) at different time points were measured.Results:1. HS treatment could significantly improve the mean arterial pressure (MAP), vascular reactivity and hemodynamics in rats with septic shock.2. HS treatment could effectively improve the arterial blood gas and lung function (PaO2/FiO2), and reduce the serum lactate level in rats with septic shock.3. HS treatment significantly improved the cardiac function (EF, FS, SV, CO), liver function (ALT, AST, TP, ALB) and renal function (BUN, Cr).4. HS treatment could significantly reduce the level of inflammatory cytokines (IL-1β, IL-6, HMGB1, NO) in serum and the activity of MPO in organ tissue (heart, lung, liver, kidney).5. HS treatment significantly reduced the expression of iNOS in organs (heart, lung, liver, kidney) of rats with septic shock.Conclusion:HS treatment can significantly improve hemodynamic, vascular reactivity and organ function of rats with septic shock, which may be associated with reduction of inflammatory response and iNOS expression. Summary1. H2inhalation improves the survival rate and organ injury of moderate and severe sepsis in mice, which is associated with the decreases of inflammatory cytokines and oxidative stress in serum and tissue via activation of Nrf2signaling pathway.2. Combination therapy with H2and hyperoxia provides enhanced therapeutic efficacy in a murine model of moderate and severe sepsis via both antioxidant and anti-inflammatory mechanisms.3. H2inhalation can dramatically alleviate the LPS-induced ALI, which might be associated with down-regulation of inflammation and apoptosis by inhibiting pulmonary NF-κB activation.4. Hydrogen-rich saline treatment has protective effects in LPS-induced ALI.5. Hydrogen-rich saline treatment can significantly improve hemodynamic, vascular reactivity and organ function of rats with septic shock, which may be associated with reduction of inflammatory response and iNOS expression.6. H2and hydrogen-rich saline treatment have no significant side effects, supporting that molecular hydrogen might be potentially a clinically feasible approach for sepsis.