Objective To investigate the effect of inhalation of hydrogen (H2) on the inflammation response during sepsis-induced acute lung injury in mice.Methods Forty-eight male ICR mice,aged 5 weeks,weighing 20-25 g,were randomly divided into 4 groups (n =12 each):sham operation group (group A),sham operation + H2 group (group B),sepsis group (group C) and sepsis + H2 group (group D).Sepsis was produced by cecal ligation and puncture (CLP).Groups B and D received 1 h inhalation of 2 ％ H2 at 1 and 6 h after CLP or sham operation.The behavior was assessed and scored at 24 h after CLP.Arterial blood samples were taken for blood gas analysis.Oxygenation index was calculated.Venous blood samples were taken for determination of the levels of serum tumor necrosis factor-α (TNF-α),interleukin-1 (IL-1β),high mobility group protein B1 (HMGB1) and interleukin-10 (IL-10).The mice were then sacrificed and lungs were removed for determination of the levels of TNF-α,IL-1β,HMGB1 and IL-10 in lung tissues.The apoptotic index was calculated.Results Compared with group A,the behavior score,apoptotic index and levels of TNF-α,IL-1β,HMGB1 and IL-10 were increased,and the oxygenation index was decreased in groups C and D,and no significant change was found in the parameters mentioned above in group B.Compare with group C,the oxygenation index and level of IL-10 were significantly increased,and the behavior score,apoptotic index and levels of TNF-α,IL-1β and HMGB1 were significantly decreased in group D.Conclusion Inhalation of H2 can regulate the balance between pro-and anti-inflammatory cytokines,inhibit the inflammation response and alleviate sepsis-induced acute lung injury in mice.

Objective To investigate the protective effects of hydrogen gas (H2) inhalation on acute renal injury in severe septic mice,and the associated mechanism.Methods Sepsis model was made by cecal ligation and puncture (CLP) operation in mice.A total of 24 mice were randomly (random number)divided into four groups:sham operation group (Sham),sham operation with H2 inhalation group (Sham +H2),sepsis group (Sepsis),and sepsis with H2 inhalation group (Sepsis + H2).The procedure for inhalation of 2％ H2 for 1 h was initiated at 1 h after CLP and 6 h after sham operation,respectively.The pathological changes and apoptosis of kidney,the blood creatinine (Cr) and blood urea nitrogen (BUN)concentration,the activities of superoxide dimutase (SOD) and catalase (CAT) in serum and kidney,as well as the levels of 8-iso-prostaglandin F2α (8-iso-PGF2α) and high mobility group box 1 (HMGB1) in serum and kidney were observed at 24 h after operation.Results H2 inhalation could significantly alleviate the renal injury of septic mice,reduce the renal cell apoptosis,increase the activities of SOD and CAT,as well as decrease the levels of 8-iso-PGF2α and HMGB1 in serum and kidney (P ＜ 0.05).Conclusions H2 inhalation can alleviate acute renal injury in septic mice,resulting from the increased activities of antioxidant enzymes and the reduced levels of oxidative products and inflammatory cytokines.

Objective To evaluate the effects of hydrogen gas (H2) on lipopolysaccharide (LPS)-induced damage to human umbilical vein endothelial cells (HUVECs) in vitro.Methods HUVECs were cultured in DMEM culture medium containing 10％ fetal bovine serum.The cells were seeded in 6-well plates (2 ml/hole) at a density of 2 × 106 cells/ml and randomly divided into 4 groups (n =35 each) using a random number table:control group (group C),group H2,LPS group and LPS + H2 group.The cells were cultured in the plain culture medium in C and LPS groups or in hydrogen-saturated culture medium in H2 and LPS + H2 groups.In addition,LPS 1 μg/ml was added simultaneously in LPS and LPS + H2 groups,and the equal volume of normal saline was added instead in C and H2 groups.The cells were incubated for 24 h.At 6,12 and 24 h of incubation,the cells of 5 wells were chosen and stained with Wright-Giemsa to observe the destruction of HUVEC barrier.At 6,12 and 24 h of incubation,the cells of 5 wells were chosen to detect the expression of VE-cadherin and β-catenin using Western blot.At 24 h of incubation,the cells of 5 wells were chosen to detect the expression of VE-cadherin and βcatenin using immunofluorescence.Results Pathological changes of endothelial cells were observed in LPS group.The pathological changes of the cells were lighter in LPS + H2 group than in LPS group.Compared with group C,VE-cadherin expression was significantly down-regulated (P ＜ 0.05),while no significant change was found in β-catenin expression in LPS and LPS + H2 groups (P ＞ 0.05).Compared with group LPS,VE-cadherin expression was significantly up-regulated (P ＜ 0.05),while no significant change was found in ~catenin expression in group LPS + H2 (P ＞ 0.05).Conclusion H2 can effectively reduce LPS-induced damage to HUVECs through inhibiting down-regulation of VE-cadherin expression.

Objective To investigate the effects of hydrogen-rich saline on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice.Methods Thirty-two male C57BL/6 mice,weighing 20-25 g,were randomly divided into 4 groups (n =8 each):control group (group C),hydrogen-rich saline group (group H2),ALI group and ALI + hydrogen-rich saline group (group ALI+ H2).ALI was induced by inhaled aerosolized LPS 25μg (dissolved in PBS) in groups ALI and ALI+ H2.Aerosolized PBS 50μl was inhaled in groups C and H2.0.6mmol/L hydrogen-rich saline 5 ml/kg was injected intraperitoneally at 1 and 12 h after aerosolized PBS or LPS was inhaled in groups H2 and ALI+ H2,respectively.At 24 h after PBS or LPS treatment,the animals were mechanically ventilation for 15 min,and arterial blood gas analysis was performed.The oxygenation index (PaO2/FiO2) was calculated.Bronchoalveolar lavage fluid (BALF) was collected for determination of total protein concentration and polymorphonuclear neutrophils (PMN) count.The concentrations of TNF-α,IL-1β,IL-6 and high mobility group box-1 (HMGB-1) protein in BALF were determined by ELISA.Then the animals were sacrificed and the lungs were obtained for determination of W/D lung weight ratio (W/D ratio),and myeloperoxidase (MPO) and caspase-3 activities.The pathological changes of the lung were scored.Apoptosis index (AI) was calculated.Results Compared with group C,no significant difference was found in PaO2/FiO2,the concentrations of protein,TNF-α,IL-1β,IL-6 and HMGB1,and PMN count in BALF,lung injury score,W/D ratio,MPO and caspase-3 activities and AI in group H2 (P ＞ 0.05),and PaO2/FiO2 was significantly decreased,the concentrations of total protein,TNF-α,IL-1β,IL-6 and HMGB1,and PMN count in BALF,lung injury score,W/D ratio,MPO and caspase-3 activities,and Al were increased in groups ALI and ALI + H2 (P ＜ 0.05).Compared with group ALI,PaO2/FiO2 was signifieantly increased,the concentrations of total protein,TNF-α,IL-1β,IL-6 and HMGB1,and PMN count in BALF,lung injury score,W/D ratio,MPO and caspase-3 activities,and AI were decreased in group ALI+ H2 (P ＜ 0.05).Conclusion Hydrogen-rich saline can alleviate LPS-induced ALI via inhibition of inflammatory response and apoptosis in mice.

ObjectiveTo investigate the effect of hydrogen (H2 ) on acute lung injury (ALI) in septic mice.MethodsOne hundred and twelve male C57BL/6 mice,aged 5 weeks,weighing 20-25 g,were randomly divided into 4 groups ( n =28 each):sham operation group (group A),sham operation + H2 group (group B),sepsis group (group C) and sepsis + H2 group (group D).Sepsis was produced by cecum ligation and puncture (CLP).Groups B and D received 1 h inhalation of 2％ H2 at 1 and 6 h after CLP operation or sham operation.Twenty animals in each group were selected and observed for the 7 d survival rate.The left 8 animals in each group were sacrificed at 24 h after CLP operation.Venous blood samples and lung tissues were obtained to determine the levels of superoxide dismutase (SOD),catalase (CAT) and 8-iso-prostaglandin F2alpha (8-iso-PGF2α) in the serum and lungs,the concentration of protein in bronchoalveolar lavage fluid (BALF),and the activity of myeloperoxidase (MPO) in the lungs.The lung injury score (LIS) was assessed and W/D lung weight ratio was calculated.ResultsCompared with group A,the 7 d survival rate and activities of SOD and CAT in the serum and lungs were significantly decreased,and LIS,W/D ratio,the concentration of protein in BALF,MPO activity and 8-iso-PGF2α level in the serum and lungs were significantly increased in group C ( P ＜ 0.05 ).Compared with group C,the 7 d survival rate and activities of SOD and CAT in the serum and lungs were significantly increased,and LIS,W/D ratio,the concentration of protein in BALF,MPO activity and 8-iso-PGF2α level in the serum and lungs were significantly decreased in group C ( P ＜ 0.05).ConclusionH2 can alleviate ALI in septic mice via inhibiting oxidative stress response.

Objective To investigate the effect of hydrogen gas on lipopolysaccharide (LPS)-induced apoptosis in human umbilical vein endothelial cells (HUVECs) in vitro.Methods HUVEC-12 cells were seeded in 96-well plates with a density of 1 × 104/ml (200 μl/hole) or in 6-well plates (2 ml/hole) with a density of 1 × 106/ml and randomly divided into 4 groups (n =30 each):control group (group C),hydrogen gas (H2) group,LPS group and LPS + H2 group.The cells were cultured in the plain culture medium in groups C and LPS or in hydrogen-saturated culture medium in groups H2 and LPS + H2.In addition,LPS 1 μg/ml was added simultaneously in groups LPS and LPS + H2 and the equal volume of normal saline was added instead in groups C and H2.The cell viability and apoptosis were measured by MTT assay and flow cytometry respectively after 24 h incubation.The concentration of high-mobility group box 1 (HMGB1) in the supernatant was determined by ELISA.Results Compared with groups C and H2,the cell viability was significantly decreased,and the apoptotic rate and concentration of HMGB1 in the supernatant were significantly increased in groups LPS and LPS + H2 (P ＜ 0.05).Compared with group LPS,the cell viability was significantly increased,and the apoptotic rate and concentration of HMGB1 in the supernatant were significantly decreased in group LPS + H2 (P ＜ 0.05).Conclusion Hhydrogen gas can effectively reduce LPS-induced apoptosis in HUVECs through inhibiting the release of HMGB1.

Objective To investigate the effects of hydrogen-rich medium on lipopolysaccharide (LPS)-induced release of inflammatory cytokines from murine macrophage RAW264.7 cells.Methods The RAW264.7cells were cultured in DMEM culture medium containing 10％ fetal bovine serum.The cells were seeded in 6-well plates (3 ml/hole) with a density of 2 × 106/ml and randomly divided into 4 groups (n =24 each):control group (group A),hydrogen-rich medium group (group B),LPS group (group C) and LPS + hydrogen-rich medium group (group D).Group A received no treatment.LPS 1 μg/ml was added to the medium in groups C and D,while the cells were incubated with 0.6 mmol/L hydrogen-rich medium simultaneously in groups B and D.Six wells in each group were chosen at 6,12.24 and 48 h of incubation,and the supernatant was collected to detect the concentrations of TNF-α,IL-1β and IL-10.Six wells in each group were chosen at 6 and 12 h of incubation for determination of heme oxygenase-1 (HO-1) expression in the cells by Western blot.Results Compared with group A,the concentrations of TNF-α,IL-1β and IL-10 in the supernatant were significantly increased,and the expression of HO-1 was up-regulated in group C (P ＜ 0.05).Compared with group C,the concentrations of TNF-α and IL-1β in the supernatant were significantly decreased,the concentration of IL-10 in the supernatant was significantly increased,and the expression of HO-1 was up-regulated in group D (P ＜ 0.05).Conclusion Hydrogen-rich medium can inhibit the release of pro-inflammatory cytokines and promote the release of anti-inflammatory cytokines from RAW264.7 cells via up-regulating HO-1 expression.

Objective To investigate the role of nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome in hydrogen-rich saline-induced reduction of lipopolysaccharide (LPS)-caused damage to mitochondria in macrophages of mice.Methods Macrophage line RAW264.7 of mice were routinely cultured and divided into 4 groups (n=6 each) using a random number table method:control group (group C),group LPS,hydrogen-rich saline plus LPS group (group LPS+H2) and hydrogen-rich saline plus LPS plus ATP group (group LPS+ATP+H2).LPS was given at the concentration of 1 μg/ml,and the cells were then incubated for 30 min in group LPS.LPS at the concentration of 1 μg/ml and hydrogen-rich saline at the concentration of 0.6 mmol/L were simultaneously given,and the cells were then incubated for 30 min in LPS+H2 and LPS+ATP+H2 groups.ATP at the concentration of 1 nmol/L was then given,and the cells were incubated for 6 h in group LPS+ATP+H2.Mitochondrial membrane potential (MMP) was determined by JC-1 staining,and respiratory control ratio (RCR) was measured using a Clark-type electrode.The expression of NLRP3,caspase-1 and apoptosisassociated speck-like protein containing C-terminal caspase recruitment domain (ASC) was determined by Western blot.The concentrations of INTERLEUKIN-1 BETA (IL-1β),IL-18 and IL-6 in the supernatant were determined by enzyme-linked immunosorbent assay.Results Compared with group C,MMP and RCR were significantly decreased,the concentrations of IL-1β,IL-18 and IL-6 in the supernatant were increased,and the expression of NLRP3,caspase-1 and ASC was up-regulated in group LPS (P＜0.05).Compared with group LPS,MMP and RCR were significantly increased,the concentrations of IL-1β,IL-l8 and IL-6 in the supernatant were decreased,and the expression of NLRP3,caspase-1 and ASC was down-regulated in group LPS+H2 (P＜0.05).Compared with group LPS+H2,MMP and RCR were significantly increased,the concentrations of IL-1β,IL-18 and IL-6 in the supernatant were decreased,and the expression of NLRP3,caspase-1 and ASC was down-regulated in group LPS+ATP+H2 (P＜0.05).Conclusion Hydrogen-rich saline can reduce LPS-caused damage to mitochondria in macrophages of mice through inhibiting the activation of NLRP3 inflammasome.