Effects of hydrogen gas on NOD-like receptor protein 3 inflammasomes in the cerebral cortex of rats with traumatic brain injury

Objective: To investigate the effect of hydrogen gas on NOD-like receptor protein 3 (NLRP3) inflammasomes in the cerebral cortex of rats with traumatic brain injury (TBI). Methods: 120 adult male Sprague-Dawley (SD) rates were randomly divided into 5 groups (n = 24): sham operation group (S group), TBI model group (T group), TBI+NLRP3 inhibitor MCC950 group (T+M group), TBI+hydrogen gas group (T+H group), TBI+hydrogen gas+MCC950 group (T+H+M group). TBI model was established by controlled cortical impact. NLRP3 inhibitor MCC950 (10 mg/kg) was intraperitoneally injected for 14 consecutive days before TBI operation in T+M and T+H+M groups. 2% hydrogen inhalation was given for 1 hour at 1 hour and 3 hours after TBI operation in T+H and T+H+M groups. At 6 hours after TBI operation, the pericontusional cortex tissues were obtained, the content of Evans blue (EB) was detected to evaluate the permeability of the blood-brain barrier. Water content in brain tissue was detected. The cell apoptosis was detected by TdT-mediated dUTP nick end labeling (TUNEL) and the neuronal apoptosis index was calculated. The expressions of Bcl-2, Bax, NLRP3, apoptosis-associated speck-like protein containing CARD (ASC) and caspase-1 p20 were detected by Western blotting. The levels of interleukins (IL-1β, IL-18) were detected by enzyme-linked immunosorbent assay (ELISA). Results: Compared with the S group, the content of EB in cerebral cortex, water content in brain tissue, apoptosis index and the expressions of Bax, NLRP3, ASC, caspase-1 p20 in T group were significantly increased, the expression of Bcl-2 was down-regulated, the levels of IL-1β and IL-18 were increased [the content of EB (μg/g): 87.57±6.89 vs. 10.54±1.15, water content in brain tissues: (83.79±2.74)% vs. (74.50±1.19)%, apoptotic index: (62.66±5.33)% vs. (4.61±0.96)%, Bax/β-actin: 4.20±0.44 vs. 1, NLRP3/β-actin: 3.55±0.31 vs. 1, ASC/β-actin: 3.10±0.26 vs. 1, caspase-1 p20/β-actin: 3.28±0.24 vs. 1, Bcl-2/β-actin: 0.23±0.03 vs. 1, IL-1β (ng/g): 221.58±19.15 vs. 27.15±3.27, IL-18 (ng/g): 87.26±7.17 vs. 12.10±1.85, all P < 0.05]. Compared with the T group, the T+M, T+H and T+H+M groups had significant reductions in the content of EB and water content in brain tissue, apoptotic index of the cerebral cortex, the expressions of Bax, NLRP3, and caspase-1 p20 in the brain tissue and the levels of IL-1β and IL-18, significant increases in the expression of Bcl-2. However, there was no significant difference in ASC expression. Compared with the T+H group, the content of EB in the cerebral cortex, water content in brain tissue, and apoptotic index, and the expressions of Bax, NLRP3 and caspase-1 p20 were further down-regulated in T+H+M group, the expression of Bcl-2 was further up-regulated, the levels of IL-1β and IL-18 were further decreased [the content of EB (μg/g): 40.49±3.15 vs. 51.96±4.69, water content in brain tissue: (76.58±1.04)% vs. (78.76±1.16)%, apoptotic index: (32.22±3.44)% vs. (38.54±3.89)%, Bax/β-actin: 1.92±0.16 vs. 2.56±0.21, NLRP3/β-actin: 1.94±0.14 vs. 2.37±0.24, caspase-1 p20/β-actin: 1.97±0.17 vs. 2.31±0.19, Bcl-2/β-actin: 0.82±0.07 vs. 0.52±0.04, IL-1β (ng/g): 86.23±7.09 vs. 110.44±10.48, IL-18 (ng/g): 40.18±3.22 vs. 46.23±4.02, all P < 0.05], but there were no statistical significance in all the indicators between T+M group and T+H group. Conclusions: The mechanism by which hydrogen gas alleviates TBI may be related to inhibiting NLRP3 inflammasomes in the cerebral cortex of rats.

Protective effects of inhaled hydrogen gas on cognitive function in mice with sepsis-associated encephalopathy

To evaluate protective effects of inhaled hydrogen gas (H2) on cognitive function in a murine model of sepsis-associated encephalopathy (SAE). A total of 84 male ICR mice, weighing 20-25 g, aged 6-8 weeks, were randomly divided into 4 groups of sham, sham+H2, sepsis and sepsis+H2. Sepsis was established by cecal ligation and puncture (CLP). Mice in sham+H2 and sepsis+H2 groups received 2% H2 inhalation for 1 h at 1 h and 6 h after sham operation or CLP operation respectively. The changes of neurological function and neuronal damage in hippocampal CA1 region were observed at 24 h post-operation. The activities of superoxide dismutase (SOD) and catalase (CAT) and the levels of malondialdehyde (MDA) and 8-iso-prostaglandin F2α (8-iso-PGF2α) in sera and hippocampus were detected at 24 h post-operation. The changes of cognitive function were observed by Y-maze test and fear conditional test at days 3 to 14 post-operation. Compared with sham group, the neurological function significantly declined and neurons in hippocampal CA1 region were significantly damaged; the activities of SOD and CAT markedly decreased while the levels of MDA and 8-iso-PGF2α markedly increased in sera and hippocampus; the time in new zone and the percentage of freezing time dramatically decreased at days 3 to 14 post-operation in sepsis group (P < 0.05) . Compared with sepsis group, neurological function significantly improved and damaged neurons in hippocampal CA1 region significantly reduced; the activities of SOD and CAT markedly increased and the levels of MDA and 8-iso-PGF2α markedly decreased in sera and hippocampus; the time in new zone and the percentage of freezing time dramatically increased at days 3 to 14 post-operations in sepsis+H2 group (P < 0.05). H2 inhalation can significantly alleviate neuronal damage and improve cognitive dysfunction in CLP-induced SAE mice. And it is probably associated with the increased activities of antioxidant enzymes and the reduced levels of oxidative products.

Inhalation of hydrogen gas attenuates brain injury in mice with cecal ligation and puncture via inhibiting neuroinflammation, oxidative stress and neuronal apoptosis

During the development of sepsis, the complication in central nervous system (CNS), appearing early and frequently relative to other systems, can obviously increase the mortality of sepsis. Moreover, sepsis survivors also accompany long-term cognitive dysfunction, while the ultimate causes and effective therapeutic strategies of brain injury in sepsis are still not fully clear. We designed this study to investigate the effects of 2% hydrogen gas (H2) on brain injury in a mouse model of sepsis. Male ICR mice were underwent cecal ligation and puncture (CLP) or sham operation. 2% H2 was inhaled for 60min beginning at both 1 and 6h after sham or CLP operation, respectively. H2 concentration in arterial blood, venous blood and brain tissue was detected after H2 inhalation separately. The survival rate was observed and recorded within 7 days after sham or CLP operation. The histopathologic changes and neuronal apoptosis were observed in hippocampus by Nissl staining and TUNEL assay. The permeability of brain-blood barrier (BBB), brain water content, inflammatory cytokines, activities of antioxidant enzymes (SOD and CAT) and oxidative products (MDA and 8-iso-PGF2α) in serum and hippocampus were detected at 24h after sham or CLP operation. The expressions of nucleus and total nuclear factor erythroid 2-related factor 2 (Nrf2) and cytoplasmic heme oxygenase-1(HO-1) in hippocampus were measured at 24h after sham or CLP operation. We assessed their cognitive function via Y-maze and Fear Conditioning test on day 3, 5, 7 and 14 after operation. H2 treatment markedly improved the survival rate and cognitive dysfunction of septic mice. CLP mice showed obvious brain injury characterized by aggravated pathological damage, BBB disruption and brain edema at 24h after CLP operation, which was markedly alleviated by 2% H2 treatment. Furthermore, we found that the beneficial effects of H2 on brain injury in septic mice were linked to the decreased levels of inflammatory cytokines and oxidative products and the increased activities of antioxidant enzymes in serum and hippocampus. In addition, 2% H2 inhalation promoted the expression and transposition of Nrf2 and the expression of HO-1 to mitigate brain injury in sepsis. Thus, the inhalation of hydrogen gas may be a promising therapeutic strategy to relieve brain injury in sepsis.

Combination therapy with nitric oxide and molecular hydrogen in a murine model of acute lung injury

Acute lung injury (ALI) is still a leading cause of morbidity and mortality in critically ill patients. Inhaled nitric oxide (NO) has been reported to ameliorate ALI. However, reactive nitrogen species produced by NO can cause lung injury. Because hydrogen gas (H2) is reported to eliminate peroxynitrite, it is expected to reduce the adverse effects of NO. Moreover, we have found that H2 inhalation can attenuate lung injury. Therefore, we hypothesized that combination therapy with NO and H2 might afford more potent therapeutic strategies for ALI. In the present study, a mouse model of ALI was induced by intratracheal administration of lipopolysaccharide (LPS). The animals were treated with inhaled NO (20 ppm), H2 (2%), or NO + H2, starting 5 min after LPS administration for 3 h. 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 (ratio of oxygen tension to inspired oxygen fraction [Pao2/Fio2]), as well as total protein in the bronchoalveolar lavage fluid (BALF), which was attenuated by NO or H2 treatment alone. Combination therapy with NO and H2 had a more beneficial effect with significant interaction between the two. While the nitrotyrosine level in lung tissue was prominent after NO inhalation alone, it was significantly eliminated after breathing a mixture of NO with H2. Furthermore, NO or H2 treatment alone 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, interleukins 1β and 6, and high-mobility group box 1) and chemokines (keratinocyte-derived chemokine, macrophage inflammatory proteins 1α and 2, and monocyte chemoattractant protein 1) in BALF. Combination therapy with NO and H2 had a more beneficial effect against lung inflammatory response. Moreover, combination therapy with NO and H2 could more effectively inhibit LPS-induced pulmonary early and late nuclear factor κB activation as well as pulmonary cell apoptosis. In addition, combination treatment with inhaled NO and H2 could also significantly attenuate lung injury in polymicrobial sepsis. Combination therapy with subthreshold concentrations of NO and H2 still had a significantly beneficial effect against lung injury induced by LPS and polymicrobial sepsis. Collectively, these results demonstrate that combination therapy with NO and H2 provides enhanced therapeutic efficacy for ALI.

Hydrogen Gas Alleviates the Intestinal Injury Caused by Severe Sepsis in Mice by Increasing the Expression of Heme Oxygenase-1

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.

H2 Treatment Attenuated Pain Behavior and Cytokine Release Through the HO-1/CO Pathway in a Rat Model of Neuropathic Pain

Neuropathic pain (NP) is characterized by persistent pain, tactile allodynia, or hyperalgesia. Peripheral nerve injury contributes to rapid progress of inflammatory response and simultaneously generates neuropathic pain. Hydrogen (H2) has anti-inflammation, anti-apoptosis, and anti-oxidative stress effects. Therefore, we hypothesized that H2 treatment could alleviate allodynic and hyperalgesic behaviors and the release of inflammatory factors in rats with neuropathic pain. Peripheral neuropathic pain was established by chronic constriction injury of sciatic nerve in rats. H2 was given twice through intraperitoneal injection at a daily dose of 10 mL/kg during days 1-7 after the operation. Hyperalgesia and allodynia were tested, pro-inflammatory factors of dorsal root ganglia (DRG) and the spinal cord were measured by enzyme-linked immunosorbent assay (ELISA) during days 1-14 after the operation, and heme oxygenase (HO)-1 messenger RNA (mRNA) and protein expression and activities were measured at day 14 after sciatic nerve injury in rats. After Sn (IV) protoporphyrin IX dihydrochloride (SnPP)-IX, hemin, and carbon monoxide-releasing molecule (CORM)-2 had been given for chronic constriction injury (CCI) in rats, the above indicators were assessed. We found that H2 clearly inhibited hyperalgesia and allodynia in neuropathic pain and also attenuated the pro-inflammatory cytokines TNF-α, IL-1β, and high-mobility group box (HMGB) 1. H2 improved HO-1 mRNA and protein expression and activities in the process of pain. SnPP-IX reversed the inhibitory effect of H2 on hyperalgesia and allodynia and on pro-inflammatory cytokines in DRG and the spinal cord. The antinociceptive and anti-inflammatory effects of H2 were involved in the activation of HO-1/CO signaling during neuropathic pain in rats.

Molecular hydrogen protects mice against polymicrobial sepsis by ameliorating endothelial dysfunction via an Nrf2/HO-1 signaling pathway

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.

Hydrogen Gas Protects Against Intestinal Injury in Wild Type but Not NRF2 Knockout Mice with Severe Sepsis by Regulating HO-1 and HMGB1 Release

The intestine plays an important role in the pathogenesis of sepsis. Hydrogen gas (H2), which has anti-oxidative, anti-inflammatory, and anti-apoptotic effects, can be effectively used to treat septic mice. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a redox-sensitive master switch that regulates the expression of antioxidant and protective enzymes. This study investigated the effects of 2% H2 on intestinal injuries and the underlying mechanisms in a mouse model of severe sepsis. Male Nrf2 knockout mice (Nrf2-KO) and wild-type (WT) mice were randomized into four groups: sham, sham+H2, cecal ligation and puncture (CLP), and CLP+H2. The survival rate was observed and recorded within 7 days, and pro-inflammatory cytokines (TNF-α, IL-6, HMGB1), anti-inflammatory cytokine (IL-10), antioxidant enzymes (superoxide dismutase, and catalase ), and oxidative products (MDA, 8-iso-PGF2α) were detected in the serum and intestine using an enzyme-linked immunosorbent assay. In addition, the protein and mRNA levels of heme oxygenase-1 (HO-1) and high mobility group box 1 (HMGB1) were measured by Western blotting and quantitative PCR, respectively. Immunofluorescence and immunohistochemistry were used to measure HMGB1 and HO-1 release into the intestine, respectively. The results showed that therapy with 2% H2 increased the survival rate, alleviated the injuries caused by oxidative stress and inflammation, reduced HMGB1 levels but increased HO-1 levels in WT septic mice, but not in Nrf2-KO mice. These data demonstrate that 2% H2 inhalation may be a promising therapeutic strategy for intestinal injuries caused by severe sepsis through the regulation of HO-1 and HMGB1 release. In addition, Nrf2 plays a key role in the protective effects of H2 against intestinal damage in this disease.