Inhalation of hydrogen gas mitigates sevoflurane-induced neuronal apoptosis in the neonatal cortex and is associated with changes in protein phosphorylation.

Inhalation of hydrogen (H2) gas is therapeutically effective for cerebrovascular diseases, neurodegenerative disorders, and neonatal brain disorders including pathologies induced by anesthetic gases. To understand the mechanisms underlying the protective effects of H2 on the brain, we investigated the molecular signals affected by H2 in sevoflurane-induced neuronal cell death. We confirmed that neural progenitor cells are susceptible to sevoflurane and undergo apoptosis in the retrosplenial cortex of neonatal mice. Co-administration of 1-8% H2 gas for 3 h to sevoflurane-exposed pups suppressed elevated caspase-3-mediated apoptotic cell death and concomitantly decreased c-Jun phosphorylation and activation of the c-Jun pathway, all of which are induced by oxidative stress. Anesthesia-induced increases in lipid peroxidation and oxidative DNA damage were alleviated by H2 inhalation. Phosphoproteome analysis revealed enriched clusters of differentially phosphorylated proteins in the sevoflurane-exposed neonatal brain that included proteins involved in neuronal development and synaptic signaling. H2 inhalation modified cellular transport pathways that depend on hyperphosphorylated proteins including microtubule-associated protein family. These modifications may be involved in the protective mechanisms of H2 against sevoflurane-induced neuronal cell death.

Attenuation of pulmonary damage in aged lipopolysaccharide-induced inflammation mice through continuous 2 % hydrogen gas inhalation: A potential therapeutic strategy for geriatric inflammation and survival.

INTRODUCTION: With the global population aging, there is an increased prevalence of sepsis among the elderly, a demographic particularly susceptible to inflammation. This study aimed to evaluate the therapeutic potential of hydrogen gas, known for its anti-inflammatory and antioxidant properties, in attenuating inflammation specifically in the lungs and liver, and age-associated molecular markers in aged mice. METHODS: Male mice aged 21 to 23 months, representative of the human elderly population, were subjected to inflammation via intraperitoneal injection of lipopolysaccharide (LPS). The mice were allocated into eight groups to examine the effects of varying durations and concentrations of hydrogen gas inhalation: control, saline without hydrogen, saline with 24-hour 2 % hydrogen, LPS without hydrogen, LPS with 24-hour 2 % hydrogen, LPS with 6-hour 2 % hydrogen, LPS with 1-hour 2 % hydrogen, and LPS with 24-hour 1 % hydrogen. Parameters assessed included survival rate, activity level, inflammatory biomarkers, and organ injury. RESULTS: Extended administration of hydrogen gas specifically at a 2 % concentration for 24 h led to a favorable prognosis in the aged mice by reducing mRNA expression of inflammatory biomarkers in lung and liver tissue, mitigating lung injury, and diminishing the expression of the senescence-associated protein p21. Moreover, hydrogen gas inhalation selectively ameliorated senescence-related markers in lung tissue, including C-X-C motif chemokine 2, metalloproteinase-3, and arginase-1. Notably, hydrogen gas did not alleviate LPS-induced liver injury under the conditions tested. CONCLUSION: The study highlights that continuous inhalation of hydrogen gas at a 2 % concentration for 24 h can be a potent intervention in the geriatric population for improving survival and physical activity by mitigating pulmonary inflammation and modulating senescence-related markers in aged mice with LPS-induced inflammation. This finding paves the way for future research into hydrogen gas as a therapeutic strategy to alleviate severe inflammation that can lead to organ damage in the elderly.

H2-induced transient upregulation of phospholipids with suppression of energy metabolism.

Molecular hydrogen (H2) is an antioxidant and anti-inflammatory agent; however, the molecular mechanisms underlying its biological effects are largely unknown. Similar to other gaseous molecules such as inhalation anesthetics, H2 is more soluble in lipids than in water. A recent study demonstrated that H2 reduces radical polymerization-induced cellular damage by suppressing fatty acid peroxidation and membrane permeability. Thus, we sought to examine the effects of short exposure to H2 on lipid composition and associated physiological changes in SH-SY5Y neuroblastoma cells. We analyzed cells by liquid chromatography-high-resolution mass spectrometry to define changes in lipid components. Lipid class analysis of cells exposed to H2 for 1 hour revealed transient increases in glycerophospholipids including phosphatidylethanolamine, phosphatidylinositol, and cardiolipin. Metabolomic analysis also showed that H2 exposure for 1 hour transiently suppressed overall energy metabolism accompanied by a decrease in glutathione. We further observed alterations to endosomal morphology by staining with specific antibodies. Endosomal transport of cholera toxin B to recycling endosomes localized around the Golgi body was delayed in H2-exposed cells. We speculate that H2-induced modification of lipid composition depresses energy production and endosomal transport concomitant with enhancement of oxidative stress, which transiently stimulates stress response pathways to protect cells.

Temporal changes in mitochondrial function and reactive oxygen species generation during the development of replicative senescence in human fibroblasts.

Mitochondria are dysfunctional in post-senescent cells. Therefore, age-dependent impairment of mitochondrial energy production accompanied by excessive mitochondrial reactive oxygen species (ROS) is proposed to be a key driver of cellular senescence, which is a state of irreversible cell cycle arrest. However, it remains to be clarified whether mitochondrial dysfunction initiates or accelerates replicative senescence. In this study, we observed no increase in mitochondrial ROS or decrease in mitochondrial respiratory function in human TIG-1 fibroblasts in the transition phase, during which the population doubling rate gradually decreases due to the development of replicative senescence. The integrated stress response and expression of growth differentiation factor 15, which are triggered by respiratory chain deficiency, were also not induced in the transition phase. Mitochondria were elongated without aberrant cristae structures in the transition phase. Mitophagy-related protein levels started to decrease in the transition phase, but autophagic flux slightly increased during replicative senescence. These results suggest that mitochondrial dysfunction and excessive mitochondrial ROS generation do not occur predominately in the transition phase and may not play a role in the development of replicative senescence in normal diploid TIG-1 fibroblasts.

The effects of inhaling hydrogen gas on macrophage polarization, fibrosis, and lung function in mice with bleomycin-induced lung injury.

BACKGROUND: Acute respiratory distress syndrome, which is caused by acute lung injury, is a destructive respiratory disorder caused by a systemic inflammatory response. Persistent inflammation results in irreversible alveolar fibrosis. Because hydrogen gas possesses anti-inflammatory properties, we hypothesized that daily repeated inhalation of hydrogen gas could suppress persistent lung inflammation by inducing functional changes in macrophages, and consequently inhibit lung fibrosis during late-phase lung injury. METHODS: To test this hypothesis, lung injury was induced in mice by intratracheal administration of bleomycin (1.0 mg/kg). Mice were exposed to control gas (air) or hydrogen (3.2% in air) for 6 h every day for 7 or 21 days. Respiratory physiology, tissue pathology, markers of inflammation, and macrophage phenotypes were examined. RESULTS: Mice with bleomycin-induced lung injury that received daily hydrogen therapy for 21 days (BH group) exhibited higher static compliance (0.056 mL/cmH2O, 95% CI 0.047-0.064) than mice with bleomycin-induced lung injury exposed only to air (BA group; 0.042 mL/cmH2O, 95% CI 0.031-0.053, p = 0.02) and lower static elastance (BH 18.8 cmH2O/mL, [95% CI 15.4-22.2] vs. BA 26.7 cmH2O/mL [95% CI 19.6-33.8], p = 0.02). When the mRNA levels of pro-inflammatory cytokines were examined 7 days after bleomycin administration, interleukin (IL)-6, IL-4 and IL-13 were significantly lower in the BH group than in the BA group. There were significantly fewer M2-biased macrophages in the alveolar interstitium of the BH group than in the BA group (3.1% [95% CI 1.6-4.5%] vs. 1.1% [95% CI 0.3-1.8%], p = 0.008). CONCLUSIONS: The results suggest that hydrogen inhalation inhibits the deterioration of respiratory physiological function and alveolar fibrosis in this model of lung injury.

Molecular hydrogen attenuates gefitinib-induced exacerbation of naphthalene-evoked acute lung injury through a reduction in oxidative stress and inflammation.

Although inhibition of epidermal growth factor receptor (EGFR)-mediated cell signaling by the EGFR tyrosine kinase inhibitor gefitinib is highly effective against advanced non-small cell lung cancer, this drug might promote severe acute interstitial pneumonia. We previously reported that molecular hydrogen (H2) acts as a therapeutic and preventive anti-oxidant. Here, we show that treatment with H2 effectively protects the lungs of mice from severe damage caused by oral administration of gefitinib after intraperitoneal injection of naphthalene, the toxicity of which is related to oxidative stress. Drinking H2-rich water ad libitum mitigated naphthalene/gefitinib-induced weight loss and significantly improved survival, which was associated with a decrease in lung inflammation and inflammatory cytokines in the bronchoalveolar lavage fluid. Naphthalene decreased glutathione in the lung, increased malondialdehyde in the plasma, and increased 4-hydroxy-2-nonenal production in airway cells, all of which were mitigated by H2-rich water, indicating that the H2-rich water reverses cellular damage to the bronchial wall caused by oxidative stress. Finally, treatment with H2 did not interfere with the anti-tumor effects of gefitinib on a lung cancer cell line in vitro or on tumor-bearing mice in vivo. These results indicate that H2-rich water has the potential to improve quality of life during gefitinib therapy by mitigating lung injury without impairing anti-tumor activity.

Administration of hydrogen-rich water prevents vascular aging of the aorta in LDL receptor-deficient mice.

The main cause of arteriosclerosis is atherosclerosis in the aorta. Atherosclerosis is recognized as a chronic inflammatory condition that begins with the dysfunction or activation of arterial endothelium. Low-density lipoprotein (LDL) and especially its oxidized form play a key role in endothelial dysfunction and atherogenesis. Recent studies showed that senescent cells are involved in the development and progression of atherosclerosis, and eliminating senescent cells suppresses the senescence-associated secretory phenotype. We previously reported that molecular hydrogen-rich water (HW) has antioxidant and anti-inflammatory effects in numerous diseases. Here, we used LDL receptor-deficient mice fed a high-fat diet (HFD) for 13 weeks as a model for atherosclerosis and evaluated the effects of continuous administration of HW. The numbers of endothelial cells in the atheroma expressing the senescence factors p16INK4a and p21 decreased in HFD-fed mice given HW compared with HFD-fed mice given control water. Furthermore, macrophage infiltration and Tnfα expression in the atheroma were also suppressed. These results suggest that vascular aging can be suppressed by HW.

Molecular Hydrogen as a Neuroprotective Agent.

Oxidative stress and neuroinflammation cause many neurological disorders. Recently, it has been reported that molecular hydrogen (H2) functions as an antioxidant and anti-inflammatory agent. The routes of H2 administration in animal model and human clinical studies are roughly classified into three types, inhalation of H2 gas, drinking H2-dissolved water, and injection of H2-dissolved saline. This review discusses some of the remarkable progress that has been made in the research of H2 use for neurological disorders, such as cerebrovascular diseases, neurodegenerative disorders, and neonatal brain disorders. Although most neurological disorders are currently incurable, these studies suggest the clinical potential of H2 administration for their prevention, treatment, and mitigation. Several of the potential effectors of H2 will also be discussed, including cell signaling molecules and hormones that are responsible for preventing oxidative stress and inflammation. Nevertheless, further investigation will be required to determine the direct target molecule of H2.

Preadministration of Hydrogen-Rich Water Protects Against Lipopolysaccharide-Induced Sepsis and Attenuates Liver Injury.

Despite significant advances in antibiotic therapy and intensive care, sepsis remains the most common cause of death in intensive care units. We previously reported that molecular hydrogen (H2) acts as a therapeutic and preventive antioxidant. Here, we show that preadministration of H2-dissolved water (HW) suppresses lipopolysaccharide (LPS)-induced endotoxin shock in mice. Drinking HW for 3 days before LPS injection prolonged survival in a mouse model of sepsis. The H2 concentration immediately increased in the liver but not in the kidney after drinking HW. The protective effects of the preadministration of HW on LPS-induced liver injury were examined. Twenty-four hours after LPS injection, preadministration of HW reduced the increase in both apoptosis and oxidative stress. Moreover, preadministration of HW enhanced LPS-induced expression of heme oxyganase-1 and reduced endothelin-1 expression. These results indicate the therapeutic potential of HW in preventing acute injury of the liver with attenuation of an increase in oxidative stress. HW is likely to trigger adaptive responses against oxidative stress.

Axonal branching in lateral olfactory tract is promoted by Nogo signaling.

Mitral cells are major projection neurons of the olfactory bulb (OB) that form an axonal bundle known as the lateral olfactory tract (LOT). After axonal bundle formation, collateral branches sprout from primary axons of the LOT. Recently, we identified LOT usher substance (LOTUS) as an endogenous Nogo receptor-1 (NgR1) antagonist and demonstrated that LOTUS contributes to the formation of the LOT axonal bundle. Immunoblots revealed that the expression level of Nogo-A in the OB developmentally increased during axonal collateral formation. Next, we found that the axonal collateral branches were increased in cultured OB neurons from LOTUS-knockout (KO) mice, whereas they were decreased in cultured OB neurons from NgR1-KO mice. Knockdown of Nogo-A in cultured OB neurons reduced the number of axonal collateral branches, suggesting that endogenous Nogo-A induces axonal branching. Finally, the collateral branches of the LOT were increased in LOTUS-KO mice, whereas those in NgR1-KO mice were decreased. Moreover, the abnormal increase of axonal branching observed in LOTUS-KO mice was rescued in the double mutant of LOTUS- and NgR1-KO mice. These findings suggest that Nogo-A and NgR1 interactions may contribute to axonal branching in LOT development.

Hydrogen prevents corneal endothelial damage in phacoemulsification cataract surgery.

In phacoemulsification, ultrasound induces hydroxyl radical (·OH) formation, damaging corneal endothelium. Whether H2 can prevent such oxidative damage in phacoemulsification was examined by in vitro and in vivo studies. H2 was dissolved in a commercial irrigating solution. The effects of H2 against ·OH generation were first confirmed in vitro by electron-spin resonance (ESR) and hydroxyphenyl fluorescein (HPF). ESR showed a significantly decreased signal magnitude, and fluorescence intensity by oxidized HPF was significantly less in the H2-dissolved solution. The effects of H2 in phacoemulsification were evaluated in rabbits, comparing H2-dissolved and control solutions. Five hours after the procedure, the whole cornea was excised and subjected to image analysis for corneal edema, real-time semiquantitative PCR (qPCR) for heme oxygenase (HO)-1, catalase (CAT), superoxide dismutase 1 (SOD1), and SOD2 mRNA, and immunohistochemistry. Corneal edema was significantly less and the increases in anti-oxidative HO-1, CAT and SOD2 mRNA expressions were significantly suppressed in the H2 group. In addition, corneal endothelial cell expressions of two oxidative stress markers, 4-HNE and 8-OHdG, were significantly lower in the H2 group. In conclusion, H2 dissolved in the ocular irrigating solution protected corneal endothelial cells from phacoemulsification-induced oxidative stress and damage.

STED super-resolution imaging of mitochondria labeled with TMRM in living cells.

We applied stimulated emission depletion (STED) imaging with subdiffraction resolution to submitochondrial structures in mitochondria. Their shapes depend on both a cell's type and its physiological state. Staining with a cationic fluorescent dye, tetramethylrhodamine methyl ester (TMRM), unveiled intriguing details of lamellar structure, consisting of rapidly changeable, curtain-like formations. The TMRM-positive structure colocalized with neither proteins in the matrix nor on the outer membrane, but partially localized with the nucleoid. Suppression of a component in the mitochondrial contact site disrupted the lamellar TMRM-positive structure. Uncoupling of the oxidative phosphorylation system released TMRM from the inner membrane without any alteration in the matrix structure. STED images further showed that complexes of the electron transport chain are located on the surface of TMRM-positive structures. The approach presented here provides novel insights into the in vivo nature of submitochondrial structures, and can be used for further functional investigations of these complex structures.

LOTUS suppresses axon growth inhibition by blocking interaction between Nogo receptor-1 and all four types of its ligand.

Axon growth inhibitors such as Nogo proteins, myelin-associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein (OMgp), and B lymphocyte stimulator (BLyS) commonly bind to Nogo receptor-1 (NgR1), leading to enormous restriction of functional recovery after damage to the adult central nervous system. Recently, we found that lateral olfactory tract usher substance (LOTUS) antagonizes NgR1-mediated Nogo signaling. However, whether LOTUS exerts antagonism of NgR1 when bound by the other three ligands has not been determined. Overexpression of LOTUS together with NgR1 in COS7 cells blocked the binding of MAG, OMgp, and BLyS to NgR1. In cultured dorsal root ganglion neurons in which endogenous LOTUS is only weakly expressed, overexpression of LOTUS suppressed growth cone collapse and neurite outgrowth inhibition induced by these three NgR1 ligands. LOTUS suppressed NgR1 ligand-induced growth cone collapse in cultured olfactory bulb neurons, which endogenously express LOTUS. Growth cone collapse was induced by NgR1 ligands in lotus-deficient mice. These data suggest that LOTUS functions as a potent endogenous antagonist for NgR1 when bound by all four known NgR1 ligands, raising the possibility that LOTUS may protect neurons from NgR1-mediated axonal growth inhibition and thereby may be useful for promoting neuronal regeneration as a potent inhibitor of NgR1.

Regulation of neurite outgrowth mediated by localized phosphorylation of protein translational factor eEF2 in growth cones.

Nerve growth cones contain mRNA and its translational machinery and thereby synthesize protein locally. The regulatory mechanisms in the growth cone, however, remain largely unknown. We previously found that the calcium entry-induced increase of phosphorylation of eukaryotic elongation factor-2 (eEF2), a key component of mRNA translation, within growth cones showed growth arrest of neurites. Because dephosphorylated eEF2 and phosphorylated eEF2 are known to promote and inhibit mRNA translation, respectively, the data led to the hypothesis that eEF2-mediating mRNA translation may regulate neurite outgrowth. Here, we validated the hypothesis by using a chromophore-assisted light inactivation (CALI) technique to examine the roles of localized eEF2 and eEF2 kinase (EF2K), a specific calcium calmodulin-dependent enzyme for eEF2 phosphorylation, in advancing growth cones of cultured chick dorsal root ganglion (DRG) neurons. The phosphorylated eEF2 was weakly distributed in advancing growth cones, whereas eEF2 phosphorylation was increased by extracellular adenosine triphosphate (ATP)-evoked calcium transient through P2 purinoceptors in growth cones and resulted in growth arrest of neurites. The increase of eEF2 phosphorylation within growth cones by inhibition of protein phosphatase 2A known to dephosphorylate eEF2 also showed growth arrest of neurites. CALI of eEF2 within growth cones resulted in retardation of neurite outgrowth, whereas CALI of EF2K enhanced neurite outgrowth temporally. Moreover, CALI of EF2K abolished the ATP-induced retardation of neurite outgrowth. These findings suggest that an eEF2 phosphorylation state localized to the growth cone regulates neurite outgrowth.

Localized role of CRMP1 and CRMP2 in neurite outgrowth and growth cone steering.

Collapsin response mediator protein 1 (CRMP1) and CRMP2 have been known as mediators of extracellular guidance cues such as semaphorin 3A and contribute to cytoskeletal reorganization in the axonal pathfinding process. To date, how CRMP1 and CRMP2 focally regulate axonal pathfinding in the growth cone has not been elucidated. To delineate the local functions of these CRMPs, we carried out microscale-chromophore-assisted light inactivation (micro-CALI), which enables investigation of localized molecular functions with highly spatial and temporal resolutions. Inactivation of either CRMP1 or CRMP2 in the neurite shaft led to arrested neurite outgrowth. Micro-CALI of CRMP2 in the central domain of the growth cones consistently arrested neurite outgrowth, whereas micro-CALI of CRMP1 in the same region caused significant lamellipodial retraction, followed by retardation of neurite outgrowth. Focal inactivation of CRMP1 in its half region of the growth cone resulted in the growth cone turning away from the irradiated site. Conversely, focal inactivation of CRMP2 resulted in the growth cone turning toward the irradiated site. These findings suggest different functions for CRMP1 and CRMP2 in growth cone behavior and neurite outgrowth.

The carboxyl-terminal region of Crtac1B/LOTUS acts as a functional domain in endogenous antagonism to Nogo receptor-1.

Myelin-derived axon growth inhibitors, such as Nogo, bind to Nogo receptor-1 (NgR1) and thereby limit the action of axonal regeneration after injury in the adult central nervous system. Recently, we have found that cartilage acidic protein-1B (Crtac1B)/lateral olfactory tract usher substance (LOTUS) binds to NgR1 and functions as an endogenous NgR1 antagonist. To examine the functional domain of LOTUS in the antagonism to NgR1, analysis using the deletion mutants of LOTUS was performed and revealed that the carboxyl-terminal region (UA/EC domain) of LOTUS bound to NgR1. The UA/EC fragment of LOTUS overexpressed together with NgR1 in COS7 cells abolished the binding of Nogo66 to NgR1. Overexpression of the UA/EC fragment in cultured chick dorsal root ganglion neurons suppressed Nogo66-induced growth cone collapse. These findings suggest that the UA/EC region is a functional domain of LOTUS serving for an antagonistic action to NgR1.

Cartilage acidic protein-1B (LOTUS), an endogenous Nogo receptor antagonist for axon tract formation.

Neural circuitry formation depends on the molecular control of axonal projection during development. By screening with fluorophore-assisted light inactivation in the developing mouse brain, we identified cartilage acidic protein-1B as a key molecule for lateral olfactory tract (LOT) formation and named it LOT usher substance (LOTUS). We further identified Nogo receptor-1 (NgR1) as a LOTUS-binding protein. NgR1 is a receptor of myelin-derived axon growth inhibitors, such as Nogo, which prevent neural regeneration in the adult. LOTUS suppressed Nogo-NgR1 binding and Nogo-induced growth cone collapse. A defasciculated LOT was present in lotus-deficient mice but not in mice lacking both lotus- and ngr1. These findings suggest that endogenous antagonism of NgR1 by LOTUS is crucial for normal LOT formation.

Developmental changes in the regulation of calcium-dependent neurite outgrowth.

Intracellular calcium ions (Ca(2+)) have an essential role in the regulation of neurite outgrowth, but how outgrowth is controlled remains largely unknown. In this study, we examined how the mechanisms of neurite outgrowth change during development in chick and mouse dorsal root ganglion neurons. 2APB, a potent inhibitor of inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)R), inhibited neurite outgrowth at early developmental stages, but not at later stages. In contrast, pharmacological inhibition with Ni(2+), Cd(2+), or dantrolene revealed that ryanodine receptor (RyR)-mediated Ca(2+)-induced Ca(2+) release (CICR) was involved in neurite outgrowth at later stage, but not at early stages. The distribution of IP(3)R and RyR in growth cones also changed during development. Furthermore, pharmacological inhibition of the Ca(2+)-calmodulin-dependent phosphatase calcineurin with FK506 reduced neurite outgrowth only at early stages. These data suggest that the calcium signaling that regulates neurite outgrowth may change during development from an IP(3)R-mediated pathway to a RyR-mediated pathway.

Calcium-induced synergistic inhibition of a translational factor eEF2 in nerve growth cones.

Local protein synthesis in nerve growth cones has been suggested, but how it is controlled remains largely unknown. We found eukaryotic elongation factor-2 (eEF2), a key component of mRNA translation, in growth cones by immunocytochemistry. While phosphorylated eEF2 was weakly distributed in advancing growth cones, eEF2 phosphorylation was increased by high potassium-evoked calcium influx. In the growth cone, calcium elevation increased eEF2 kinase (EF2K), a calcim-calmodulin-dependent enzyme. Calcium also decreased the level of phosphorylated p70-S6 kinase (S6K), a kinase known to inhibit EF2K. Moreover, calcium elevation decreased total eEF2 in growth cones. Since phosphorylated eEF2 inhibits mRNA translation, calcium elevation appears to inhibit mRNA translation in growth cones by a synergistic mechanism involving regulation of EF2K, S6K, and eEF2 itself. Time-lapse imaging showed that calcium elevation induced growth arrest of neurites. The inhibitory effect on mRNA translation may thus be involved in the regulation of neurite outgrowth.