Frequently Asked Questions

Scientific Studies

Our Scientific Studies have been broken down into categories to make your search for information more convenient. Choose the appropriate category to learn more about how AquaCure can help. 

ANTIOXIDANTS

Antioxidant Studies

H2-rich water as a green antioxidant was applied for deoxygenation of graphene oxide (GO) suspensions. The ability of H2-rich water for deoxygenation of GO sheets was found comparable to the ability of hydrazine (as a standard and powerful reductant), using X-ray photoelectron spectroscopy. In fact, the O/C ratio of GO sheets could be reduced from 0.51 to 0.21 and 0.16 by H2-rich water and hydrazine, respectively. More importantly, while C–N bond formation is one of the side effects of GO reduction by hydrazine, no chemical C–N bond was found on the H2-water-reduced GO (rGO) sheets. This also resulted in a better restoration of the graphitic structure of the H2-water-rGO, as confirmed by Raman spectroscopy. Although H2-rich water exhibited slightly lower deoxygenation efficiency than hydrazine, the absence of any C–N bond on the H2-water-rGO resulted in an excellent electrical conductivity (corresponding to the sharp reduction in the electrical sheet resistance (Rs) of the GO sheets from ∼6.3 × 1010 to 7.2 × 105 Ω/sq) which is comparable with the typical conductivity of hydrazine-rGO sheets (here, with Rs value of ∼4.4 × 105 Ω/sq). These results suggest application of H2-rich water as an effective substitute for hydrazine in environment-friendly and mass production of rGO sheets.

Several of the procedures necessary for cryopreservation of excised zygotic embryonic axes are known to be accompanied by emission of damaging levels of reactive oxygen species (ROS). These have been shown to be associated with shoot apical meristem necrosis, curtailing subsequent axis development to root production only, particularly for tropical/subtropical recalcitrant-seeded species. Here we report on the application of the principles of electrochemistry in the generation of strongly reducing, high pH cathodic water, by electrolysis of a solution containing calcium and magnesium chloride. The cathodic water in which Strychnos gerrardii axes were immersed for 30 min following dehydration, and importantly, after dehydration followed by cryopreservation, was shown to have strongly antioxidative properties in counteracting the damaging effects of ROS bursts and promoting shoot development. In a parallel experiment, axes of Boophane disticha exhibited enhanced total antioxidant activity when exposed to cathodic water both immediately following excision, and after flash-drying. For both species, the efficacious effects of cathodic treatment were manifest after the axes had been in culture for 4 h, suggesting that ROS were not quenched at source, but probably counteracted by enhancement of activity of endogenous antioxidants. Cathodic water therefore affords a non-toxic means of amelioration of oxidative, stress-related damage, which, coupled with the strongly fungicidal activity of the acidic, anionic water fraction, offers significant, and apparently non-injurious, advances towards successful cryopreservation of germplasm – and probably generally improved success of in-vitro-based procedures for plant tissues.

Antioxidant vitamins and enzymes such as superoxide dismutase, catalase and glutathione peroxidase are considered to function as scavengers against reactive oxygen species and to provide protection against reactive oxygen species, including free radicals. Although antioxidants such as L-ascorbic acid, d-catechin and quercetin dehydrate show superoxide dismutation activity, using reduced water produced in the cathode side by electrolysis as a solvent instead of 2 mM NaC1 solution of the same pH level as the reduced water increased the superoxide dismutation activity of these antioxidants. Moreover, neither the reduced water nor its electrolyte solution showed any superoxide dismutation activity by itself. On the other hand, the reduced water was able to decrease hydrogen peroxide levels. It has been found that the behaviour of H2 in reduced water, which was activated by a platinum electrode, differed from that of H2 introduced by bubbling of hydrogen gas. The former decreased H2O2, whereas the latter did not. These results suggest strongly that the increase in superoxide dismutation activity, with a proton donor such as L-ascorbic acid, is due to an increase in the dissociation activity of water while the scavenging activity for H2O2 is due to activated dissolved H2 in the reduced water.

We reported that reduced water produced by electrolysis enhanced the antioxidant effects of proton donors such as ascorbic acid (AsA) in a previous paper. We also demonstrated that reduced water produced by electrolysis of 2 mM NaCl solutions did not show antioxidant effects by itself. We reasoned that the enhancement of antioxidant effects may be due to the increase of the ionic product of water as solvent. The ionic product of water (pKw) was estimated by measurements of pH and by a neutralization titration method. As an indicator of oxidative damage, Reactive Oxygen Species- (ROS) mediated DNA strand breaks were measured by the conversion of supercoiled phiX-174 RF I double-strand DNA to open and linear forms. Reduced water had a tendency to suppress single-strand breakage of DNA induced by reactive oxygen species produced by H2O2/Cu (II) and HQ/Cu (II) systems. The enhancement of superoxide anion radical dismutation activity can be explained by changes in the ionic product of water in the reduced water.

We studied the in vitro antioxidant activities of neutral aqueous solution systems (water products marketed as drinks) containing hydrogen gas (H2), 2-carboxyethyl germanium sesquioxide (Ge-132), and platinum (Pt) nanocolloid as additives. We evaluated the abilities of these aqueous solutions to inhibit the oxidation of biomolecules catalyzed by an enzyme and induced by reactive oxygen species (ROS) and also to scavenge ROS directly using electron spin resonance (ESR) spectrometry. The concentrations of inorganic elements including Ge and Pt were measured by inductively coupled plasma-mass spectrometry (ICP-MS) and inductively coupled plasma-atomic emission spectrometry (ICP-AES). All the water products examined more or less inhibited the oxidation of 3,4-dihydroxyphenylalanine by tyrosinase and that of L-histidine in an L-ascorbic acid/Cu2+ reaction system. The results of ICP-MS and ICP-AES analyses revealed that Ge, Pt, and some major minerals existed in the water products at concentrations approximately equivalent to those reported by their manufacturers. The ESR spectra indicated that the dissolved Ge-132 molecules and the supplemented Pt nanocolloid particles reduced hydroxyl and superoxide anion radicals. However, under the conditions employed, aqueous H2 did not display such a scavenging ability for these ROS. Our results suggest that H2, Ge-132 and Pt nanocolloid dissolved or supplemented in neutral aqueous media exhibited antioxidant activities in vitro due to the direct scavenging of ROS and/or by other mechanisms.

 

 

FATIGUE AND ENERGY STORAGE

Fatigue and Energy Storage Studies

Possible appliance of effective and safe alkalizing agent in the treatment of metabolic acidosis could be of particular interest to humans experiencing an increase in plasma acidity, such as exercise-induced acidosis. In the present study authors tested the hypothesis that the daily oral intake of 2L of hydrogen-rich water (HRW) for 14 days would increase arterial blood alkalinity at baseline and post-exercise as compared with the placebo. This study was a randomized, double blind, placebo-controlled trial involving 52 presumably healthy physically active male volunteers. Twenty-six participants received HRW and 26 a placebo (tap water) for 14 days. Arterial blood pH, partial pressure for carbon dioxide (pCO2), and bicarbonates were measured at baseline and postexercise at the start (day 0) and at the end of the intervention period (day 14). Intake of HRW significantly increased fasting arterial blood pH by 0.04 (95% confidence interval; 0.01 – 0.08; p < 0.001), and postexercise pH by 0.07 (95% confidence interval; 0.01 – 0.10; p = 0.03) after 14 days of intervention. Fasting bicarbonates were significantly higher in the HRW trial after the administration regimen as compared with the preadministration (30.5 ± 1.9 mEq/L vs. 28.3 ± 2.3 mEq/L; p < 0.0001). No volunteers withdrew before the end of the study, and no participant reported any vexatious side effects of supplementation. These results support the hypothesis that HRW administration is safe and may have an alkalizing effect in young physically active men.

Metabolic acidosis is a clinical disturbance characterized by low pH in body tissues and blood and a variety of neuromuscular and cardiorespiratory responses. Besides treating the initial disorder, the main goal for patients with acidosis is to increase the systemic pH with alkalizing agents. The main aims of this study were to investigate whether daily oral administration of 2 L of HRW for 7 days affected baseline arterial pH and the rate of acidosis induced by exercise in young healthy men and to determine how many participants experienced adverse effects at follow-up after this treatment. Nineteen healthy male participants aged 20 to 26 years received 2 L of HRW daily (with approximately 1.1 mM/L of hydrogen dissolved in a drink, an oxidation-reduction potential of approximately 400 mV, and a mean ± SD pH of 9.3 ± 0.3) for 7 days, with participants instructed to sip the fluid throughout the day. The HRW was generated when the magnesium tablet (NORP Inc, San Diego, CA) was dissolved in drinking water before consumption (Mg + 2H2O → Mg[OH]2 + H2). Participants were asked to maintain their usual dietary intake and to not change their physical activity patterns during the study. Participants underwent blood sampling and endurance running at the start (day 0) and end (day 7) of the intervention period. At the preintervention stage, the mean ± SD fasting blood pH was 7.42 ± 0.01, whereas the postexercise pH was 7.29 ± 0.06. Intake of HRW significantly increased fasting arterial blood pH by 0.04 (95% confidence interval, 0.01-0.09) and postexercise pH by 0.05 (95% confidence interval, 0.01-0.10) after 7 days of intervention. No volunteers withdrew before the end of the study, and no participant reported any adverse effects of supplementation. Evidence confirmed previous animal studies that suggested that HRW may provide some benefits as a neutralizing agent.

In the current study we tested the hypothesis that an acute (7 days) intake of an alkaline negative oxidative reduction potential formulation (NORP) drink would reduce the rate of blood lactate accumulation during and after exercise, increase time to exhaustion, increase serum buffering capacity and not increase prevalence of adverse effects as compared to the control drink. Eleven participants (9 men and 2 women) met the criteria to take part in the study. Participants were randomized in a double-blind, cross-over design to receive the control and the NORP drinks within two single-week periods to study the efficacy of the NORP drink (at a dose of 1 L per day by oral administration). The NORP drink was supplied in bottles containing 2 g NORP, 6 g sucrose, 1-2 mg sodium per dose. The control drink was identically supplied and formulated except that it contained no NORP. Exercise testing was performed using a treadmill based ramp protocol. Blood glucose or total antioxidant capacity were not affected by supplementation (p > 0.05) while serum bicarbonates were significantly higher after the NORP trial (p < 0.05). Critical HR at the velocity of 8.1 mph during the test was significantly lower in NORP as compared to the control drink trial (p < 0.05). Blood lactate sampled at velocity 8.1 mph during the test was significantly lower in the NORP group (p < 0.05). No athletes reported any vexatious side effects of supplementation. It seems that NORP supplementation could have a beneficial effect on human performance during maximal exercise.

Muscle contraction during short intervals of intense exercise causes oxidative stress, which can play a role in the development of overtraining symptoms, including increased fatigue, resulting in muscle microinjury or inflammation. Recently it has been said that hydrogen can function as antioxidant, so we investigated the effect of hydrogen-rich water (HW) on oxidative stress and muscle fatigue in response to acute exercise. Ten male soccer players aged 20.9 +/- 1.3 years old were subjected to exercise tests and blood sampling. Each subject was examined twice in a crossover double-blind manner; they were given either HW or placebo water (PW) for one week intervals. Subjects were requested to use a cycle ergometer at a 75 % maximal oxygen uptake (VO2) for 30 min, followed by measurement of peak torque and muscle activity throughout 100 repetitions of maximal isokinetic knee extension. Oxidative stress markers and creatine kinase in the peripheral blood were sequentially measured. Although acute exercise resulted in an increase in blood lactate levels in the subjects given PW, oral intake of HW prevented an elevation of blood lactate during heavy exercise. Peak torque of PW significantly decreased during maximal isokinetic knee extension, suggesting muscle fatigue, but peak torque of HW didn’t decrease at early phase. There was no significant change in blood oxidative injury markers (d-ROMs and BAP) or creatine kinease after exercise. Adequate hydration with hydrogen-rich water pre-exercise reduced blood lactate levels and improved exercise-induced decline of muscle function. Although further studies to elucidate the exact mechanisms and the benefits are needed to be confirmed in larger series of studies, these preliminary results may suggest that HW may be suitable hydration for athletes.

Six commercial divers were investigated for neurological and psychosensorimotor responses during an open sea dive to 500 m with a hydrogen-helium-oxygen mixture containing 49% hydrogen. Results showed only moderate neurological symptoms of high-pressure nervous syndrome, whereas the narcotic effect of hydrogen was detectable, as investigated by psychosensorimotor tests. Nevertheless, the divers successfully carried out the main purpose of the operational dive, which was to prove the feasability of such diving methods by connecting specific elements of an offshore oil installation. Finally, these data support the hypothesis that hydrogen can alleviate some of the symptoms of the high-pressure nervous syndrome and can constitute a useful gas for commercial diving, as it decreases the density of the breathing mixture and therefore improves the living conditions, work, and comfort of the divers. Nevertheless, the present results underscore the relevance of research on individual susceptibility to pressure environment regardless of the composition of the breathing mixture.

 

 

GASTROINTESTINAL FUNCTION

Gastrointestinal Function Studies

Molecular hydrogen reacts with the hydroxyl radical, a highly cytotoxic species produced in inflamed tissues. It has been suggested therefore to use gaseous hydrogen in a new anti-inflammatory strategy. We tested this idea, with the aid of the equipment and skills of COMEX SA in Marseille, a group who experiments with oxygen-hydrogen breathing mixtures for professional deep-sea diving. The model used was schistosomiasis-associated chronic liver inflammation. Infected animals stayed 2 weeks in an hyperbaric chamber in a normal atmosphere supplemented with 0.7 MPa hydrogen. The treatment had significant protective effects towards liver injury, namely decreased fibrosis, improvement of hemodynamics, increased NOSII activity, increased antioxidant enzyme activity, decreased lipid peroxide levels and decreased circulating TNF-alpha levels. Under the same conditions, helium exerted also some protective effects, indicating that hydroxyl radical scavenging is not the only protective mechanism. These findings indicate that the proposed anti-inflammatory strategy deserves further attention.

Molecular hydrogen ameliorates oxidative stress-associated diseases in animal models. We found that oral intake of hydrogen-rich water abolishes an immediate-type allergic reaction in mice. Using rat RBL-2H3 mast cells, we demonstrated that hydrogen attenuates phosphorylation of the FcepsilonRI-associated Lyn and its downstream signal transduction, which subsequently inhibits the NADPH oxidase activity and reduces the generation of hydrogen peroxide. We also found that inhibition of NADPH oxidase attenuates phosphorylation of Lyn in mast cells, indicating the presence of a feed-forward loop that potentiates the allergic responses. Hydrogen accordingly inhibits all tested signaling molecule(s) in the loop. Hydrogen effects have been solely ascribed to exclusive removal of hydroxyl radical. In the immediate-type allergic reaction, hydrogen exerts its beneficial effect not by its radical scavenging activity but by modulating a specific signaling pathway. Effects of hydrogen in other diseases are possibly mediated by modulation of yet unidentified signaling pathways. Our studies also suggest that hydrogen is a gaseous signaling molecule like nitric oxide.

It is well known that some intestinal bacteria, such as Escherichia coli, can produce a remarkable amount of molecular hydrogen (H(2)). Although the antioxidant effects of H(2) are well documented, the present study examined whether H(2) released from intestinally colonized bacteria could affect Concanavalin A (ConA)-induced mouse hepatitis. Systemic antibiotics significantly decreased the level of H(2) in both liver and intestines along with suppression of intestinal bacteria. As determined by the levels of AST, ALT, TNF-alpha and IFN-gamma in serum, suppression of intestinal bacterial flora by antibiotics increased the severity of ConA-induced hepatitis, while reconstitution of intestinal flora with H(2)-producing E. coli, but not H(2)-deficient mutant E. coli, down-regulated the ConA-induced liver inflammation. Furthermore, in vitro production of both TNF-alpha and IFN-gamma by ConA-stimulated spleen lymphocytes was significantly inhibited by the introduction of H(2). These results indicate that H(2) released from intestinal bacteria can suppress inflammation induced in liver by ConA.Biochem Biophys Res Commun, 2009. 386(2): p. 316-21.

Liver fibrosis is the universal consequence of chronic liver diseases. Sustained hepatocyte injury initiates an inflammatory response, thereby activating hepatic stellate cells, the principal fibrogenic cells in the liver. Reactive oxygen species are involved in liver injury and are a promising target for treating liver fibrosis. Hydrogen water is reported to have potential as a therapeutic tool for reactive oxygen species-associated disorders. This study aimed to investigate the effects of hydrogen water on liver fibrogenesis and the mechanisms underlying these effects.

METHODS:

C57BL/6 mice were fed with hydrogen water or control water, and subjected to carbon tetrachloride, thioacetamide and bile duct ligation treatments to induce liver fibrosis. Hepatocytes and hepatic stellate cells were isolated from mice and cultured with or without hydrogen to test the effects of hydrogen on reactive oxygen species-induced hepatocyte injuries or hepatic stellate cell activation.

RESULTS:

Oral intake of hydrogen water significantly suppressed liver fibrogenesis in the carbontetrachloride and thioacetamide models, but these effects were not seen in the bile duct ligation model. Treatment of isolated hepatocyte with 1 μg/mL antimycin A generated hydroxyl radicals. Culturing in the hydrogen-rich medium selectively suppressed the generation of hydroxyl radicals in hepatocytes and significantly suppressed hepatocyte death induced by antimycin A; however, it did not suppress hepatic stellate cell activation.

CONCLUSION:

We conclude that hydrogen water protects hepatocytes from injury by scavenging hydroxyl radicals and thereby suppresses liver fibrogenesis in mice.

AIM:

Liver fibrosis is the universal consequence of chronic liver diseases. Sustained hepatocyte injury initiates an inflammatory response, thereby activating hepatic stellate cells, the principal fibrogenic cells in the liver. Reactive oxygen species are involved in liver injury and are a promising target for treating liver fibrosis. Hydrogen water is reported to have potential as a therapeutic tool for reactive oxygen species-associated disorders. This study aimed to investigate the effects of hydrogen water on liver fibrogenesis and the mechanisms underlying these effects.

METHODS:

C57BL/6 mice were fed with hydrogen water or control water, and subjected to carbon tetrachloride, thioacetamide and bile duct ligation treatments to induce liver fibrosis. Hepatocytes and hepatic stellate cells were isolated from mice and cultured with or without hydrogen to test the effects of hydrogen on reactive oxygen species-induced hepatocyte injuries or hepatic stellate cell activation.

RESULTS:

Oral intake of hydrogen water significantly suppressed liver fibrogenesis in the carbon tetrachloride and thioacetamide models, but these effects were not seen in the bile duct ligation model. Treatment of isolated hepatocyte with 1 μg/mL antimycin A generated hydroxyl radicals. Culturing in the hydrogen-rich medium selectively suppressed the generation of hydroxyl radicals in hepatocytes and significantly suppressed hepatocyte death induced by antimycin A; however, it did not suppress hepatic stellate cell activation.

CONCLUSION:

We conclude that hydrogen water protects hepatocytes from injury by scavenging hydroxyl radicals and thereby suppresses liver fibrogenesis in mice.

 

 

DIABETES AND OBESITY

Diabetes and Obesity

Recent extensive studies have revealed that molecular hydrogen (H(2)) has great potential for improving oxidative stress-related diseases by inhaling H(2) gas, injecting saline with dissolved H(2), or drinking water with dissolved H(2) (H(2)-water); however, little is known about the dynamic movement of H(2) in a body. First, we show that hepatic glycogen accumulates H(2) after oral administration of H(2)-water, explaining why consumption of even a small amount of H(2) over a short span time efficiently improves various disease models. This finding was supported by an in vitro experiment in which glycogen solution maintained H(2). Next, we examined the benefit of ad libitum drinking H(2)-water to type 2 diabetes using db/db obesity model mice lacking the functional leptin receptor. Drinking H(2)-water reduced hepatic oxidative stress, and significantly alleviated fatty liver in db/db mice as well as high fat-diet-induced fatty liver in wild-type mice. Long-term drinking H(2)-water significantly controlled fat and body weights, despite no increase in consumption of diet and water. Moreover, drinking H(2)-water decreased levels of plasma glucose, insulin, and triglyceride, the effect of which on hyperglycemia was similar to diet restriction. To examine how drinking H(2)-water improves obesity and metabolic parameters at the molecular level, we examined gene-expression profiles, and found enhanced expression of a hepatic hormone, fibroblast growth factor 21 (FGF21), which functions to enhance fatty acid and glucose expenditure. Indeed, H(2) stimulated energy metabolism as measured by oxygen consumption. The present results suggest the potential benefit of H(2) in improving obesity, diabetes, and metabolic syndrome.

We previously reported that molecular hydrogen (H2) acts as a novel antioxidant to exhibit multiple functions. Moreover, long-term drinking of H2-water (water infused with H2) enhanced energy expenditure to improve obesity and diabetes in db/db mice accompanied by the increased expression of fibroblast growth factor 21 (FGF21) by an unknown mechanism. H2 was ingested by drinking of H2-water or by oral administration of an H2-producing material, MgH2. The comprehensive gene expression profile in the liver of db/db mice was analyzed by DNA microarray. The molecular mechanisms underlying the gene expression profile was investigated using cultured HepG2 cells. Moreover, the effects on lifespan of drinking H2-water were examined using wild-type mice that were fed a fatty diet. Pathway analyses based on comprehensive gene expression revealed the increased expression of various genes involved in fatty acid and steroid metabolism. As a transcription pathway, the PPARα signaling pathway was identified to upregulate their genes by ingesting H2. As an early event, the gene expression of PGC-1α was transiently increased, followed by increased expression of FGF21. The expression of PGC-1α might be regulated indirectly through sequential regulation by H2, 4-hydroxy-2-nonenal, and Akt/FoxO1 signaling, as suggested in cultured cell experiments. In wild-type mice fed the fatty diet, H2-water improved the level of plasma triglycerides and extended their average of lifespan. H2 induces expression of the PGC-1α gene, followed by stimulation of the PPARα pathway that regulates FGF21, and the fatty acid and steroid metabolism.

 

 

HEART AND CARDIOVASCULAR HEALTH

Heart and Cardiovascular Health

Until the early 1950’s there did not exist any effective treatment for airway obstruction or cardiac arrest for laypersons. In the late 50’s isolated steps were described to establish a patent airway (A), provide mouth-to-mouth breathing (B) and restore circulation (C) with chest compressions. Tying those steps together into an A-B-C sequence became the basis of physiologically effective cardiopulmonary-cerebral resuscitation (CPCR), as the method was called originally.1

Although single steps proved to be effective, the outcomes of out-of-hospital CA treated by CPCR were not encouraging from the very start. The efforts provided by bystanders and medical personnel came often “too little too late”,2 and there was lack of specific therapies to treat the underlying causes or complications. The community-wide efforts of the public health organizations focused on the promotion of the “cardio-pulmonary resuscitation” (CPR), a newly coined term, now lacking the cerebral component.

It is thus not surprising that the mainstay of further medical research focused mainly on the heart. Restoration of cardiac rhythm became an essential centerpiece of resuscitation efforts. Significant improvements in survival of cardiac arrest victims were enabled by technological developments generally aimed to support the failing heart. The emergence of defibrillators in the 1960’s, followed by percutaneous coronary interventions and mechanical devices supporting the failing heart granted the extra time to recover cardiac function in patients who would not have the same chance several years ago. The brain as the key and target organ seemed somewhat left behind, at least in these early years. Indeed, there was very little that medicine could offer to protect, or restore, the brain function.

Compared to the orchestrated full-front industry-sponsored research aimed at supporting the failing heart, only a few research centers remained interested in the brain. Negovsky’s Institute of Reanimatology in Moscow, Hossmann at the Max Planck Institute in Cologne and Peter Safar’s Resuscitation Research Center in Pittsburgh were pioneers of brain-oriented resuscitation science that systematically explored the limits of restoring brain function, looking beyond the traditional horizons of the restoration of heart function. One of the areas of exploration in these investigations was the use of cerebral blood-flow promoting therapies includes hypertension and hemodilution (“H-H”) designed to better support post-resuscitation brain metabolism.3, 4

Even if the most effective methods to preserve the circulation are employed, there are often insufficient reserves to combat evolving brain ischemia. These hemodynamic manipulations were complemented by contemporaneous explorations of the benefits of post-resuscitative therapeutic hypothermia (“TH”),5, 6 previously well documented in cardiac surgery. The extensive work of Busto, Colbourne, and Corbett et al. documented the short and long-term benefits of hypothermia in small animal models of brain ischemia.7 A major breakthrough in resuscitation science was achieved when two seminal papers showed that prolonged mild hypothermia improved survival and neurologic outcome in comatose survivors from cardiac arrest in a clinical setting.8, 9 Therapeutic hypothermia has become an integral part of the resuscitation guidelines and despite recent challenges to specific details to its application,10 targeted temperature management seems to have become an established paradigm of post-resuscitative care.

The use of pharmacologic adjuncts to prevent or ameliorate the deleterious effects of ischemia-reperfusion injury is a highly appealing concept. Different mechanistic strategies and cell signaling pathways were targeted, including delaying energy failure, protecting cell membrane integrity, preventing structural degradation, regulating protein synthesis, preventing re-oxygenation injury, and/or preserving mitochondria. Surprisingly, multiple established and promising novel drugs that seemed to have a potential to protect the brain in ischemia or restore post-resuscitation brain function have failed to deliver a breakthrough effect.11 None of the drugs that may have yielded positive results in pre-clinical models has translated to successful clinical use.

In this issue of Circulation, Hayashida et al. report on a salutary effect of hydrogen (H2) gas on outcome from experimental cardiac arrest in rats.12 Inhalation of H2 gas initiated upon resuscitation from six minutes of ventricular fibrillation cardiac arrest resulted in improved survival rate, neurologic outcome, and attenuation of histological damage. These results were comparable to the effects achieved with therapeutic hypothermia, while the best results were achieved when both techniques were combined.

These results are even more impressive when put into perspective with their previously published studies. The benefits of H2 gas were documented in their prior work using a similar rat model of cardiac arrest resuscitated with 100% oxygen. Improvement of cardiac function with hydrogen inhalation was highlighted. The salutary impact of H2 gas was at least partially attributed to its radical-scavenging effect.13 However, prolonged administration of 100% oxygen in post-cardiac arrest victims could be deleterious in experimental setting and is not recommended in the clinical setting. In this study, the authors used resuscitation with “room air” to eliminate the potential harmful effects of post-resuscitative hyperoxia.14 The benefits were sustained – moreover, the attenuation of CNS damage was now also documented. The authors should be applauded for their continuous efforts to explore the effects of H2 on both the heart and the brain.

The average response time of urban EMS is around 7–10 minutes, and resuscitation efforts lead to restoration of spontaneous circulation after ~ 25 min.10 The rather short duration of the insult used in this experimental scenario – five or six minutes of ventricular fibrillation – with also rather short resuscitation efforts may not seem to be clinically relevant. These doubts are most likely unsubstantiated. Even as short experimental insults as these result in a significant post-resuscitative hemodynamic compromise and a substantial delayed neuronal degeneration in selectively vulnerable brain regions, as documented by multiple researchers worldwide.15–17 Increased durations of the ischemic insult result in significant mortality, preventing systematic exploration of long-term outcome and complicating data interpretation with mortality bias. Thus, the paradigm used in this study is clinically relevant and well suited for testing promising therapeutic strategies.

The first report on the protective effects of H218 has been subsequently confirmed in various animal models, including limiting the infarct volume of brain18 and heart19 by reducing ischemia–reperfusion injury and providing protection against multiple-organ failure induced by sepsis.20 These mechanisms could be shared with post-cardiac arrest syndrome which is often linked to sepsis-like state.21,22

Several other studies explored the potential of H2 therapy in different paradigms. Intraperitoneal administration of H2 improved survival rate and neurological scores, reduced neuronal injury and inhibited neuronal apoptosis after ventricular fibrillation cardiac arrest in rabbits.23 Intravenous treatment with hydrogen-enriched saline improved survival and neurological outcome after asphyxial cardiac arrest in rats, which were partially mediated by reducing oxidative stress, inflammation, and apoptosis.24 The ostensibly subtle difference between the two types of cardiac arrests – ventricular fibrillation vs. asphyxial – could translate in significant differences in treatment strategies in the clinical setting. The field is beginning to recognize the fact that “not all cardiac arrests are created equal”. Differences in underlying pathophysiological mechanisms25–27 and outcomes between these two insults have been reported. Different regions of the brain show unique reaction even to the same insult, including different tissue oxygen levels28 or neuroinflammation,29 both purported targets of H2 therapy. This is further underscored by the different efficacy of selected therapies in these respective insults, or even between cardiac arrest presenting with ventricular fibrillation vs. asystole.30 It is thus reassuring that H2 was protective in multiple scenarios. The fact that H2 was effective even in an intravenous formulation makes the drug even more potentially appealing.

The high dose of H2 tested in this study was limited by administrative regulations. The dose-finding studies aimed at identifying the optimal therapeutical protocol were not yet completed. However, we are enthused that H2 therapy – either inhalational or intravenous – exerts its benefits on both the heart and brain, providing a potential to put back the cerebral “C” into the CPCR concept. It is also important of paramount importance that the effects of H2 are exerted independently of the effects of therapeutic hypothermia, and in fact the combined effects of these therapies appear to be synergistic. The exact underpinning mechanisms of these two therapies remain to be unveiled in future studies.

The exciting results with H2 gas reported in the current study, put into perspective with multiple other reports, spark an enthusiasm for its future explorations in other experimental settings and potential translation into clinical settings. A clinical trial of hydrogen therapy in patients after cardiac arrest is currently underway.31 We are eagerly awaiting the results.

Systemic inflammatory responses in patients receiving cardiac surgery with the use of the cardiopulmonary bypass (CPB) significantly contribute to CPB-associated morbidity and mortality. We hypothesized that insufflated hydrogen gas (H₂) would provide systemic anti-inflammatory and anti-apoptotic effects during CPB, therefore reducing proinflammatory cytokine levels. In this study, we examined the protective effect of H₂ on a rat CPB model. Rats were divided into three groups: the sham operation (SHAM) group, received sternotomy only; the CPB group, which was initiated and maintained for 60 min; and the CPB + H₂ group in which H₂ was given via an oxygenator during CPB for 60 min. We collected blood samples before, 20 min, and 60 min after the initiation of CPB. We measured the serum cytokine levels of (tumor necrosis factor-α, interleukin-6, and interleukin-10) and biochemical markers (lactate dehydrogenase, aspartate aminotransferase, and alanine aminotransferase). We also measured the wet-to-dry weight (W/D) ratio of the left lung 60 min after the initiation of CPB. In the CPB group, the cytokine and biochemical marker levels significantly increased 20 min after the CPB initiation and further increased 60 min after the CPB initiation as compared with the SHAM group. In the CPB + H₂ group, however, such increases were significantly suppressed at 60 min after the CPB initiation. Although the W/D ratio in the CPB group significantly increased as compared with that in the SHAM group, such an increase was also suppressed significantly in the CPB + H₂ group. We suggest that H₂ insufflation is a possible new potential therapy for counteracting CPB-induced systemic inflammation.

Sleep apnea syndrome increases the risk of cardiovascular morbidity and mortality. We previously reported that intermittent hypoxia increases superoxide production in a manner dependent on nicotinamide adenine dinucleotide phosphate and accelerates adverse left ventricular (LV) remodeling. Recent studies have suggested that hydrogen (H(2)) may have an antioxidant effect by reducing hydroxyl radicals. In this study, we investigated the effects of H(2) gas inhalation on lipid metabolism and LV remodeling induced by intermittent hypoxia in mice. Male C57BL/6J mice (n = 62) were exposed to intermittent hypoxia (repetitive cycle of 1-min periods of 5 and 21% oxygen for 8 h during daytime) for 7 days. H(2) gas (1.3 vol/100 vol) was given either at the time of reoxygenation, during hypoxic conditions, or throughout the experimental period. Mice kept under normoxic conditions served as controls (n = 13). Intermittent hypoxia significantly increased plasma levels of low- and very low-density cholesterol and the amount of 4-hydroxy-2-nonenal-modified protein adducts in the LV myocardium. It also upregulated mRNA expression of tissue necrosis factor-α, interleukin-6, and brain natriuretic peptide, increased production of superoxide, and induced cardiomyocyte hypertrophy, nuclear deformity, mitochondrial degeneration, and interstitial fibrosis. H(2) gas inhalation significantly suppressed these changes induced by intermittent hypoxia. In particular, H(2) gas inhaled at the timing of reoxygenation or throughout the experiment was effective in preventing dyslipidemia and suppressing superoxide production in the LV myocardium. These results suggest that inhalation of H(2) gas was effective for reducing oxidative stress and preventing LV remodeling induced by intermittent hypoxia relevant to sleep apnea.

BACKGROUND:

All clinical and biological manifestations related to postcardiac arrest (CA) syndrome are attributed to ischemia–reperfusion injury in various organs including brain and heart. Molecular hydrogen (H2) has potential as a novel antioxidant. This study tested the hypothesis that inhalation of H2 gas starting at the beginning of cardiopulmonary resuscitation (CPR) could improve the outcome of CA.

METHODS AND RESULTS:

Ventricular fibrillation was induced by transcutaneous electrical epicardial stimulation in rats. After 5 minutes of the subsequent CA, rats were randomly assigned to 1 of 4 experimental groups at the beginning of CPR: mechanical ventilation (MV) with 2% N2 and 98% O2 under normothermia (37°C), the control group; MV with 2% H2 and 98% O2 under normothermia; MV with 2% N2and 98% O2 under therapeutic hypothermia (TH), 33°C; and MV with 2% H2 and 98% O2 under TH. Mixed gas inhalation and TH continued until 2 hours after the return of spontaneous circulation (ROSC). H2 gas inhalation yielded better improvement in survival and neurological deficit score (NDS) after ROSC to an extent comparable to TH. H2 gas inhalation, but not TH, prevented a rise in left ventricular end-diastolic pressure and increase in serum IL-6 level after ROSC. The salutary impact of H2 gas was at least partially attributed to the radical-scavenging effects of H2 gas, because both 8-OHdG- and 4-HNE- positive cardiomyocytes were markedly suppressed by H2 gas inhalation after ROSC.

CONCLUSION:

Inhalation of H2 gas is a favorable strategy to mitigate mortality and functional outcome of post-CA syndrome in a rat model, either alone or in combination with TH.

 

 

BRAIN AND NEURODEGENERATION

BRAIN AND NEURODEGENERATION

Hydrogen is the most abundant chemical element in the Universe, but is seldom regarded as a therapeutic agent. Recent evidence has shown that hydrogen is a potent antioxidative, antiapoptotic and anti-inflammatory agent and so may have potential medical applications in cells, tissues and organs. There are several methods to administer hydrogen, such as inhalation of hydrogen gas, aerosol inhalation of a hydrogen-rich solution, drinking hydrogen dissolved in water, injecting hydrogen-rich saline (HRS) and taking a hydrogen bath. Drinking hydrogen solution (saline/pure water/other solutions saturated with hydrogen) may be more practical in daily life and more suitable for daily consumption. This review summarizes the findings of recent studies on the use of hydrogen in emergency and critical care medicine using different disease models.

BACKGROUND:

Hydrogen, a popular antioxidant gas, can selectively reduce cytotoxic oxygen radicals and has been found to protect against ischemia-reperfusion (I/R) injury of multiple organs. Acute neuronal death during I/R has been attributed to loss of mitochondrial permeability transition coupled with mitochondrial dysfunction. This study was designed to investigate the potential therapeutic effect of hydrogen-rich saline on neuronal mitochondrial injury from global cerebral I/R in rats.

MATERIALS AND METHODS:

We used a four-vessel occlusion model of global cerebral ischemia and reperfusion, with Sprague-Dawley rats. The rats were divided randomly into six groups (n = 90): sham (group S), I/R (group I/R), normal saline (group NS), atractyloside (group A), hydrogen-rich saline (group H), and hydrogen-rich saline + atractyloside (group HA). In groups H and HA, intraperitoneal hydrogen-rich saline (5 mL/kg) was injected immediately after reperfusion, whereas the equal volume of NS was injected in the other four groups. In groups A and HA, atractyloside (15 μL) was intracerebroventricularly injected 10 min before reperfusion, whereas groups NS and H received equal NS. The mitochondrial permeability transition pore opening and mitochondrial membrane potential were measured by spectrophotometry. Cytochrome c protein expression in the mitochondria and cytoplasm was detected by western blot. The hippocampus mitochondria ultrastructure was examined with transmission electron microscope. The histologic damage in hippocampus was assessed by hematoxylin and eosin staining.

RESULTS:

Hydrogen-rich saline treatment significantly improved the amount of surviving cells(P ≤ 0.05). Furthermore, hydrogen-rich saline not only reduced tissue damage, the degree of mitochondrial swelling, and the loss of mitochondrial membrane potential but also preserved the mitochondrial cytochrome c content (P ≤ 0.05).

CONCLUSIONS:

Our study showed that hydrogen-rich saline was able to attenuate neuronal I/R injury, probably by protecting mitochondrial function in rats.

Traumatic brain injury (TBI) in its various forms has emerged as a major problem for modern society. Acute TBI can transform into a chronic condition and be a risk factor for neurodegenerative diseases such as Alzheimer's and Parkinson's diseases, probably through induction of oxidative stress and neuroinflammation. Here, we examined the ability of the antioxidant molecular hydrogen given in drinking water (molecular hydrogen water; mHW) to alter the acute changes induced by controlled cortical impact (CCI), a commonly used experimental model of TBI. We found that mHW reversed CCI-induced edema by about half, completely blocked pathological tau expression, accentuated an early increase seen in several cytokines but attenuated that increase by day 7, reversed changes seen in the protein levels of aquaporin-4, HIF-1, MMP-2, and MMP-9, but not for amyloid beta peptide 1-40 or 1-42. Treatment with mHW also reversed the increase seen 4 h after CCI in gene expression related to oxidation/carbohydrate metabolism, cytokine release, leukocyte or cell migration, cytokine transport, ATP and nucleotide binding. Finally, we found that mHW preserved or increased ATP levels and propose a new mechanism for mHW, that of ATP production through the Jagendorf reaction. These results show that molecular hydrogen given in drinking water reverses many of the sequelae of CCI and suggests that it could be an easily administered, highly effective treatment for TBI.

Hydrogen (H2) has been reported to neutralize toxic reactive oxygen species. Oxidative stress is an important mechanism of neuronal damage after perinatal asphyxia. We examined whether 2.1% H2-supplemented room air (H2-RA) ventilation would preserve cerebrovascular reactivity (CR) and brain morphology after asphyxia/reventilation (A/R) in newborn pigs. Anesthetized, ventilated piglets were assigned to one of the following groups: A/R with RA or H2-RA ventilation (A/R-RA and A/R-H2-RA; n = 8 and 7, respectively) and respective time control groups (n = 9 and 7). Asphyxia was induced by suspending ventilation for 10 min, followed by reventilation with the respective gases for 4 h. After euthanasia, the brains were processed for neuropathological examination. Pial arteriolar diameter changes to graded hypercapnia (5-10% CO2 inhalation), and NMDA (10(-4) M) were determined using the closed cranial window/intravital microscopy before and 1 h after asphyxia. Neuropathology revealed that H2-RA ventilation significantly reduced neuronal injury induced by A/R in virtually all examined brain regions including the cerebral cortex, the hippocampus, basal ganglia, cerebellum, and the brainstem. Furthermore, H2-RA ventilation significantly increased CR to hypercapnia after A/R (% vasodilation was 23 ± 4% versus 41 ± 9%, p ≤ 0.05). H2-RA ventilation did not affect reactive oxygen species-dependent CR to NMDA. In summary, H2-RA could be a promising approach to reduce the neurologic deficits after perinatal asphyxia.

 

 

LUNGS AND ASTHMA

LUNGS AND ASTHMA

Hydrogen is considered to be a novel antioxidant as it inhibits inflammation, removes oxygen-derived free radicals and reduces oxidative damage. This study investigated the effects of hydrogen-rich saline on plasma interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), superoxide dismutase (SOD) and malondialdehyde (MDA) in rats with uncontrolled hemorrhagic shock (UHS). The UHS model was induced by arterial bleeding and tail amputation. The rats were randomly divided into: Group A (sham-operated group), Group B [shock + intravenously (IV) injected saline], Group C (shock + IV-injected hydrogen-rich saline), Group D [shock + intraperitoneally (IP) injected saline] and Group E (shock + IP-injected hydrogen-rich saline). The survival rate 24 h after successful resuscitation was calculated. The mean arterial pressure and heart rate were recorded at 0, 30, 90 and 210 min. The plasma levels of IL-6, TNF-α, SOD and MDA were measured at 0, 90 and 210 min. The survival rate of each group was 100% and the hemodynamics among the experimental groups were not significantly different. At 90 and 210 min, the levels of IL-6, TNF-α and MDA in Groups C and E were lower than those of Groups B and D, while the SOD levels were higher than those of Groups B and D (P ≤ 0.01). At 90 min, the levels of IL-6, TNF-α and MDA in Groups B and C were lower than those of Groups D and E, respectively (P ≤ 0.01). Hydrogen-rich saline has anti-inflammatory and anti-oxidative effects in UHS. In conclusion, the results showed that itravenous injection of hydrogen-rich saline is more effective than intraperitonal injection.

Hydrogen has been reported to selectively quench detrimental reactive oxygen species, particularly hydroxyl radical, and to prevent myocardial or hepatic ischemia/reperfusion injury in multiple models. The aim of this study is to investigate whether hydrogen protects against severe burn-induced acute lung injury in rats. Rats were divided into four groups: sham plus normal saline, burn injury plus normal saline, burn injury plus hydrogen-rich saline, and burn injury plus edaravone. Animals were given full-thickness burn wounds (30% TBSA) using boiling water, except the sham group that was treated with room temperature water. The rats in hydrogen group received 5 ml/kg of hydrogen-rich saline, sham and burn controls obtained the same amount of saline, and the edaravone group was treated with 9 mg/kg of edaravone in saline. Lactated Ringer's solution was given at 6 hours postburn. The lungs were harvested 12 hours postburn for laboratory investigations. Severe burns with delayed resuscitation rapidly caused lung edema and impaired oxygenation in rats. These dysfunctions were ameliorated by administration of hydrogen-rich saline or edaravone. When compared with the burn injury plus normal saline group, hydrogen-rich saline or edaravone group significantly attenuated the pulmonary oxidative products, such as malondialdehyde, carbonyl, and 8-hydroxy-2'-deoxyguanosine. Furthermore, administration of hydrogen-rich saline or edaravone dramatically reduced the pulmonary levels of pulmonary inflammation mediators and myeloperoxidase. Intraperitoneal administration of hydrogen-rich saline improves pulmonary function by reducing oxidative stress and inflammatory response in severe burn-induced acute lung injury.

 

OBJECTIVES:

Lung transplantation is a well-established treatment of end-stage lung disease; however, it is limited by a shortage of donor lungs. To overcome this problem, donation after cardiac death (DCD) and ex vivo lung perfusion (EVLP) are being widely investigated. In this study, the effect of hydrogen gas, a known antioxidant, was investigated on a DCD lung model during EVLP.

METHODS:

Ten pigs were randomized into either a control (n = 5) or a hydrogen group (n = 5). After fibrillation by electric shock, no further treatment was administered in order to induce warm ischaemic injury for 1 h. The lungs were then procured, followed by 4 h of EVLP. During EVLP, the lungs were ventilated with room air in the control group, and with 2% hydrogen gas in the hydrogen group. Oxygen capacity (OC), pulmonary vascular resistance (PVR) and peak airway pressure (PAP) were measured every hour, and the expressions of interleukin-1 beta (IL-1β), IL-6 (IL-6), IL-8 (IL-8) and tumour necrosis factor-alpha (TNF-α) were evaluated in lung tissue after EVLP. Pathological evaluations were performed using lung injury severity (LIS) scores and the wet/dry ratio was also measured.

RESULTS:

The OC in the hydrogen group was higher than in the control group, but the difference was not statistically significant (P = 0.0862). PVR (P = 0.0111) and PAP (P = 0.0189) were statistically significantly lower in the hydrogen group. Compared with the control group, the hydrogen group had a statistically significantly lower expression of IL-1β (P = 0.0317), IL-6 (P = 0.0159), IL-8 (P = 0.0195) and TNF-α (P = 0.0159). The LIS scores (P = 0.0358) and wet/dry ratios (P = 0.040) were also significantly lower in the hydrogen group.

CONCLUSIONS:

Hydrogen gas inhalation during EVLP improved the function of DCD lungs, which may increase the utilization of DCD lungs.

INTRODUCTION:

Mechanical ventilation (MV) can provoke oxidative stress and an inflammatory response, and subsequently cause ventilator-induced lung injury (VILI), a major cause of mortality and morbidity of patients in the intensive care unit. Inhaled hydrogen can act as an antioxidant and may be useful as a novel therapeutic gas. We hypothesized that, owing to its antioxidant and anti-inflammatory properties, inhaled hydrogen therapy could ameliorate VILI.

METHODS:

VILI was generated in male C57BL6 mice by performing a tracheostomy and placing the mice on a mechanical ventilator (tidal volume of 30 ml/kg without positive end-expiratory pressure, FiO(2) 0.21). The mice were randomly assigned to treatment groups and subjected to VILI with delivery of either 2% nitrogen or 2% hydrogen in air. Sham animals were given same gas treatments for two hours (n = 8 for each group). The effects of VILI induced by less invasive and longer exposure to MV (tidal volume of 10 ml/kg, 5 hours, FiO(2) 0.21) were also investigated (n = 6 for each group). Lung injury score, wet/dry ratio, arterial oxygen tension, oxidative injury, and expression of pro-inflammatory mediators and apoptotic genes were assessed at the endpoint of two hours using the high-tidal volume protocol. Gas exchange and apoptosis were assessed at the endpoint of five hours using the low-tidal volume protocol.

RESULTS:

Ventilation (30 ml/kg) with 2% nitrogen in air for 2 hours resulted in deterioration of lung function, increased lung edema, and infiltration of inflammatory cells. In contrast, ventilation with 2% hydrogen in air significantly ameliorated these acute lung injuries. Hydrogen treatment significantly inhibited upregulation of the mRNAs for pro-inflammatory mediators and induced antiapoptotic genes. In the lungs treated with hydrogen, there was less malondialdehyde compared with lungs treated with nitrogen. Similarly, longer exposure to mechanical ventilation within lower tidal volume (10 mg/kg, five hours) caused lung injury including bronchial epithelial apoptosis. Hydrogen improved gas exchange and reduced VILI-induced apoptosis.

CONCLUSIONS:

Inhaled hydrogen gas effectively reduced VILI-associated inflammatory responses, at both a local and systemic level, via its antioxidant, anti-inflammatory and antiapoptotic effects.

 

 

METABOLIC SYNDROME

METABOLIC SYNDROME

Electrolyzed reduced water (ERW) containing hydrogen molecules and Pt nanoparticles is expected as a new antioxidant. Thiobarbituric acid reactive substances (TBARS) assay revealed that ERW had an inhibitory effect on the oxidation of low-density lipoproteins (LDL). In addition, ERW significantly suppressed LDL oxidation in the medium cultured J.774.A1 macrophage like cells. ERW inhibited Cu2+ ion-catalyzed oxidation of LDL ex vivo. ERW lowered lipid peroxide level in blood red cells and plasma triglyceride in rats fed a basal diet containing 2% cholesterol. These results suggest that ERW has anti-LDL oxidation and anti-hyperlipidemia effects.

Hydrogen (H(2)) acts as a therapeutic antioxidant. However, there are few reports on H(2) function in other capacities in diabetes mellitus (DM). Therefore, in this study, we investigated the role of H(2) in glucose transport by studying cultured mouse C2C12 cells and human hepatoma Hep-G2 cells in vitro, in addition to three types of diabetic mice [Streptozotocin (STZ)-induced type 1 diabetic mice, high-fat diet-induced type 2 diabetic mice, and genetically diabetic db/db mice] in vivo. The results show that H(2) promoted 2-[(14)C]-deoxy-d-glucose (2-DG) uptake into C2C12 cells via the translocation of glucose transporter Glut4 through activation of phosphatidylinositol-3-OH kinase (PI3K), protein kinase C (PKC), and AMP-activated protein kinase (AMPK), although it did not stimulate the translocation of Glut2 in Hep G2 cells. H(2) significantly increased skeletal muscle membrane Glut4 expression and markedly improved glycemic control in STZ-induced type 1 diabetic mice after chronic intraperitoneal (i.p.) and oral (p.o.) administration. However, long-term p.o. administration of H(2) had least effect on the obese and non-insulin-dependent type 2 diabetes mouse models. Our study demonstrates that H(2) exerts metabolic effects similar to those of insulin and may be a novel therapeutic alternative to insulin in type 1 diabetes mellitus that can be administered orally.

 

 

BONE

BONE STUDIES

Tumor necrosis factor-alpha (TNFα) plays a crucial role in inflammatory diseases such as rheumatoid arthritis and postmenopausal osteoporosis. Recently, it has been demonstrated that hydrogen gas, known as a novel antioxidant, can exert therapeutic anti-inflammatory effect in many diseases. In this study, we investigated the effect of treatment with hydrogen molecule (H(2)) on TNFα-induced cell injury in osteoblast. The osteoblasts isolated from neonatal rat calvariae were cultured. It was found that TNFα suppressed cell viability, induced cell apoptosis, suppressed Runx2 mRNA expression, and inhibited alkaline phosphatase activity, which was reversed by co-incubation with H(2). Incubation with TNFα-enhanced intracellular reactive oxygen species (ROS) formation and malondialdehyde production increased NADPH oxidase activity, impaired mitochondrial function marked by increased mitochondrial ROS formation and decreased mitochondrial membrane potential and ATP synthesis, and suppressed activities of antioxidant enzymes including SOD and catalase, which were restored by co-incubation with H(2). Treatment with H(2) inhibited TNFα-induced activation of NFκB pathway. In addition, treatment with H(2) inhibited TNFα-induced nitric oxide (NO) formation through inhibiting iNOS activity. Treatment with H(2) inhibited TNFα-induced IL-6 and ICAM-1 mRNA expression. In conclusion, treatment with H(2) alleviates TNFα-induced cell injury in osteoblast through abating oxidative stress, preserving mitochondrial function, suppressing inflammation, and enhancing NO bioavailability.

The objectives of this paper were to determine the level of oxidative stress in atrophied gastrocnemius, and to verify the effect of molecular hydrogen (H2) saturated alkaline electrolyzed water (HSW) on gastrocnemius atrophy by modifying the redox status, indicated by 8-hydroxy-2'-deoxyguanosine (8-OHdG), malondialdehyde (MDA), and superoxide dismutase (SOD)-like activity. Female Wistar rats were divided into four groups: (1) the control (CONT); (2) the Hindlimb unloading (HU, for 3 weeks) given purified normal water (HU-NW); (3) the HU given alkaline electrolyzed reduced water (HU-AEW); and (4) the HU given HSW (HU-HSW). We showed that 8-OHdG, but not MDA, significantly increased by 149% and 145% in HU-NW and HU-AEW, respectively, when compared with CONT. In contrast, there was a trend toward suppression in 8-OHdG levels (increased by 95% compared with CONT) by treatment of HSW, though this effect was not prominent. Additionally, SOD-like activity significantly increased in both HU-NW (184%) and HU-AEW (199%) when compared with CONT. This result suggests the elevation of O2-· in the atrophied gastrocnemius. However, upregulation of SOD-like activity in the HU-HSW was increased by only 169% compared with CONT, though this difference is too small to detect statistical significance. HU led to 13% and 15% reduction of gastrocnemius wet weights in HU-NW and HU-AEW, respectively, compared with CONT. And the reduction of gastrocnemius wet weights in HU-HSW was attenuated by 7% compared with CONT. The gastrocnemius wet weights in the HU-HSW group were significantly greater than those in the HU-AEW, but not statistically significant with HU-NW. These results indicate that HU causes an increase in oxidative stress, but, in this experimental protocol, continuous consumption of HSW during HU does not demonstrate successful attenuation of oxidative stress and HU-mediated gastrocnemius atrophy.

BACKGROUND AND PURPOSE:

Accumulating evidence indicates an important role of oxidative stress in the progression of osteoporosis. Recently, it was demonstrated that hydrogen gas, as a novel antioxidant, could selectively reduce hydroxyl radicals and peroxynitrite anion to exert potent therapeutic antioxidant activity. The aim of the present work was to investigate the effect of hydrogen water (HW) consumption on ovariectomy-induced osteoporosis.

EXPERIMENTAL APPROACH:

Ovariectomized rats were fed with HW (1.3 ± 0.2 mg·L⁻¹) for 3 months. Then, blood was collected and femur and vertebrae were removed for evaluation of the effect of HW on bone.

KEY RESULTS:

HW consumption in ovariectomized rats had no significant effect on oestrogen production, but prevented the reduction of bone mass including bone mineral content and bone mineral density in femur and vertebrae, and preserved mechanical strength including ultimate load, stiffness, and energy, and bone structure including trabecular bone volume fraction, trabecular number, and trabecular thickness in femur, and preserved mechanical strength including ultimate load and stiffness, and bone structure including trabecular bone volume fraction and trabecular number in vertebrae. In addition, treatment with HW abated oxidative stress and suppressed IL-6 and TNF-α mRNA expressions in femur of ovariectomized rats; treatment with HW increased femur endothelial NOS activity and enhanced circulating NO level in ovariectomized rats.

CONCLUSIONS AND IMPLICATIONS:

HW consumption prevents osteopenia in ovariectomized rats possibly through the ablation of oxidative stress induced by oestrogen withdrawal.

 

 

CHRONIC PAIN

CHRONIC 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.

BACKGROUND:

Reactive oxygen species (ROS) are often associated with persistent pains such as neuropathic and inflammatory pain. Hydrogen gas can reduce ROS and alleviate cerebral, myocardial, and hepatic ischemia/reperfusion injuries. In the present study, we aim to investigate whether hydrogen-rich saline can reduce neuropathic pain in a rat model of chronic constriction injury (CCI).

METHODS:

Thirty SD rats were randomly divided into three groups: sham group was administered sodium chloride by intrathecal injection (n=10); control groups underwent CCI surgery and were administered sodium chloride by intrathecal injection (n=10); vehicle group underwent CCI surgery and was administered hydrogen-rich saline by intrathecal injection (n=10). Drugs were administered in the dose of 100 ul/kg once a day at 0.5 hours before and 1-7 day after CCI surgery. The mechanical thresholds were tested at one day before and 3-14 day after CCI surgery.

RESULTS:

We found that hydrogen-rich saline significantly elevated the mechanical thresholds of neuropathic pain compared to vehicle (physiologic saline) control in CCI rats (p < 0.05); it also decreased the levels of myeloperoxidase, maleic dialdehyde, and protein carbonyl in spinal cord by 7 days post-chronic constriction injury(p < 0.05). In addition, hydrogen-rich saline also suppressed the expression of p38-mitogen-activated protein kinase (p38MAPK) and brain-derived neurotrophic factor (BDNF) in the spinal cord by 7 days post-chronic constriction injury (p < 0.01, p < 0.01, respectively), but had no effect on P2X4R (p > 0.05), an ATP receptor.

CONCLUSION:

Intrathecal injection of hydrogen-rich saline can decrease oxidative stress and the expression of p38MAPK and BDNF that may contribute to the elevated threshold of neuropathic pain in rat CCI model.

BACKGROUND:

Reactive oxygen and nitrogen species are key molecules that mediate neuropathic pain. Although hydrogen is an established antioxidant, its effect on chronic pain has not been characterized. This study was to investigate the efficacy and mechanisms of hydrogen-rich normal saline induced analgesia.

METHODOLOGY/PRINCIPAL FINDINGS:

In a rat model of neuropathic pain induced by L5 spinal nerve ligation (L5 SNL), intrathecal injection of hydrogen-rich normal saline relieved L5 SNL-induced mechanical allodynia and thermal hyperalgesia. Importantly, repeated administration of hydrogen-rich normal saline did not lead to tolerance. Preemptive treatment with hydrogen-rich normal saline prevented development of neuropathic pain behavior. Immunofluorochrome analysis revealed that hydrogen-rich normal saline treatment significantly attenuated L5 SNL-induced increase of 8-hydroxyguanosine immunoreactive cells in the ipsilateral spinal dorsal horn. Western blot analysis of SDS/PAGE-fractionated tyrosine-nitrated proteins showed that L5 SNL led to increased expression of tyrosine-nitrated Mn-containing superoxide dismutase (MnSOD) in the spinal cord, and hydrogen-rich normal saline administration reversed the tyrosine-nitrated MnSOD overexpression. We also showed that the analgesic effect of hydrogen-rich normal saline was associated with decreased activation of astrocytes and microglia, attenuated expression of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the spinal cord.

CONCLUSION/SIGNIFICANCE:

Intrathecal injection of hydrogen-rich normal saline produced analgesic effect in neuropathic rat. Hydrogen-rich normal saline-induced analgesia in neuropathic rats is mediated by reducing the activation of spinal astrocytes and microglia, which is induced by overproduction of hydroxyl and peroxynitrite.

 

 

INFLAMMATION

INFLAMMATION

Rheumatoid arthritis (RA) is a chronic inflammatory disease in which the progressive destruction of joint causes morbidity. It is also associated with an increased risk of atherosclerosis, which can result in cardiovascular disease and mortality. The therapeutic goal is to control the systemic inflammation to obtain not only the remission of symptoms, but also improve general state of health. Although recent biologic immunosuppressive therapies targeting pro-inflammatory cytokines have spawned a paradigm shift regarding the prognosis of RA, these therapies possess inherent side effects. Also, early diagnosis of the disease remains confounded by uncertainty. While the mechanisms responsible for the onset of RA remain unclear, reactive oxygen species (ROS) play a significant role in the pathogenesis of RA. ROS play a central role both upstream and downstream of NF-κB and TNFα pathways, which are located at the center of the inflammatory response. Among the ROS, the hydroxyl radical is the most harmful, and molecular hydrogen (H2) is a selective scavenger for this species. Recently, it has been shown that H2 is useful when administered along with the conventional therapy in RA as it acts to reduce oxidative stress in the patients. Especially in the early stage, H2 showed significant therapeutic potential, which also seemed to assist diagnosis and treatment decisions of RA. The possible expectations regarding the potential benefits of H2 by reducing the oxidative stress, resulting from inflammatory factors, are raised and discussed here. They include prevention of RA and related atherosclerosis, as well as therapeutic validity for RA

BACKGROUND:

Molecular hydrogen (H2) as a new medical gas has an anti-inflammatory effect. In the present study, we investigated whether heme oxygenase-1 (HO-1) contributes to the anti-inflammatory effect of H2 in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages.

METHODS:

RAW 264.7 macrophages were stimulated by LPS (1 μg/mL) with presence or absence of different concentrations of H2. Cell viability and injury were tested by 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay and lactate dehydrogenase (LDH) release, respectively. The cell culture supernatants were collected to measure inflammatory cytokines [TNF-α, IL-1β, HMGB1 (high mobility group box-1) and IL-10] at different time points. Moreover, HO-1 protein expression and activity were tested at different time points. In addition, to further identify the role of HO-1 in this process, zinc protoporphyrin (ZnPP)-IX, an HO-1 inhibitor, was used.

RESULTS:

H2 treatment had no significant influence on cell viability and injury in normally cultured RAW 264.7 macrophages. Moreover, H₂ treatment dose-dependently attenuated the increased levels of pro-inflammatory cytokines (TNF-α, IL-1β, HMGB1), but further increased the level of anti-inflammatory cytokine IL-10 at 3 h, 6 h, 12 h and 24 h after LPS stimulation. Furthermore, H₂ treatment could also dose-dependently increase the HO-1 protein expression and activity at 3 h, 6 h, 12 h and 24 h in LPS-activated macrophages. In addition, blockade of HO-1 activity with ZnPP-IX partly reversed the anti-inflammatory effect of H₂ in LPS-stimulated macrophages.

CONCLUSION:

Molecular hydrogen exerts a regulating role in the release of pro- and anti-inflammatory cytokines in LPS-stimulated macrophages, and this effect is at least partly mediated by HO-1 expression and activation.

OBJECTIVE:

To investigate the effect of hydrogen gas inhalation on survival rate and serum high mobility group box 1 (HMGB1) levels in severe septic mice.

METHODS:

Severe sepsis was induced by cecal ligation and puncture (CLP) operation in mice.A total of 248 mice were randomly divided into four groups: sham operation group (sham), sham operation with hydrogen gas inhalation group (sham+H2), severe CLP group (severe CLP) and severe CLP with hydrogen gas inhalation group (severe CLP+H2). Hydrogen gas inhalation was given for 1 h at 1st and 6th h after CLP or sham operation, respectively. The survival rates and serum HMGB1 levels of all groups at different time points were measured.

RESULT:

The 7-d survival rates of severe CLP mice was 0 % (Compared with Sham group, P ❬0.05), and the serum HMBG1 levels from h2 to h32 after CLP operation were significantly increased in severe CLP mice (Compared with Sham group, P ❬0.05). Hydrogen gas treatment increased the 7-d survival rate of severe CLP mice to 60 % (Compared with severe sepsis group, P ❬0.05) and significantly reduced the serum HMGB1 levels at different time points (Compared with severe sepsis group, P ❬0.05).

CONCLUSION:

Hydrogen gas inhalation can decrease the serum HMGB1 levels and increase the survival rate of rats with severe sepsis.

 

 

EYE AND EAR

Eye and Ear

The purpose of the current study was to evaluate hydrogen-saturated saline protecting intensive narrow band noise-induced hearing loss. Guinea pigs were divided into three groups: hydrogen-saturated saline; normal saline; and control. For saline administration, the guinea pigs were given daily abdominal injections (1 ml/100 g) 3 days before and 1 h before narrow band noise exposure (2.5-3.5 kHz 130 dB SPL, 1 h). The guinea pigs in the control group received no treatment. The hearing function was assessed by the auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) recording. The changes of free radicals in the cochlea before noise exposure, and immediately and 7 days after noise exposure were also examined. By Scanning electron microscopy and succinate dehydrogenase staining, we found that pre-treatment with hydrogen-saturated saline significantly reduced noise-induced hair cell damage and hearing loss. We also found that the malondialdehyde, lipid peroxidation, and hydroxyl levels were significantly lower in the hydrogen-saturated saline group after noise trauma, indicating that hydrogen-saturated saline can decrease the amount of harmful free radicals caused by noise trauma. Our findings suggest that hydrogen-saturated saline is effective in preventing intensive narrow band noise-induced hearing loss through the antioxidant effect.

AIM:

To explore the effect of saturated hydrogen saline on blue light-induced retinal damage in rats.

METHODS:

The retinal damage of rats was induced by blue light exposure for 6 hours and examined 8 hours, 16 hours and 24 hours after the exposure. One hundred female Sprague-Dawley rats were randomly divided into four groups. Group 1 included 30 rats received light exposure without any other treatment. Group 2 included 30 rats received light exposure with intraperitoneal injection of normal saline. Group 3 included 30 rats received light exposure with intraperitoneal injection of saturated hydrogen saline. And Group 4 included the other 10 rats which did not receive any treatment. The amount of intraperitoneal injection of saturated hydrogen saline and normal saline was calculated in the ratio of 1ml/100g of rat weight. Specimens were collected and processed by H-E staining, ultrastructure observation, biochemical measurement. Morphological changes were observed by light microscope and transmission electron microscope (TEM) and the retinal outer nuclear layer (ONL) thickness was measured by IPP 6.0, while the malondialdehyde (MDA) was measured by colorimetric determination at 532 nm.

RESULTS:

Although the structure of retina in Group 1 and Group 2 was injured heavily, the injury in Group 3 was mild. The differences between Group 1 and Group 2 were not significant. Compared with the rats in Group 1 and Group 2, the ones in Group 3 had more clearly demarcated retina structure and more ordered cells by light microscope and TEM observation. The ONL thicknesses (400 times) of four groups at each time point except between Group 1 and Group 2 were significantly different (P❰0.05). The thicknesses of the ONL in Group 1 at three time points were 30.41±4.04µm, 26.11±2.82µm and 20.63±1.06µm, in Group 2 were 31.62±4.54µm, 25.08±3.63µm and 19.07±3.86µm, in Group 3 were 29.75±3.62µm, 28.83±1.97µm and 27.61±1.83µm. In Group 4 the mean of the thickness was 37.35±1.37µm. As time went by, the damage grew more severely. At 24h point, the differences were most significant. Compared with Group 4, the thickness was 46.23% thinner in Group 1, 50.29% thinner in Group 2 and 28.04% thinner in Group 3. The stack structures of membranous disc in Group 3 were injured slightly, but in Group 1 and Group 2 the damage was more obvious by TEM. Compared with Group 4 at each time point, the content of MDA in Group 1 was higher (P❰0.05). The content of MDA in Group 3 was significantly lower than those of Group 1 (P❰0.05) and Group 2 (P❰0.05). Between the Group 1 and Group 2, the MDA concentration at each time point was no significant difference (P>0.05).

CONCLUSION:

Saturated hydrogen saline could protect the retina from light-induced damage by attenuating oxidative stress.

OBJECTIVE:

Retinal neovascularization or retinopathy is a proliferative disorder of the retinal capillaries and is the primary cause of blindness. Some studies have shown that oxidative stress plays an important role in hyperoxia-induced retinal neovascularization. Previous reports have indicated that hydrogen has a therapeutic, antioxidant activity by selectively reducing hydroxyl radicals. This study examined the therapeutic effect of hydrogen saline on retinopathy in an established mouse model of hyperoxia-induced retinopathy.

METHODS:

Mouse pups were exposed to 75% O(2) from postnatal day 7 (P7) to P12. Hydrogen saline was administered by intraperitoneal injection (5 ml/kg) daily for 5 days. On P17, the pups were decapitated, and retinal neovascularization was assessed using fluorescence imaging and histopathological examination. Vascular endothelial growth factor (VEGF) expression was evaluated using real-time polymerase chain reaction and fluorescence immunohistochemistry. Oxidative stress was quantified based on the malondialdehyde (MDA) level.

RESULTS:

Hydrogen saline decreased retinal neovascularization, reduced the mRNA and protein expression of VEGF, and suppressed the MDA levels.

CONCLUSIONS:

Hydrogen saline may be a potential treatment for hyperoxia-induced retinopathy that acts via the inhibition of oxidative stress and the reduction of VEGF expression.

 

 

CANCER

Cancer

In order to erase reactive oxygen species (ROS) related with the proliferation of tumor cells by reducing activity of hydrogen, we developed functional water containing nano-bubbles (diameters: ❰900 nm for 71%/population) hydrogen of 1.1-1.5 ppm (the theoretical maximum: 1.6 ppm) with a reducing ability (an oxidation-reduction potential -650 mV, normal water: +100-200 mV) using a microporous-filter hydrogen-jetting device. We showed that hydrogen water erased ROS indispensable for tumor cell growth by ESR/spin trap, the redox indicator CDCFH-DA assay, and was cytotoxic to Ehrlich ascites tumor cells as assessed by WST-8 assay, crystal violet dye stain and scanning electron microscopy, after 24-h or 48-h incubation sequent to warming at 37°C or 42°C. Hydrogen water supplemented with platinum colloid (0.3 ppm Pt in 4% polyvinylpyrrolidone) had more antitumor activity than hydrogen water alone, mineral water alone (15.6%), hydrogen water plus mineral water, or platinum colloid alone as observed by decreased cell numbers, cell shrinkage and pycnosis (nuclear condensation)/karyorrhexis (nuclear fragmentation) indicative of apoptosis, together with cell deformation and disappearance of microvilli on the membrane surface. These antitumor effects were promoted by combination with hyperthermia at 42°C. Thus, the nano-bubble hydrogen water with platinum colloid is potent as an anti-tumor agent.

H2 is a therapeutic agent for tumors because it could scavenge free radicals, which is one of the causes for this disease in the human body. Biomedical magnesium (Mg) could release H2 in the biodegradation process, thus it might have antitumor properties. In this study, Mg metal (P-Mg) was subjected to anodic oxidation plus heat treatment to get AO-HT-Mg covered with MgO. In SBF experiments AO-HT-Mg showed bioactivity as it could induce calcium phosphate deposition. The MgO layer played a protective role in the biodegradation process and controlled the H2 releasing rate. In MRMT-1 rat breast carcinoma cell culture experiments, both P-Mg and AO-HT-Mg could inhibit free radical expression in the cells, and AO-HT-Mg showed higher inhibiting ability. In the animal experiments with 72 mice divided into 4 groups, both P-Mg and AO-HT-Mg could inhibit tumor growth. After implantation in the animals, P-Mg showed higher inhibiting ability at the initial stage, and AO-HT-Mg showed higher inhibiting ability after 26 days. The tumor inhibiting properties depended on H2 releasing rates. The results confirm Mg metal has antitumor properties in vivo, and it is possible to optimize its antitumor properties by surface modification.

Hairless albino mice with squamous cell carcinoma were exposed to a mixture of 2.5 percent oxygen and 97.5 percent hydrogen at a total pressure of 8 atmospheres for periods up to 2 weeks in order to see if a free radical decay catalyzer, such as hydrogen, would cause a regression of the skin tumors. Marked aggression of the tumors was found, leading to the possibility that hyperbaric hydrogen therapy might also prove to be of significance in the treatment of other types of cancer.

It has been demonstrated that hydrogen peroxide (H2O2) is directly associated with elevated matrix metalloproteinase-2 (MMP-2) expression in several cell lines. Electrochemically reduced water (ERW), produced near the cathode during electrolysis, and scavenges intracellular H2O2 in human fibrosarcoma HT1080 cells. RT-PCR and zymography analyses revealed that when HT1080 cells were treated with ERW, the gene expression of MMP-2 and membrane type 1 MMP and activation of MMP-2 was repressed, resulting in decreased invasion of the cells into matrigel. ERW also inhibited H2O2-induced MMP-2 upregulation. To investigate signal transduction involved in MMP-2 downregulation, mitogen-activated protein kinase (MAPK)-specific inhibitors, SB203580 (p38 MAPK inhibitor), PD98059 (MAPK/extracellular regulated kinase kinase 1 inhibitor) and c-Jun NH2-terminal kinase inhibitor II, were used to block the MAPK signal cascade. MMP-2 gene expression was only inhibited by SB203580 treatment, suggesting a pivotal role of p38 MAPK in regulation of MMP-2 gene expression. Western blot analysis showed that ERW downregulated the phosphorylation of p38 both in H2O2-treated and untreated HT1080 cells. These results indicate that the inhibitory effect of ERW on tumor invasion is due to, at least in part, its antioxidative effect.

It has been demonstrated that hydrogen peroxide (H(2)O(2)) is directly associated with elevated matrix metalloproteinase-2 (MMP-2) expression in several cell lines. Electrochemically reduced water (ERW), produced near the cathode during electrolysis, and scavenges intracellular H(2)O(2) in human fibrosarcoma HT1080 cells. RT-PCR and zymography analyses revealed that when HT1080 cells were treated with ERW, the gene expression of MMP-2 and membrane type 1 MMP and activation of MMP-2 was repressed, resulting in decreased invasion of the cells into matrigel. ERW also inhibited H(2)O(2)-induced MMP-2 upregulation. To investigate signal transduction involved in MMP-2 downregulation, mitogen-activated protein kinase (MAPK)-specific inhibitors, SB203580 (p38 MAPK inhibitor), PD98059 (MAPK/extracellular regulated kinase kinase 1 inhibitor) and c-Jun NH(2)-terminal kinase inhibitor II, were used to block the MAPK signal cascade. MMP-2 gene expression was only inhibited by SB203580 treatment, suggesting a pivotal role of p38 MAPK in regulation of MMP-2 gene expression. Western blot analysis showed that ERW downregulated the phosphorylation of p38 both in H(2)O(2)-treated and untreated HT1080 cells. These results indicate that the inhibitory effect of ERW on tumor invasion is due to, at least in part, its antioxidative effect.

 

 

SKIN AND RADIATION

Skin and Radiation

Our recent studies suggest that H2 (hydrogen) has a potential as a novel radioprotector without known toxic side effects. The present study was designed to examine the underlying radioprotective mechanism of H2 and its protective role on irradiated germ cells. Produced by the Fenton reaction and radiolysis of H2O, hydroxyl radicals (•OH) were identified as the free radical species that were reduced by H2. We used a H2 microelectrode to dynamically detect H2 concentration in vivo, and found H2 significantly reduced in situ fluorescence intensity of hydroxyphenyl fluorescein; however, as we treated the mice with H2 after irradiation, the decrease is not significant. We found that pre-treatment of H2 to IR (ionizing radiation) significantly suppressed the reaction of •OH and the cellular macromolecules which caused lipid peroxidation, protein carbonyl and oxidatively damaged DNA. The radioprotective effect of H2 on male germ cells was supported by ameliorated apoptotic findings examined by morphological changes and TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling) in testicular tissue, and by preserved viability of stem spermatogonia examined for testicular histological parameters, daily sperm production and sperm quality; we used WR-2721 [S-2-(3-aminopropylamino)ethyl phosphorothioic acid] as a reference compound. Our results represent the first in vivo evidence in support of a radioprotective role of H2 by neutralizing •OH in irradiated tissue with no side effects.

BACKGROUND:

Recent studies show that molecular hydrogen (dihydrogen, H2) has potential as an effective and safe radioprotective agent through reducing oxidative stress. The aim of this study was to investigate whether H2 is able to protect spermatogenesis and hematopoiesis from radiation-induced injuries.

MATERIAL/METHODS:

H2 was dissolved in physiological saline using an apparatus produced by our department. -60Co-gamma rays in the irradiation centre were used for irradiation. Spermatid head counts and histological analysis were used to evaluate spermatogenesis. Endogenous hematopoietic spleen colony formation (endoCFUs), bone marrow nucleated cells (BMNC) and peripheral blood (PB) leukocytes were used to evaluate hemopoiesis.

RESULTS:

This study demonstrates that treating mice with H2 before ionizing radiation (IR) can increase the spermatid head count and protect seminiferous epithelium from IR. This study also demonstrates that H2 could significantly increase the number of endoCFUs, BMNC and PB leukocyte.

CONCLUSIONS:

This study suggests that hydrogen-rich saline could partially protect spermatogenesis and hematopoiesis in irradiated mice.

INTRODUCTION:

Deep burn wounds undergo a dynamic process known as wound progression that results in a deepening and extension of the initial burn area. The zone of stasis is more likely to develop more severe during wound progression in the presence of hypoperfusion. Hydrogen has been reported to alleviate injury triggered by ischaemia/reperfusion and burns in various organs by selectively quenching oxygen free radicals. The aim of this study was to investigate the possible protective effects of hydrogen against early burn-wound progression.

METHODS:

Deep-burn models were established through contact with a boiled, rectangular, brass comb for 20 s. Fifty-six Sprague-Dawley rats were randomly divided into sham, burn plus saline, and burn plus hydrogen-rich saline (HS) groups with sacrifice and analysis at various time windows (6 h, 24 h, 48 h) post burn. Indexes of oxidative stress, apoptosis and autophagy were measured in each group. The zone of stasis was evaluated using immunofluorescence staining, ELISA, and Western blot to explore the underlying effects and mechanisms post burn.

RESULTS:

The burn-induced increase in malondialdehyde was markedly reduced with HS, while the activities of endogenous antioxidant enzymes were significantly increased. Moreover, HS treatment attenuated increases in apoptosis and autophagy postburn in wounds, according to the TUNEL staining results and the expression analysis of Bax, Bcl-2, caspase-3, Beclin-1 and Atg-5 proteins. Additionally, HS lowered the level of myeloperoxidase and expression of TNF-α, IL-1β, and IL-6 in the zone of stasis while augmenting IL-10. The elevated levels of Akt phosphorylation and NF-κB p65 expression post burn were also downregulated by HS management.

CONCLUSION:

Hydrogen can attenuate early wound progression following deep burn injury. The beneficial effect of hydrogen was mediated by attenuating oxidative stress, which inhibited apoptosis and inflammation, and the Akt/NF-κB signalling pathway may be involved in regulating the release of inflammatory cytokines.

 

 

SPINE AND PANCREAS

Spine and Pancreas

In the present study, we examined the mechanisms of hydrogen-rich saline, a reported therapeutic antioxidant, in the treatment of acute spinal cord contusion injury. Male Sprague-Dawley rats were used to produce a standardized model of contuses spinal cord injury (125 kdyn force). Hydrogen-rich saline was injected intraperitoneally (5 ml/kg) immediately, and at 24 and 48 h after injury. All rats were sacrificed at 72 h after spinal cord injury (SCI). Apoptotic cell death, oxidative stress, inflammation, level of Brain derived neurotrophic factor (BDNF) were evaluated. In addition, locomotor behavior was assessed using the Basso, Beattice and Bresnahan (BBB) scale. We observed that administration of hydrogen-rich saline decreased the number of apoptotic cells, suppressed oxidative stress, and improved locomotor functions. Hydrogen-rich saline increased the release of BDNF. In conclusion, hydrogen-rich saline reduced acute spinal cord contusion injury, possibly by reduction of oxidative stress and elevation of BDNF.

Molecular hydrogen, which reacts with the hydroxyl radical, has been considered as a novel antioxidant. Here, we evaluated the protective effects of hydrogen-rich saline on the l-arginine (l-Arg)-induced acute pancreatitis (AP). AP was induced in Sprague-Dawley rats by giving two intraperitoneal injections of l-Arg, each at concentrations of 250mg/100g body weight, with an interval of 1h. Hydrogen-rich saline (>0.6mM, 6ml/kg) or saline (6ml/kg) was administered, respectively, via tail vein 15min after each l-Arg administration. Severity of AP was assessed by analysis of serum amylase activity, pancreatic water content and histology. Samples of pancreas were taken for measuring malondialdehyde and myeloperoxidase. Apoptosis in pancreatic acinar cell was determined with terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling technique (TUNEL). Expression of proliferating cell nuclear antigen (PCNA) and nuclear factor kappa B (NF-kappaB) were detected with immunohistochemistry. Hydrogen-rich saline treatment significantly attenuated the severity of l-Arg-induced AP by ameliorating the increased serum amylase activity, inhibiting neutrophil infiltration, lipid oxidation and pancreatic tissue edema. Moreover, hydrogen-rich saline treatment could promote acinar cell proliferation, inhibit apoptosis and NF-kappaB activation. These results indicate that hydrogen treatment has a protective effect against AP, and the effect is possibly due to its ability to inhibit oxidative stress, apoptosis, NF-kappaB activation and to promote acinar cell proliferation.

Oxidative stress plays a pivotal role in the pathogenesis of varied nervous system diseases. Recent studies have demonstrated that hydrogen has selective antioxidative effect. It selectively reduces the hydroxyl radical (*OH) and peroxynitrite (ONOO(-)), the most cytotoxic of reactive oxygen species (ROS); however, it does not affect other ROS, which play important physiological roles at low concentrations. A large body of experimental studies has proved that hydrogen, through anti-oxidation, anti-inflammatory and inhibiting apoptosis, has a significant therapeutic effect in various neurological diseases, such as ischemia, hypoxia, degeneration and spinal cord contusion. It provides us with a new clinical method for the prevention and treatment of neurological diseases.

BACKGROUND:

Currently, little evidence exists to support whether the therapeutic approaches for treating ordinary acute pancreatitis (AP) are effective in trauma-induced pancreatitis. Hydrogen-rich (H2) saline is an antioxidant treatment capable of ameliorating the severity of L-arginine-induced AP. In this study, we attempted to validate its protective role against traumatic pancreatitis (TP).

METHODS:

A previously established experimental rat model of TP was generated by controlled delivery of high pressure air impact. The protective effects of H2 saline against TP were evaluated in this model system by measuring survival rate and determining changes in histopathology, plasma enzymes, cytokines, and oxidative stress-associated molecules.

RESULTS:

Intraperitoneal administration of H2-rich saline produced a pronounced protection against TP in rats. Significant improvements were observed in survival rate and histopathological findings. In addition, plasma cytokines concentrations were reduced in H2 saline-treated TP rats. Although no marked inhibitory effect on plasma amylase and lipase activities was observed, H2 saline caused considerable suppression of pancreatic malondialdehyde level and recruitment of endogenous pancreatic antioxidants, such as glutathione and superoxide dismutase.

CONCLUSIONS:

H2-rich saline has beneficial effects on TP, presumably because of its detoxification activities against excessive reactive oxygen species. Our findings highlight the potential of H2-rich saline as a therapeutic agent of trauma-induced AP.

 

 

KIDNEY

Kidney

BACKGROUND:

Renal ischemia-reperfusion (I/R) injury is unavoidable in kidney transplantation and frequently influences both short- and long-term allograft survival rates. One of the major events in I/R injury is the generation of cytotoxic oxygen radicals. Recently, hydrogen gas has been reported to display antioxidant properties and protective effects against organ dysfunction induced by various I/R injuries. We investigated whether hydrogen-rich University of Wisconsin (HRUW) solution attenuates renal cold I/R injury.

METHODS:

We prepared HRUW solution by a novel method involving immersion of centrifuge tubes containing UW solution into hydrogen-saturated water. Hydrogen readily permeates through the centrifuge tubes, and thus, the hydrogen concentration of the UW solution gradually increases in a time-dependent manner. Syngeneic rat kidney transplantation was performed, and the animals were divided into three groups: recipients with nonpreserved grafts (control group), recipients with grafts preserved in UW solution for 24 to 48 hr (UW group), and recipients with grafts preserved in HRUW solution for 24 to 48 hr (HRUW group).

RESULTS:

In the early phases, HRUW solution decreased oxidative stress, tubular apoptosis, and interstitial macrophage infiltration in the kidney grafts. Consequently, HRUW solution improved renal function and prolonged recipient survival rate compared with simple cold storage using UW solution. Histopathologically, HRUW treatment alleviated tubular injury and suppressed development of interstitial fibrosis.

CONCLUSIONS:

HRUW solution improved graft function and prolonged graft survival compared with simple cold storage using UW solution by protecting tubular epithelial cells from inflammation and apoptosis. Our new method of organ preservation is a groundbreaking, safe, and simple strategy that may be applied in the clinical setting.

Reactive oxygen species (ROS) contribute to the development of interstitial fibrosis and tubular atrophy seen in chronic allograft nephropathy (CAN). As molecular hydrogen gas can act as a scavenger of ROS, we tested the effect of treatment with hydrogen water (HW) in a model of kidney transplantation, in which allografts from Lewis rats were orthotopically transplanted into Brown Norway recipients that had undergone bilateral nephrectomy. Molecular hydrogen was dissolved in water and recipients were given HW from day 0 until day 150. Rats that were treated with regular water (RW) gradually developed proteinuria and their creatinine clearance declined, ultimately leading to graft failure secondary to CAN. In contrast, treatment with HW improved allograft function, slowed the progression of CAN, reduced oxidant injury and inflammatory mediator production, and improved overall survival. Inflammatory signaling pathways, such as mitogen-activated protein kinases, were less activated in renal allografts from HW-treated rats as compared with RW-treated rats. Hence, oral HW is an effective antioxidant and antiinflammatory agent that prevented CAN, improved survival of rat renal allografts, and may be of therapeutic value in the setting of transplantation.

Background: The present study aimed at investigating the effect of a novel antioxidant, hydrogen (H2) gas, on the severity of contrast-induced acute kidney injury (CIAKI) in a rat model. Methods: CIAKI was induced in rats by intravenous injection of a contrast medium, Ioversol, in addition to reagents inhibiting prostaglandin and nitric oxide synthesis. During the injection of these reagents, the rats inhaled H2 gas or control gas. Results: One day after the injection, serum levels of urea nitrogen were significantly lower in H2 gas-inhaling CIAKI rats (17.6 ± 2.3 mg/dl) than those in control gas-treated CIAKI rats (36.0 ± 7.3 mg/dl), although they both were elevated as compared to untreated rats (14.9 ± 0.9 mg/dl). Consistently, creatinine clearance in H2 gas-treated CIAKI rats was higher than that in control gas-treated counterparts. Renal histological analysis revealed that the formation of proteinaceous casts and tubular necrosis was improved by H2 gas inhalation. Mechanistic analyses showed that inhalation of H2 gas significantly reduced renal cell apoptosis, expression of cleaved caspase 3, and expression of an oxidative stress marker, 8-hydroxydeoxyguanosine, in injured kidneys. Conclusion: Results suggest that H2 gas inhalation is effective in ameliorating the severity of CIAKI in rats by reducing renal cell apoptosis and oxidative stress. © 2015 S. Karger AG, Basel.

BACKGROUND:

Rhabdomyolysis is a leading cause of acute kidney injury. The pathophysiological process involves oxidative stress and inflammation. Hydrogen-rich saline (HRS) is an antioxidant and anti-inflammatory. This study explored the protective effect of pretreatment with HRS on the development of glycerol-induced rhabdomyolysis acute kidney injury.

MATERIALS AND METHODS:

Forty-eight rats were randomly divided into four equal groups. Group 1 served as the control, group 2 was given 50% glycerol (10 mL/kg, intramuscular), group 3 was given glycerol after 7 d pretreatment with high dose HRS (10 mL/kg/d, intraperitoneal), and group 4 was given glycerol after 7 d pretreatment with low dose HRS (5 mL/kg/d, intraperitoneal). Renal health was monitored by serum creatinine (Cr), urea, and histologic analysis; rhabdomyolysis was monitored by creatine kinase (CK) levels; and oxidative stress was monitored by kidney tissue reactive oxygen species (ROS), malondialdehyde, 8-hydroxydeoxyguanosine (8-OH-dG), superoxide dismutase (SOD), and glutathione peroxidase (GSH-PX) levels. Inflammation was monitored by interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) evaluation.

RESULTS:

Glycerol administration resulted in an increase in the mean histologic damage score, serum Cr, urea and CK, kidney tissue ROS, malondialdehyde, 8-OH-dG, GSH-PX, IL-6, and TNF-α, and a decrease in kidney tissue superoxide dismutase activity. All these factors were significantly improved by both doses of HRS, but the mean histologic damage score, urea, Cr, CK, ROS, 8-OH-dG, GSH-PX, IL-6, and TNF-α for the high dose HRS treatment group were even lower.

CONCLUSIONS:

Pretreatment by HRS ameliorated renal dysfunction in glycerol-induced rhabdomyolysis by inhibiting oxidative stress and the inflammatory response.

 

 

FEMALE REPRODUCTIVE ORGANS

Female Reproductive Organs

Oxidative stress is generated during the pathophysiology of endometriosis (EMT). Hydrogen (H2) has been demonstrated as a gas antioxidant. The aim of the present study is to evaluate the protective effect of H2 on EMT in rats. Sprague Dawley rats with surgically induced EMT were randomly received the inhalation of 67% H2-33% oxygen (O2) mixture (1 h/d, 4 weeks) immediately after the EMT surgery or 4 weeks after the operation. The mixture of 67% N2-33% O2 was also used to exclude the possible influence of the increased O2. Eight weeks after the operation, the endometrial tissues were weighted and analyzed using histology, immunohistochemistry, and real-time polymerase chain reaction. Several antioxidant enzymes and malondialdehyde were also measured in serum and tissue. The estrous cycles were monitored for H2 safety. The results showed that both profiles of high-dose H2 breathing reduced the size of the endometrial explants, inhibited cell proliferation, improved superoxide dismutase, glutathione peroxidase, malondialdehyde, and catalase activities, and regulated the expression of matrix metalloproteinase 9 and cyclooxygenase 2. However, inhalation of the same dose of nitrogen failed to show the protection. High-dose H2 breathing did not change the normal estrous cyclicity. These results suggest that 67% H2-33% O2 breathing has a beneficial effect on EMT model rats, and inhalation of a high dose of H2 could be a potential method applied in clinical practice.

Premature ovarian failure (POF) is a disease that affects female fertility but has few effective treatments. Ovarian reserve function plays an important role in female fertility. Recent studies have reported that hydrogen can protect male fertility. Therefore, the authors explored the potential protective effect of hydrogen-rich water on ovarian reserve function through a mouse immune POF model. To set up immune POF model, fifty female BALB/c mice were randomly divided into four groups: Control (mice consumed normal water, n = 10), hydrogen (mice consumed hydrogen-rich water, n = 10), model (mice were immunized with zona pellucida glycoprotein 3 [ZP3] and consumed normal water, n = 15), and model-hydrogen (mice were immunized with ZP3 and consumed hydrogen-rich water, n = 15) groups. After 5 weeks, mice were sacrificed. Serum anti-Müllerian hormone (AMH) levels, granulosa cell (GC) apoptotic index (AI), B-cell leukemia/lymphoma 2 (Bcl-2), and BCL2-associated X protein (Bax) expression were examined. Analyses were performed using SPSS 17.0 (SPSS Inc., Chicago, IL, USA) software. Immune POF model, model group exhibited markedly reduced serum AMH levels compared with those of the control group (5.41 ± 0.91 ng/ml vs. 16.23 ± 1.97 ng/ml, P = 0.033) and the hydrogen group (19.65 ± 7.82 ng/ml, P = 0.006). The model-hydrogen group displayed significantly higher AMH concentrations compared with that of the model group (15.03 ± 2.75 ng/ml vs. 5.41 ± 0.91 ng/ml, P = 0.021). The GC AI was significantly higher in the model group (21.30 ± 1.74%) than those in the control (7.06 ± 0.27%), hydrogen (5.17 ± 0.41%), and model-hydrogen groups (11.24 ± 0.58%) (all P ❰0.001). The GC AI was significantly higher in the model-hydrogen group compared with that of the hydrogen group (11.24 ± 0.58% vs. 5.17 ± 0.41%, P = 0.021). Compared with those of the model group, ovarian tissue Bcl-2 levels increased (2.18 ± 0.30 vs. 3.01 ± 0.33, P = 0.045) and the Bax/Bcl-2 ratio decreased in the model-hydrogen group. Hydrogen-rich water may improve serum AMH levels and reduce ovarian GC apoptosis in a mouse immune POF model induced by ZP3.

The present study aimed to investigate the effects of hydrogen rich saline solution (HRSS) in a rat model of ovarian ischemia-reperfusion injury. Thirty-six female Wistar-albino rats were grouped randomly, into six groups of six rats. The groups were classified as: sham (S), hydrogen (H), torsion (T), torsion/detorsion (TD), hydrogen-torsion (HT), and hydrogen-torsion/detorsion (HTD). Bilateral adnexal torsion was performed for 3h in all torsion groups. HRSS was given 5ml/kg in hydrogen groups intraperitoneally. Malondialdehyde (MDA) and glutathione-S-transferase (GST) levels were measured in both the plasma and tissue samples. Tissue sections were evaluated histopathologically, and the apoptotic index was detected by TUNEL assay. The results were analyzed by Kruskal-Wallis and Pearson chi-square tests using computer software, SPSS Version 20.0 for Windows. The MDA levels were higher and GST levels were lower in the torsion and detorsion groups when compared to other groups, but the differences were insignificant (P>0.05). The MDA levels were lower and GST levels were higher in the HT and HTD groups compared with the T and TD groups (P>0.05). Follicular injury, edema, vascular congestion, loss of cohesion and apoptotic index were higher in the torsion groups but decreased in the groups that received HRSS. According to histopathological and biochemical examinations, HRSS is effective in attenuating ischemia-reperfusion induced ovary injury.