Series Studies Of The Biological Effects Of Hydrogen Gas

Hydrogen, the simplest elements in the universal, is colorless,odorless,tasteless and reducing agent composed of two atoms. Hydrogen has low solubility, which cannot easily been absorbed by human bodies. So the biological effects of hydrogen on higher organisms have been overlooked. In the realm of radiochemistry, it has been reported that hydrogen could react with hydroxyl radical, which received no attention from biologists. Scientists tried to demonstrate that hydrogen could react with high reactive free radicals in hyperbaric conditions, but no direct results can prove this reaction. Therefore, most biologists think hydrogen is an inert gas. However, in 2007, it was first reported that inhalation of 2% hydrogen gas confer protection in focal brain ischemia reperfusion injury in an animal model. Subsequently, our primary study proved neuroprotective effects of intraperitoneal injection of hydrogen saline in a neonatal hypoxia-ischemia rat model in the first place. Though, selective antioxidation is one possible mechanism of hydrogen treatment, the exactly role in vivo by hydrogen remains to be elucidated, and still whether hydrogen has protective effect on other kinds of ischemia injury is yet to be discovered. Hence, this project will apply three kinds of ischemia models (myocardial ischemia reperfusion, hyperoxia lung injury and CO poisoning delayed neurologic sequelae) to investigate the possibly evidence of selective antioxidation of hydrogen in vivo. Accordingly, I will study on the intensive mechanism underlines in the protective effect of hydrogen on ischemic diseases, exploit hydrogen from basic research to clinical application. The most important of all, establishing the foundation for further evaluation of the biology function of hydrogen.Currently, restoring blood flow in an acutely occluded vessel represents the most effective, long-term clinical therapy for acute myocardial infarction (AMI). Although restoration of blood flow is critical, the reintroduction of molecular oxygen triggers a cytotoxic cascade during which reactive oxygen species (ROS) are generated by the mitochondria. This burst of reactive oxygen species irrevocably drives downstream signaling networks that lead to cellular necrosis and apoptosis, which is named lethal reperfusion injury. Reperfusion injury accounts for up to 50% of the final size of the infarct, providing an important potential target for protection of the heart. In the current study, we tested whether hydrogen-rich saline, which is easy to use, safe, and economical, confers cardioprotection against myocardial ischemia reperfusion injury in rats. We evaluated specifically the reduction of infarction and improvement of functions at 24 hrs after intraperitoneal injection of hydrogen-rich saline.Hyperoxic acute lung injury (HALI) is caused by prolonged supplement of very high concentrations of oxygen (fractional concentrations of oxygen greater than 50%). It is generally accepted that increased generation of reactive oxygen species (ROS) plays an important role in lung injury during exposure to hyperoxia. Our previous study has demonstrated that hydrogen-rich saline is a novle antioxidant, which could reduce oxidative stess and inflammation. Therefore, the present study was to investigate the possible therapeutic effects of hydrogen-rich saline on lung injury induced by hyperoxia in rats. We applying rat HALI model, evaluated specifically lung injury related markers, oxidative markers and inflammatory factors to confirm the effect of hydrogen and preliminary investigate the mechanisms, providing the new evidences of prevention hyperoxia lung injury.Carbon monoxide (CO) poisoning is one of the most common gas intoxications confronting clinical toxicologists, intensivists and emergency physicians worldwide. The clinical symptoms of CO poisoning are often nonspecific and diverse (2). Among survivors, more than half of those with serious poisoning develop a cognitive sequelae between 3 days and 4 weeks after CO poisoning. The pathophysiology of CO poisoning is related not only to hypoxia but also to an elevated excitatory amino acids and oxidative stress (1, 4-7). Reactive oxygen species produced by activated neutrophils, mitochondria, and xanthine oxidase lead to lipid peroxidation which render delayed neurological injuries. The aim of this study is to examine our above mentioned hypothesis and to evaluate the feasibility and efficacy of hydrogen therapy for severe acute CO poisoning encephalopathy in an animal model. PartⅠHydrogen-Rich Saline Protects Myocardium against Ischemia/Reperfusion Injury in RatsMethods:1. To establish the in vivo myocardial I/R model, a suture was tightened around the proximal left anterior descending (LAD) coronary artery, after 30 min of ischemia, the tube for the myocardial reperfusion was removed and the heart were reperfusion for 24h.2. 24 h after ischemia reperfusion, a PE 50 catheter was introduced into the right carotid artery to measure the hemodynamic parameters.3. 24 h after ischemia reperfusion, heart sections were thawed and incubated in a 1% tetrazolium chloride (TTC) phosphate buffered solution (pH 7.4) at 37oC for 15 min and fixed in 10% formalin to measure the infarct size.4. Histological analysis of heart sections stained with H&E were scored 24 hrs after reperfusion5. Extent of lipid peroxidation in the AAR (area at risk zones) myocardium and plasma were examined,caspase-3 activity was also examined in AAR.6. The numbers and locations of 8-OHdG positive cells were detected by immunohistochemistry, TUNEL staining was to detect apoptotic cell death.Results:1. The intraperitoneal injection of hydrogen-rich saline (5ml/Kg)5min before reperfusion, significantly decreased I/R induced degrading of hemodynamic parameters (P<0.05).2. The infarct size in the H2 group was significantly smaller than in the Control group.3. MDA concentration was significantly reduced in plasma and AAR. H2 comparably reduced caspase-3 activity relative to the Control group in AAR.4. Hydrogen treatment comparably decreased the DNA marker, 8-OHdG.PartⅡ: Hydrogen-rich saline provides protection against hyperoxic lung injuryMethod:1. To establish hyperoxia lung injury model, the animals were placed in chambers exposed continueously to hyperoxia (>98% O2) for 60 h. 2. Pleural fluid volume and the wet-to-dry weight ratio were measured, morphological changes were observed by H&E staining.3. Oxidative stress markers MDA and SOD were examined in lung tissues.4. TNF-a, IL-1b, MPO and 8-OHdG levels in the lung tissues were determined by ELISA.5. TUNEL staining was to detect apoptotic cell death in lung tissues.Results:1. Hydrogen could effectively prevent HALI, reduce volume of pleural effusion and level of W/D. H&E staining showed that hydrogen treatment could significantly reduce lung edema, alveolar hemorrhage and extensive inflammatory cell infiltration.2. Hydrogen treatment significantly decreased the SOD, MDA and 8-OHdG levels,indicating the inhibition of oxidative stress.3. Hydrogen treatment significantly reduced the elevation of TNF-a, IL-1b and MPO in the lung tissues.4. Hydrogen treatment comparably decreased the percent of TUNEL-positive epithelial and endothelial cells relative to the control group and physiological saline group.PartⅢHydrogen-rich saline reduces delayed neurologic sequelae in experimental carbon monoxide toxicityMethod:1. To establish CO poisoning model, rats breathed 1000 ppm CO for 40 min then 3000ppm for up to 20 min, until they lost consciousness, then they were removed to breathe room air and regain consciousness.2. 9 days after CO poisoning, animals were sacrificed, and fixed with 4% paraformaldehyde, brain segment was removed, Nissl staining was applied to indicate the morphology changes of cerebral cortex and hippocampus.3. The activation of microglias was measured by immunostaining of Iba1, MBP degradation was detected by immunohistochemistry.4. Concentrations of TNF-α, IL-1βand IL-6 in the cerebral cortex and hippocampus were measured by ELISA.5. Morris Water maze testing was performed to test cognitive function differences among difference groups. Results:1. The number of Nissl staining positive cells in cortex and hippocampus of control animals was lower than that of hydrogen treated animals.2. Hydrogen treatment significantly reduced the expression of microglia after CO poisoning. MBP degradation was reduced in hydrogen treated group.3. Levels of TNF-α, IL-1β, IL-6 and 8-OHdG protein in the cerebral cortex and hippocampus were significantly lowered by hydrogen treatment when compared with control group.4. Hydrogen treatment could protect against decrements in learning function after CO poisoning obviously through Morris Water maze testing.Conclusion:1. Hydrogen-rich saline significantly improved post-ischemic functional recovery of rat hearts. This cardiac improvement may result from radical oxygen species (ROS) scavenging effect of molecular H2.2. Hydrogen-rich saline ameliorated hyperoxia induced acute lung injury by reducing oxidative stress and inflammatory cascades in lung tissue.3. Hydrogen-rich saline peritoneal injection improves histological and functional assessment in a rat model of CO encephalopathy. Hydrogen saline has potentials as a novel and alternative therapy for severely CO-poisoned patients with delayed neurologic sequelae. The therapeutic effects of hydrogen-rich saline may be related to antioxidant and anti-inflammatory actions.4. Hydrogen is an important physiological regulatory factor with antioxidant, anti-inflammatory and anti-apoptotic protective effects on cells and organs-warrants further clinical evaluation in oxidative stress-related diseases.