Protective Effects And Mechanisms Of Hydrogen-rich Saline On Intestinal Ischemia-reperfusion Injury And Carbon Tetrachloride Induced Hepatic Injury In Rats

Protective Effects and Mechanisms of Hydrogen-rich Saline on Intestinal Ischemia-reperfusion Injury and Carbon Tetrachloride Induced Hepatic Injury in RatsBackground:Intestinal ischemia/reperfusion (II/R) injury and drug/chemical induced liver injury are two common clinical problems faced in burn department and critical care medicine. II/R injury often occurs in severe burns, acute hemorrhagic, shock, sepsis, intestinal obstruction, mesenteric arterial occlusion by thrombi or embolisms, and so on. II/R is accompanied by decreased contractile activity, increased microvascular permeability and dysfunction of mucosal barrier, which could eventually cause systemic inflammatory response syndrome and remote organ damage, and even to multiple organ dysfunction syndrome (MODS). Drug or chemical induced liver injury is also a major health problem faced in clinical practice and there is still lack of effective therapeutic strategies or specific medicines for such liver diseases. The prevalence of drug or chemicals induced hepatotoxicity went up over the past decades following the drug development. In addition, liver is the main metabolic and detoxifying organ. It is prone to be injured as it is often exposed to environmental toxicants, drugs, and other xenobiotics or endogenous agents, so the protection of liver is the first task for us to conduct cyto-preservation measures. CCl4 is a potent toxicant for producing model of drug or chemical induced hepatic injury. CCl4 is also a common compound used in industry, and the treatment of CCl4 toxicity is an important subject in critical care and emergency medicine.Oxidative stress plays a very important role in the pathological process of intestinal ischemia/reperfusion injury and CCl4 induced hepatic injury. The excessive production and activation of radical oxygen species is the mechanism in common in these two models of injury. Reactive oxygen species (ROS) are reactive molecules that contain the oxygen atom. They are highly reactive due to the presence of unpaired valence shell electrons. Major ROS includes hydrogen peroxide, superoxide, and hydroxyl radical. Excessive ROS will damage DNA, lipids and proteins in cells. As we know, the hydroxyl radical is the most reactive product of ROS generated in cells. It is biologically important to eliminate hydroxyl radicals, because superoxide anion and hydrogen peroxide are detoxified by antioxidant defense enzymes, superoxide dismutase, and peroxidase or glutathione-peroxidase, respectively; however, no endogenous enzymatic pathway is known to neutralize hydroxyl radicals. Recently, Ohsawa et al. found that molecular hydrogen could selectively reduce·OH in vitro and inhalation of hydrogen could exert therapeutic antioxidant activity in a rat middle cerebral artery occlusion model. Therefore, in this study, we developed hydrogen saturated physiological saline (hydrogen-rich saline), which is easy and safe to apply. We investigated whether administration of hydrogen-rich saline exerted protective effect in the models of intestinal ischemia/reperfusion injury and CCl4 induced hepatic injury.Methods:1. The SD rats were randomly divided into four groups:(1) sham-operated plus vehicle physiological saline treatment; (2) intestinal ischemia/reperfusion plus vehicle physiological saline treatment; (3) intestinal ischemia/reperfusion plus hydrogen-rich saline treatment; (4) intestinal ischemia/reperfusion plus nitrogen-rich saline treatment. A rat small intestinal I/R model was adapted by clamping the superior mesenteric artery maintaining for 45 minutes and removing the clamp for 120 minutes reperfusion. Physiological saline, hydrogen-rich saline or nitrogen-rich saline (5 ml/kg) was given via jugular venous cannula infusion at 10 minutes before reperfusion initiation, respectively. 120 minutes after reperfusion initiation, blood was drawn and intestinal tissue samples were collected. The following markers are measured:1) HE staining and histopathological observation;2)Serum diamine oxidase (DAO), tissue malondialdehyde (MDA) and myeloperoxidase (MPO)level;3) Serum tumor necrosis factor-a (TNF-a), interleukin-1β(IL-1β), and interleukin-6 (IL-6).2. The SD rats were randomly divided into three groups:Control, CCl4+ nitrogen-rich saline and CC14+ hydrogen-rich saline. CC14 mixed with olive oil (1:1 v/v, 2 ml/kg) was injected intraperitoneally for hepatic injury model. (For survival analysis experiment, Rats were injected with a lethal dose of CCl4 mixed with olive oil (1:1 v/v,7.5 ml/kg) intraperitoneally.) Hydrogen-rich saline (5 ml/kg) or nitrogen-rich saline of equivalent volume was given intraperitoneally 10 minutes prior to the injection of CCl4 and every 3 hours after the administration of CCl4. The blood and liver tissue samples were collected 12 hours after CCl4 administration (For MAP kinase tested liver tissues were collected 3 hours after CCl4 administration). The following markers are measured:1) HE staining and histopathological observation;2) Serum AST, ALT, LDH activities and DBIL, TBIL levels;3) TUNEL histochemical assay and tissue caspase-3 activity;4) Tissue malondialdehyde (MDA) and glutathione (GSH) level;5) Serum tumor necrosis factor-α(TNF-α);6) Tissue MAP kinase signal molecules:JNK, ERK and p38 MAPK phosphorylation.Result:1. The intestine damage was detected microscopically and was assessed by Chiu score system after I/R injury. In addition, serum DAO activity, TNF-α, IL-1βand IL-6 levels, tissue MDA, protein carbonyl and MPO activity were all increased significantly by I/R injury. Hydrogen-rich saline reduced these markers and relieved morphological intestinal injury while no significant reduction was observed in the nitrogen-rich saline treated animals.2. Hydrogen-rich saline administration significantly attenuated the CCl4-induced hepatic injury, with the alleviation of pathological hepatic lesions and the reduction of elevated serum hepatic enzyme activities and bilirubin levels, and prolonged the survival time of CCl4-intoxicated rats. In addition, hydrogen-rich saline ameliorated the hepatocytes apoptosis, as well as lowered the increase of hepatic caspase-3 activity in CCl4-treated rats. Furthermore, the increases of malondialdehyde concentrations as well as the reduction of glutathione levels in liver induced by CCl4 were diminished by hydrogen-rich saline treatment. Moreover, the elevated serum TNF-a levels and the activation of JNK, ERK and p38 MAPK induced by CCl4 was downregulated by hydrogen-rich saline treatment.Conclusion:1) Hydrogen-rich saline protected the small intestine against I/R injury possibly by relief of oxidative stress, attenuating PMN infiltration and reduction of inflammatory mediators elevation.2) Hydrogen-rich saline has a protective effect on CCl4 induced hepatotoxicity in rats and the hepatoprotective effects of hydrogen-rich saline may be mediated through anti-oxidative, anti-inflammatory and anti-apoptotic pathways, as well as the inhibition of MAP kinase activation.3) This study developed and applied hydrogen-rich saline for intravenous and intraperitoneal delivery. It supplies evidence for effects, mechanisms, delivery approach, and potential for clinical application of hydrogen. And it supplies potential measurement for treating critical illness patients in burn department.