Hydrogen-rich Saline And Hyperbaric Oxygen Preconditioning Ameliorates Intestinal Ischemia-Reperfusion Injury In Rats

BackgroundThe intestinal ischemia-reperfusion (I/R) injury is a devastating syndrome which occurs in a variety of clinical settings including abdominal aortic aneurysm, small intestinal transplantation, cardiopulmonary bypass, strangulated hernia, and neonatal necrotizing enterocolitis. I/R injury of the intestine also occurs in septic and hypovolemic shock. The consequence of intestinal I/R injury not only alternates absorptive function of intestine, but also may cause intestinal bacteria colony shifting, and even lead to the multiple organ failure (MOF). Overproduction of toxic hydroxyl radicals has been shown to play an important role in the pathogenesis of intestinal I/R injury, which thereby promoting the lipid peroxidation, DNA oxidation, thiyl radical formation and mitochondrial depolarization and eventually leading to cellular apoptosis and necrosis. Toxic hydroxyl radicals can damage cellular membrane and subcellular structures, which contain large amounts of phospholipids and protein, resulting in lipid peroxidation and sequentially structural and metabolic alterations, and leading to cell death and necrosis.Hydrogen (H2), which could react with hydroxyl radical to produce water, has been considered as a novel antioxidant and has been demonstrated recently to have high protective properties in human, animal, and in vivo and in vitro studies, including the protective effect on lipid and glucose metabolism in patients with type 2 diabetes or impaired glucose tolerance, transplantation induced intestinal graft injury, chronic liver inflammation, arteriosclerosis, myocardial ischemia-reperfusion (I/R) injury, acute oxidative stress and focal brain I/R injury, and chronic allograft nephropathy. A recent study provided evidence that inhaled H2 gas markedly protect the brain against I/R injury and stroke in cell-free systems by selectively reducing the hydroxyl radical, the most cytotoxic one of reactive oxygen species (ROS), which is commonly produced under various pathological conditions including in intestinal I/R injury.Hyperbaric oxygenation (HBO) has been widely used as a primary therapy in patients with carbon monoxide poisoning, decompression sickness, and arterial gas embolism, and it has been used as an adjunctive therapy for the treatment of various diseases accompanied by impaired oxygen delivery. Interestingly, HBO preconditioning has also shown promising results in some models of ischemia, including I/R injury of brain, spinal cord, liver, and myocardium. However, protective effects of HBO preconditioning has not been verified concerning intestinal ischemia-reperfusion injury.Objective1. To determine the protective effect of hydrogen-rich saline on intestinal I/R injury and the underlying related mechanisms using a rat mesenteric ischemia-reperfusion injury model.2. To investigate whether HBO preconditioning plays a role in intestinal I/R injury and its possible mechanisms.Methods1. Intestinal I/R injury was induced in Sprague-Dawley rats using bulldog clamps in superior mesenteric artery by 45 min ischemia followed by 1 h reperfusion.2. Following reperfusion, segments of terminal jejunum were rapidly taken and transferred into isolated organ bath and responses to KCl were recorded.3. Samples of terminal jejunum were taken for measuring malondialdehyde(MDA) and myeloperoxidase(MPO).4. Cytokine levels in serum were determined by highly sensitive enzyme-linked immunosorbent assay(ELISA) kits.5. Apoptosis in intestinal epithelium was determined with terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling technique (TUNEL).6. Expression and distribution of proliferating cell nuclear antigen (PCNA) were detected with immunohistochemistry.Results1. In part one, compared with I/R or I/R + saline group, the intestinal smooth muscle contractility in the hydrogen-rich saline treatment animals recovered more significantly (P < 0.01). The intestinal MDA content and MPO activity increased significantly in intestinal I/R and I/R + saline groups when compared with sham-operated group (P< 0.05). More importantly, treatment of rats with hydrogen-rich saline at a dose of 6 ml/kg significantly attenuated the increasing in intestinal MDA level and MPO activity (P< 0.05). Serum TNF-αand IL-6 concentrations were significantly elevated in intestinal I/R and I/R + saline animals when compared with those in sham-operated animals. Hydrogen-rich saline administration lowered the I/R-induced elevation of serum TNF-a and IL-6 concentrations. In intestinal crypts, hydrogen-rich saline administration significantly elevated the positive rate of PCNA, compared with I/R or I/R + saline group (P< 0.01). There were few apoptotic cells in sham-operated group. The apoptotic index in I/R and saline-treated groups markedly increased compared with sham-operated group (P< 0.01). When rats treated with hydrogen-rich saline, fewer TUNEL-positive cells were seen and the apoptotic index significantly decreased as compared to the I/R or I/R + saline-treated group (P< 0.01).2. In part two, compared with I/R group, the intestinal smooth muscle contractility in the HBO preconditioning animals recovered more significantly (P< 0.01). The intestinal MDA content and MPO activity increased significantly in intestinal I/R group when compared with sham-operated group (P< 0.05). More importantly, treatment of rats with HBO preconditioning significantly attenuated the increasing in intestinal MDA level and MPO activity (P< 0.05). There were few apoptotic cells in sham-operated group. The apoptotic index in I/R group markedly increased compared with sham-operated group (P< 0.01). When rats treated with HBO preconditioning, fewer TUNEL-positive cells were seen and the apoptotic index significantly decreased as compared to the I/R group(P< 0.01).Conclusions1. Hydrogen treatment has a protective effect against intestinal I/R injury. This protective effect is possibly due to its ability to inhibit I/R-induced oxidative stress, inflammation, apoptosis and to promote epithelial cell proliferation.2. HBO preconditioning protects the small intestine against I/R injury mediated by anti-oxidative, anti-inflammatory, and anti-apoptotic activities.