In this study we have shown that the administration of hydrogen-enriched saline prior to the reperfusion stage in a surgical model of partial ischemia-reperfusion injury attenuates hepatic damage and dysfunction. This is associated with reduced HMGB1 and pro-inflammatory cytokine production. We hypothesise that this is secondary to reduced ROS generation and oxidative stress in the hydrogen-enriched saline treatment group.
Although reperfusion after sustained ischemia salvage tissue, the reperfusion itself paradoxically induces injury (“reperfusion injury”). It is now well recognized that a protective stimulus can be applied at the onset of reperfusion, thereby attenuating reperfusion injury. This is known as postconditioning. It has been investigated most extensively in the heart but has also been described in the liver
. The aetiology is complex and multifactorial involving tissue damage secondary to ATP depletion during hypoxia, followed by further cell injury occurring after the resolution of hypoxia and return of perfusion
. Although controversial, both stages are considered to independently mediate tissue damage via the production of directly injurious reactive oxygen species (ROS)
, as well as substances that modulate a local and systemic inflammatory response.
Oxidative stress can be defined as a disturbance in the balance between the production of ROS (with strong cellular oxidizing potential) and antioxidant defences
. It plays an important role in the pathogenesis of various hepatic disorders
, including I/R injury. ROS generated intracellularly include the superoxide anion radical (O2
), hydrogen peroxide (H2O2), hypochlorous acid (HClO), hydroxyl radical (OH
), and singlet oxygen (1O2). These agents are produced as a consequence of normal mitochondrial processes
, and some behave as endogenous intracellular signaling molecules at low concentrations. However, the strongest of the oxidant species, the hydroxyl radical (
OH), is highly toxic and is not formed by any enzymatic process, but rather from H2O2 in the presence of divalent metal ions via the Fenton reaction. It reacts almost instantaneously with many cellular components, including the polyunsaturated fatty acids of membrane lipids, nucleic acids, and proteins. No known detoxification system exists and scavenging (
OH) is critical to prevent nuclear DNA and protein disorganization as well as lipid peroxidation
. Lipid peroxidation can disrupt cellular membrane integrity leading to changes in its fluidity and permeability
. In addition, lipid peroxides degrade to reactive aldehyde products, including malondialdehyde (MDA) and 4-hydroxyl-2-nonenal (HNE)
The hydrogen molecule has antioxidant properties. It has been demonstrated previously that liver IRI can trigger a cascade of innate-dominated pro-inflammatory immune responses that activate an adaptive immune response, culminating in systemic inflammation
[3, 32]. TLR4 activation by endogenous and exogenous ligands has been confirmed to stimulate the production of pro-inflammatory cytokines including TNF-α and IL-6. These mediate cell death and can further enhance the pro-inflammatory response
[33, 34]. Indeed, recent studies have shown that endogenous TLR4 ligands, including HMGB1 generated during liver IRI, can trigger a local inflammatory reaction that culminates in hepatocellular damage. Similarly, blocking HMGB1 production and release can effectively minimize liver damage from ischemia
However, recent studies using hydrogen treatment in liver IRI have mainly focused on its anti-oxidative rather than anti-inflammatory action, with little published work having explored HMGB1's role in this process
. We have shown in the current study that hepatic IR injury triggers the release of HMGB1 in liver tissue and its subsequent release into serum, and that intraperitoneal hydrogen-rich saline can modulate this.
There is increasing evidence that extracellular HMGB1 acts as an inflammatory mediator in ischemia, hemorrhagic shock, noninfectious hepatitis, and peripheral tissue trauma
[35, 36]. HMGB1 is actively secreted by activated macrophages
 and passively released through the porous membrane of cells undergoing necrosis. It has been shown to mediate lethality in sepsis models
[38, 39]. Recent studies show that HMGB1 is mobilized and released in response to hypoxia, suggesting that the actions in IR occur before cell death
. HMGB1 release from cultured hepatocytes is an active process regulated by ROS including H2O2. Furthermore, HMGB1 release from hepatic cells can occur without causing measurable cell death, and HMGB1 release is mediated by NADPH oxidase or TLR4 signal transduction in a ROS-dependent manner
. However, the exact mechanism of ROS regulating HMGB1 release remains unknown. In our study, the results of immunohistochemistry showed that HMGB1 was not detected in the sham group, while it is found both in nucleus and cytoplasm of hepatocytes induced by ischemia reperfusion. Meanwhile, much less HMGB1 was detected in hydrogen-enriched saline treated animals compared with saline group. The results indicated that the
OH attacked the DNA and lead to exposure antigen recognition site of HMGB1. The freed HMGB1 moved from nucleus to cytoplasm through increased permeability nuclear membrane induced by peroxidation. Finally, they were released to extracellular and acted as an initiator of inflammation. Therefore, our results suggest that, during hepatic I/R, systemic HMGB1 levels are associated with the degree of hepatocellular peroxidation, indicating that HMGB1 is a marker of cell damage that reflects the integrity of cellular structure.
Hydrogen has a powerful ability to penetrate biomembranes and diffuse into the cytosol, mitochondria and nucleus thereby effectively reducing the hydroxyl radical, the most cytotoxic of reactive oxygen species. Its ability to protect nuclear DNA and cell membrane suggests that it can reduce oxidative stress induced cellular injury and the subsequent inflammatory response
. Hydrogen gas cannot be produced by the human body since mammalian cells lack the hydrogenase activity. However, it is continuously produced by colonic bacteria in the body and normally circulates in the blood, so it is physiologically safe for humans to inhale hydrogen at a relatively low concentration. Medical use of hydrogen in the past was limited to test the effects of antibiotic therapy. Previously, other therapeutic strategies for scavenging reactive oxygen species seemed promising in animal models but most of them failed in human clinical trials
. This study demonstrates that hydrogen-rich saline protects the liver against cellular injury and organ dysfunction through a mechanism that reduces the impact of oxidative stress and associated inflammation. Its ease of preparation and administration, and its favourable safety profile make hydrogen-enriched saline an attractive and potentially clinically useful tool.
Certain limitations to our study should be considered. The intraperitoneal administration of our treatment agent is not a clinically used modality, although intravenous administration has been safely employed by other researchers in animal models of organ I-R (9). In addition, blood and tissue levels of hydrogen were not determined so we do not know the bioavailability of this route of administration.