Protective Mechanism Of Hydrogen-enriched Saline In Hepatic Ischemia-reperfusion Injury

Backgroud:Hepatic ischemia-reperfusion injury is common in liver surgery, particularly in hepatic transplantation, part lobes of liver resection, hemorrhagic shock and trauma. HIRI is a dynamic process, the mechanism of which has not been clear now. Free oxygen radical release, active pro-inflammatory cytokines expression, calcium overload, microcirculation dysfuction, mitochondrial damage as well as energy metabolism disruption were considered to involve the HIRI pathophysiological procedure. As the most important defensive barrier, hepatic injury induced the endotoxemia with high probability, which in turn aggravate the injured liver to nonfunction, with the result of sepsis as well as multiple organ failure or even to death. The current papers reported that the therapeutic strategies on HIRI mainly aimed at anti-inflammation and antioxidation were proved to be available. Therefore, seeking for an agent with the characteristic of high specificity, mild side reaction, potential clinical application prospect and effective anti-inflammatory and antioxidative action is requisite and significant. Recent papers point out that hydrogen molecule act as an oxygen free radical scavenger to neutralize the cytotoxic hydroxyl radical selectively and hydrogen gas inhalation was demonstrated to protect liver, brain, kindey against ischemia reperfusion injury. Otherwise, for explosion danger of mixed gas, special equipment for inhalation, complex operating sequence, inhalation hydrogen gas is difficult to generalize in clinical. Thus, the suitable therapeutic method need to be further studied. It is calculated theoretically that hydrogen gas could be dissolved to saturation in saline under 0.4 KPa, which is named as hydrogen-enrich saline. The current study was designed to evaluate the protection of this novel agent in hepatic ischemia reperfusion injury and probe the underlying mechanism.Methods:â‘ Serum alanine aminotransferase level as well as morphological change was detected in rats treated with sham, hepatic ischemia reperfusion only, saline before hepatic IR and hydrogen-enrich saline before hepatic IR respectively to determine the protection of this novel agent in HIRI.â‘¡Malondialdehyde level in hepatic tissue was also detedcted to estimate the antioxidation of hydrogen-enrich saline. To further determine the underlying mechanism of cytoprotection of novel fluid, the HNE and 8-OH-G location was detected through immunohistochemistry and the positive percentage was compared among sham, control and therapeutic groups.â‘¢Pro-inflammative cytokines levels including TNF-a and IL-6 mRNA levels in hepatic tissue after 2,6,12,24 h reperfusion between control and therpeutic groups was measured. HMGB1 protein expression in hepatic tissue and plasma were detected through westernblot analysis between two groups. Immunohistochemistry was performed to determine HMGB1 location in hepatic tissue.Results:1. Study of animal models:Significantly lower serum alanine aminotransferase as well as tissue malondialdehyde level were observed in hydrogen-enrich saline treated animals than controls. Moreover, morphological detetion results showed little necrosis in hydrogen-enrich saline treated animals than controls. Furthermore, HNE and 8-OH-G positive cells were lower in therpeutic animals than controls. Accordingly, the results suggested this that protection is asscoiated with low oxidation result from hydrogen molecule.2. Study of underlying mechanism:â‘ Compared with controls, animals treated with hydrogen-enrich saline showed the low proinflammatory cytokines mRNA levels in tissue including TNF-aå’ŒIL-6.â‘¡HMGB1 protein expression both in tissue and blood decreased significantly in compared with controls. Moreover, only a little of HMGB1 was detected inside cells, while abundant positive cells with brown precipitation both in cytoplasm as well as nucleus were detected in controls.Conclusion:Intraperitoneal injection of hydrogen-enriched saline attenuates hepatic ischemia reperfusion injury, with the characteristic of low serum ALT and little necrotic hepatic cells. Hydrogen-enriched saline play an antioxidative role in hepatic ischemia reperfusion injury, characterized by little peroxdiative hepatocytes and low level of tissue MDA. Moreover, hydrogen-enrich saline prevent HMGB1 and cytokines from expressing as well as releaseing during the period of ischemia reperfusion, with the subsequent of inhibiting inflammatory response.