Methane Reduce DCD Liver Cold Storage-Reperfusion Injury In Rat By Protecting The Mitochondrial Function

Background and objectiveLiver transplantation is the only effective treatment for a variety of end-stage liver disease.However,as the number of patients with liver disease are increasing,more and more patients are waiting for transplantation,which makes the contradiction between the donor and recipient become increasingly prominent.Because of Chinese laws and regulations,donation after cardiac death(DCD)becomes the main trend of the donor liver.DCD donor liver transplant has to experience three stages : the warm ischemia?cold preservation and reperfusion.Compared to the traditional one,DCD donor is obligated to undergo a longer warm ischemia and the time is difficult to control.So the recovery ability of postoperative DCD donor will be worse,which even will significantly increase the rate of transplantation failure.The subsequent stage cold preservation and reperfusion,which can be controlled by technique,has also important influence on the function of the organ.The cell dysfunction and energy exhaustion in the cold preservation stage can reduce the organ function,which have a great influence on the subsequent reperfusion.The injury mechanism in reperfusion stage is very complex,which mainly includes oxygen free radical injury,calcium overload,neutrophil accumulation injury,inflammatory cytokines,mitochondrial dysfunction and so on,and these various damaging factors also influence each other mutually.In recent years,because of the constant research on injury mechanism in cold preservation-reperfusion stage,the role of mitochondria in the injury have been paid more and more attention.Mitochondria is the main place of oxidative phosphorylation and energy production,so mitochondrial dysfunction will have adverse effects on cold preservation and reperfusion stage.Mitochondrial dysfunction at cold preservation stage can not only cause cell energy synthesis disorders,but also increase cell edema and apoptosis;and the loss of mitochondrial function at reperfusion stage can cause oxidative stress increased,which even makes the mitochondrial function more difficult to recover.In addition,mitochondrial injury at early reperfusion stage will produces a small amount of ROS and further leads to the outbreak of ROS generation.The organ preservation solution,which plays an important role in the stage of cold preservation,provides a great impetus to the development of organ transplantation technology.It can not only protect the cell function in cold preservation stage by reducing the cell swelling and necrosis and energy depletion,but also reduce the injury factors in reperfusion process,and ultimately mitigate the dysfunction of transplanted organ.With the development of the research and understanding of organ preservation mechanism,people gradually attenmpt to change the composition of UW solution to improve the efficiency of preservation,which has achieved good results.Improvement of effective components in the organ preservation solution,which makes it more effective to protect the donor organs,has become a hot issue in the field of organ transplantation research in recent years.Methane is a colorless,odorless,flammable gas.It is just a simple suffocating gas and has no toxicity.Methane,which is abundant in the atmosphere,is often used as a fuel because of its flammability.With the study of the physical and chemical properties of methane,its value in the medical field are gradually recognized.It has been reported that methane can enhance the intestinal peristalsis amplitude and increase the intestinal contractile activity.Boros confirmed that methane could reduce intestinal ischemia-reperfusion injury because of its anti-oxidant and anti-inflammatory effects.In another study,ischemia-reperfusion injury of liver was reduced by injecting the methane-rich saline injected into rats.Methane,which itself has a good reduction,also plays an important role in the balance of oxidation and reduction.Some studies confirm that in the case of lack of oxygen,mitochondria can produce methane to mitigate peroxide mitochondria injury.Hence,we hypothesize that methane can be join in cold preservation stage to decrease mitochondrial damage,and further reduce reperfusion injury.Our study intends to use liver transplantation model of SD rats to investigate the effects of methane on donor liver injury and mitochondrial function in cold preservation and reperfusion stage,and reveal its protective effects and relative mechanism.Methods and ResultsPart 1 Effects of methane on mitochondrial function of DCD donor in cold preservation stage1.Effects of methane on liver HE staining in cold preservation stage;The donor liver of SD rat was coldly preserved for 4 hours to simulate the DCD donor liver model.HE staining was performed to observe the morphological changes of the donor liver.We can observe that compared with ordinary UW group,the donor liver injury are more ease in methane-rich UW group.2.Effects of methane on mitochondrial phosphorus and oxygen ratio(ADP/O)and respiratory control rate(RCR)in cold preservation stage;Mitochondria were purified from donor liver experienced cold preservation by using differential centrifugation.Clark oxidation electrode were used to detect the phosphorus and oxygen ratio(ADP/O)and respiratory control rate(RCR).The result showed that the mitochondrial function of the treated methane-rich UW was significantly increased compared with that of treated with the conventional UW solution.3.Effects of methane on mitochondrial function of producing ATP in cold preservation stage;In order to further understand the differences between all groups in the function of mitochondria after cold preservation,the level of ATP in liver tissue was examined by ELISA kits.We found that the level of ATP in liver tissue of the methane-rich UW group was higher than that of the common UW group.Part 2 Effects of methane on mitochondrial function of DCD donor in reperfusion stage1.Effects of methane on morphology of donor liver in reperfusion stageIn order to verify methane-rich UW solution is better to tolerate reperfusion injury than ordinary UW solution,the differences of morphology in liver cells are observed by HE staining.It can be seen that after 6 hours' reperfusion of methane-rich UW solution,the cell injury of DCD donor liver was significantly worse than that before transplantation,but the damage was more reduced compared to the same treatment by conventional UW solution.2.Effects of methane on apoptosis of donor liver in reperfusion stageThe apoptosis of liver cells after reperfusion in each group was detected by TUNEL staining.The apoptotic proportion of DCD donor liver saved by methane-rich UW solution is lower than that by conventional UW solution.3.Effects of methane on ALT/AST in reperfusion stageTo learn more about liver damage in reperfusion groups after reperfusion,we examined the level of ALT and AST and found that liver injury index of methane-rich UW solution group decreased more deeply than that of conventional UW solution group.4.Effects of methane on mitochondrial phosphorus and oxygen ratio(ADP/O)and respiratory control rate(RCR)in reperfusion stageOur previous studies have described a simple cold preservation stage methane can reduce the injury of mitochondrial function,in order to learn more about the relationship between mitochondrial dysfunction and oxidative stress,we examined the various oxidation of mitochondrial function by Clark electrode method.We found that the injury of mitochondrial function of the treated with methane–rich UW was reducted compared with that of treated with the conventional UW.5.Effects of methane on mitochondrial function of producing ATP in reperfusion stageThe main function of mitochondria is to provide oxidative phosphorylation sites,and to produce ATP,in order to further clarify the difference between each group of mitochondrial function,we examined ATP levels of the donor liver tissue 6 hours after reperfusion by ELISA kit,which showed that the level of ATP in liver tissue of the methane-rich UW group was higher than that of the common UW group.6.Effects of methane on parameters(MDA,SOD,ROS,GSH)of liver donor stress oxidation in reperfusion stageRelated studies have shown that low concentration of methane has a good antioxidant effect.According to our assumptions,low concentration of methane can play a antioxidant effect via reducing mitochondrial injury.We examined the parameters(MDA,SOD,ROS,GSH)of liver donor stress oxidation and the results showed that the parameters of the treat with methane-rich UW are effectively reduced compared with that with conventional UW,which is consistent with mitochondrial function changes.7.Effects of methane on survival rate and time of SD rats after transplantationThe ultimate goal of improving UW component is to optimize the quality of the donor liver.We created liver transplantation model of rat in order to observe the function recovery of rats after transplantation with methane-rich UW or conventional UW solution.The results showed that the use of methane-rich UW can prolong the survival time and rates after transplantation in rats.ConclusionOur research have provided evidences that methane can protect DCD donor liver in cold preservation-reperfusion stage,further reduce the mitochondria injury in cold preservation stage,and ultimately improve survival time of transplantation in rats by creating DCD donor liver transplantation model in rat.