| Background and objectivesIschemia and reperfusion injury(I/R)is one kind of classical pathologically injury.It is commonly seen in some clinical disease such as liver transplantation,stroke and so on.Ischemia injury happens when tissue suffer with deficiency of blood and oxygen.Myocardial ischemia injury caused by obstracted coronary artery is one major kind of ischemia injury.All these diseases are mentally and physically damaged to human health.By far,the treatment typically dealing with I/R injury and ischemia injury is not developed yet and not well applied to bedside.Therefore,the research focus on the treatment for I/R injury and ischemia is necessary.Augment of oxidative injury,increase of inflammation level and magnification of cell death are concentrated as the major pathological mechanism during the I/R injury and ischemia injury by now.And targeting these illness factors is the gravity that the research project should grasp and would be the most promising strategy.Previous work demonstrated that inhibition one or the other factors would bring protective effects but none of these strategies are successfully applied in clinical application.For these years,methane was considered only as greenhouse gas but its biochemical feature was not well recognized.Researches supported that mammal body could utilize methane and constitute organic compound with it.Methane was also detected in injured tissue and the reason for this phenomenon was not clear.Further,researchers suggested that methane respiration could attenuates intestine I/R injury and improve physical function.However,in consideration of protection of intestinal I/R injury with methane is not well defined,the protective effects of methane on I/R and ishcemia injury needs further confirmation.Furthermore,the protective effect of methane on other organ I/R injury and ischmia injury is not explored and molecular mechanism for methane treatment on I/R injury needs further exploration.Hence,this project explored the protective effect of methane on liver,brain I/R injury and myocardial ischemia injury and its probably molecular mechanism.This project first receive positive methane saline(MS)dosage and best application time through research of methane treatment on hepatic I/R injury,exploring methane mechanism on inflammation,oxidative injury and cell apoptosis;then this project searches the possibility of methane protective effect on cerebral I/R injury and the possibility of methane in protection of brain I/R injury through inhibition of apoptosis;the best application time and does were second time testified in the myocardial ischemia injury treated with methane saline research,more comprehensively detection on the underlying mechanism of methane protective effects targeting on three aspects such as cell apoptosis,inflammation and oxidative injury were also conducted.This project is majorly divided into three parts:Part I The protective effect of methane on hepatic I/R injury and its mechanismMethod: This sub project explored best dose of methane saline with five different doses treating on hepatic I/R injury using liver function detector after establishment of stable animal injury model.Liver suffered ischemia for 60 mins and reperfusion for 6 hours.MS was administrated once at the beginning of the reperfusion.And divided another group of animals randomly into three group: Sham,I/R injury group and I/R+10ml/kg MS treatment group.ALT,AST,LDH were detected to illustrate liver function.IL-1?,IL-6,TNF-? were detected as inflammation level detector;8-OHG,SOD and MDA were detected as oxidative detector and caspase-3 was detected to illustrate apoptosis level.HE staining and TUNEL positive cell staining were also conducted.Results: Administration of 10 ml/kg methane saline significantly decreased inflammation level,attenuated oxidative injury and inhibited cell apoptosis.There is significant difference between I/R injury group and I/R+MS treatment group.Conclusion: Methane saline with intraperitoneal administration is a practical way of treating I//R injury.Methane saline with dose of 10 ml/kg may improve hepatic I/R injury via its anti-inflammation,anti-oxidative injury and anti-apoptosis mechanism.Part II The protective effects of methane on cerebral I/R injury and its mechanismMethod: Stable cerebral ischemia and reperfusion injury model was established and animals were randomly divided into three groups: Sham group,cerebral I/R injury + Saline group and cerebral I/R injury + Methane Saline group.MS was administrated with 10 ml/kg once at beginning of reperfusion.Behavior tests were also detected to demonstrate methane effect on I/R injury.HE staining,TUNEL positive cell staining and protein expression of caspase-3 were also detected to explore the anti-apoptosis role of methane saline on I/R injury.Results: Animals suffered with cerebral I/R injury but administrated with MS have significantly improved behavior test score,reduced cerebral infarction volume,reduced HE staining infarction size,lessen TUNEL positive neuron cell and decreased protein expression of caspase-3.Conclusion: Methane effectively protects cerebral I/R injury through anti-apoptosis mechanism.Part III The protective effects of methane on myocardial ischemia injury and its mechanismMethod: The sub-project established stable myocardial ischemia injury rat model and randomly divided animal into three groups: Sham group,myocardial ischemia injury + saline group and myocardial ischemia injury + methane saline group.MS was administrated after 30 minutes' ligation once with certain dose respectably.Increase of protective effect of methane with does usage was testified.The concentration of methane in blood and in situ heart tissue were also detected after methane administration.TTC was applied to demonstrate volume of myocardial infarction.Heart remodeling and ejection fraction were also detected to show the protective of methane on ischemia injury.SOD,MDA,8-OHG and MPO were detected as oxidative detector,IL-1?,IL-6,TNF-? and Xanthine were detected as inflammation level detector.Caspase-3,cleaved caspase-3,caspase-9,cleaved caspase-9,Bax/Bcl-2,cytochrome C were detected for apoptosis research.Bax,Bcl-2 and caspase-3 positive cells were also detected using immunocytochemistry method.Results: Method for producing methane saline make methane concentration stable in 4 weeks and administration with methane saline significantly increase in situ tissue methane concentration.Compared with myocardial ischemia injury + saline group,10ml/kg methane saline significantly attenuated myocardial infarction,released heart remodeling,decreased inflammation level,reduced oxidative injury,inhibited caspase-3,caspase-9,Bax/Bcl-2 and cytochrome C activation and increase.Conclusion: Methane saline protects myocardial ischemia injury through anti-oxidative injury,anti-inflammation and anti-apoptosis mechanism.Statement: Tested within multiple animal injury model,supported with abundant results,this project first demonstrated that methane saline has the possibility for treatment of liver,brain ischemia and reperfusion injury and heart ischemia injury.Methane could attenuate hepatic I/R injury and improve liver function;methane could treat cerebral I/R injury,protected brain tissue and improved brain function;methane also could treat myocardial ischemia injury,reduced cell death and delayed ventricular remodeling.The protective effects of methane could be realized by anti-inflammation,anti-oxidative and anti-cell apoptosis mechanism.This project applied methane-rich saline in the study which has received patent of invention from Chinese government,endorsed and followed by other scientist in other project and offer theoretically evidence for future clinical application. |
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