Effects Of Methane Saline On Liver Mitochondria In Mice With Hepatic Ischemia-Reperfusion

As early as 1955,the dog’s coronary artery was ligated for the first time in an ischemic dog model.It was found that if the ligature was immediately released to restore blood supply,some dogs would suddenly die of ventricular fibrillation.The concept of myocardial reperfusion injury was first proposed in 1960.Hepatic ischemia-reperfusion injury is common in liver transplantation,shock,and liver interventional therapy,and is one of the important factors affecting the prognosis of patients.The pathogenesis of hepatic ischemia-reperfusion injury is complicated,and most studies focus on oxidative stress,calcium overload,inflammatory response,mitochondrial damage,membrane permeability changes,etc.The interrelation and interaction among these mechanisms eventually lead to cell death.For critical diseases,hepatic ischemia-reperfusion injury is extremely common.Unlike alternative treatments for renal failure or respiratory failure,alternative treatments for liver diseases are extremely lacking currently.The existing alternative treatments cannot replace the synthetic function of the liver.Therefore,the research on the pathogenesis of hepatic ischemia-reperfusion injury is of great scientific significance for finding and discovering therapeutic drugs.Methane（CH₄）is a common organic gas in the atmosphere,the main component of biogas,a raw material consuming ozone,and a kind of greenhouse gas.It is flammable and has low solubility in water.Methane is traditionally considered to be produced by anaerobic bacteria in an anaerobic environment.It is traditionally believed that in addition to the combustion of organic matter or petroleum,the largest source of methane in nature is the production by anaerobic bacteria in an anaerobic environment.As for human body,methane is mainly produced in the colon,and methanobrevibacter smithii accounts for 94%.However,the research published by Kelpper on“Nature”in 2006 ruled out the participation of anaerobic bacteria and found the release of methane was increased when the living bodies of corn and ryegrass were exposed to sunlight.The subsequent researches further found that animal and plant cells could self-produce methane in mitochondria under the stimulation of oxidative stress.These researches prove that cells can self-produce methane without the participation of anaerobic bacteria,but the mechanism of how mitochondria produce methane remains unsolved so far.Some researches speculate that methyl such as phosphatidylcholine may be a potential source of methane,and some suggest that the methylation of mitochondrial DNA changes the expression of genes under oxidative stress conditions,affects the expression and activity of s-adenosyl homocysteine hydrolase,and changes the metabolism of methionine,during which methane can be released as a mesostate.The research of Althoff provides support for this theory and proposes that under high oxidation,normal temperature and atmospheric pressure,organic sulfides such as methionine can be oxidized to corresponding sulfoxide,which then produces methane by demethylation through bond dissociation.Methane is generally considered to be an inert gas that does not participate in the body’s metabolism.While as early as 1967,Dougherty et al.injected methane labeled with C¹⁴ into the sheep’s circulatory system,and found carbon dioxide（CO₂）containing C¹⁴ in the expiratory air of the sheep.It was initially found that methane might participate in in the body’s carbon cycle.In 2012,Boros et al.found that the added exogenous methane had an anti-inflammatory effect in the intestinal IRI model of dogs.In a number of subsequent researches,it was found that methane had protective effects on IRI of several organs（liver,kidney,heart,retina,spinal cord,etc.）.These researches found that methane had anti-oxidative stress effect,anti-inflammatory effect,and antiapoptotic effect.Methane has potential to become a gas in treating organ ischemia-reperfusion injury.In summary,methane is produced by mitochondria under oxidative stress.Meanwhile,mitochondria produce 95%of the ROS.The released cytochrome c is a key step in apoptosis.Since mitochondria and oxidative stress are closely related to each other and are methane-producing organelles,it is possible that mitochondria are the targets of methane’s antioxidant effect.Therefore,in this paper,the model of hepatic ischemia-reperfusion injury is used to verify the antioxidant effect of methane and study the effects of exogenous methane on mitochondria.This paper gives a preliminary understanding of mitochondrial changes and finds a way to study the mechanism of methane’s resistance effect in organ ischemia-reperfusion injury.Objectives:To verify the protective effect of methane normal saline,verify the anti-oxidative stress effect of methane,and further detect the mitochondrial respiratory chain complex I～V activity and protein expressions such as subunit,PGC-1α,DLP1,Mfn1,Mfn2,OPA1,and PINK1 protein expressions with the model of the mouse’s hepatic ischemia-reperfusion injury,so as to give a preliminary understanding of the effects of methane on mitochondria and provide directions for further research.Methods:A 70%mouse hepatic ischemia-reperfusion injury model was used and three groups（SHAM,IR and IR+CH₄,n=6）were randomly assigned with 6 mice in each group.Sham Group was a blank control group;IR Group was subjected to ischemia for 1 hour,reperfusion for 6 hours,and intraperitoneal injection of 0.5ml saline before reperfusion;IR+CH₄ Group was subjected to ischemia for 1 hour,reperfusion for 6 hours,and intraperitoneal injection of 0.5ml saline before reperfusion.After reperfusion,0.3～1 ml of mouse blood was collected from eyeball blood.The left middle lobe of the liver was taken to detect the mitochondrial respiratory chain complex I～IV activity and protein expressions such as subunit,PGC-1α,DLP1,Mfn1,Mfn2,OPA1,and PINK1 protein expressions.The data was analyzed and processed using Graph Pad Prism 7.0 software.The measurement data was expressed in the form of mean±standard deviation（?x±s）.Unpaired t-test was used for the comparison between groups.One-way analysis of variance was used for the comparison among multiple groups.If there was any difference,further comparisons would be performed by Tukey method,and P<0.05 was considered statistically significant.Results:1.AST and ALT in IR Group was significantly increased（P<0.05）.Methane normal saline reduced AST/ALT in IR+CH₄ Group（P<0.05）.2.In IR Group,MDA and ROS was increased and GSH and SOD was decreased（P<0.05）,while in IR+CH₄ Group,MDA and ROS was decreased and GSH and SOD was increased（P<0.05）.3.There was no difference in mitochondrial respiratory chain complex IV activity among the three groups.In IR Group,the expressions of subunit proteins of respiratory chain complex I,IV,and V were increased（P<0.05）;there was no statistically significant difference in the expression of PGC-1α,suggesting that the mitochondrial complex is not the effect target of methane.4.Compared with SHAM Group,the expressions of Mfn1 and OPA1 were decreased（P<0.05）and the expression of DLP1 was increased（P<0.05）in IR Group;the increase in Mfn2 may be compensatory for the decrease of Mfn1.Compared with IR Group,Mfn1 was increased（P<0.05）and DLP1 expression was decreased（P<0.05）in IR+CH₄ Group.Methane normal saline can promote mitochondrial fusion,reduce mitochondrial division,and recover the dynamic equilibrium of mitochondria.5.Compared with SHAM Group,the expression of PINK1 was increased（P<0.05）,the expression of Parkin in mitochondria was increased（P<0.01）,and the expression of Parkin outside of mitochondria was decreased（P<0.01）in IR Group.Compared with IR Group,the expressions of PINK1 and Parkin in mitochondria were both decreased（P<0.05）in IR+CH₄ Group.These results indicate that cells attempt to promote the transfer of Parkin to mitochondria through the high expression of PINK1 during IRI,thereby initiating mitophagy and clearing the damaged mitochondria.While,CH₄ can effectively reverse the up-regulation of PINK1（P<0.05）and Parkin in mitochondria,suggesting that methane normal saline may promote the recovery of mitochondrial function and the clearance of damaged mitochondria.Conclusions:1.Methane has the effect of protecting liver function against ischemia-reperfusion injury and resisting oxidative stress.2.Methane has a positive effect on mitochondrial homeostasis,and can promote mitochondrial fusion,reduce mitochondrial division,restore mitophagy,and recover the dynamic equilibrium of mitochondria.