| As the largest gland in the body,liver not only take part in important material metabolism such as carbohydrate,lipid and protein,but also has the important function of excretion,secretion and biological transformation.So liver function damage not only lead to the metabolic disorders,but also cause dyfunction of other vital organs in body.The hepatic blood flow is temporary blocked during liver transplantation,liver resection and treatment of severe liver trauma.Hepatic ischemia reperfusion injury(HIRI)will happen when its blood supply is returned.HIRI is a pathological physiological phenomena frequently encountered in liver surgery,which is also an important reason for liver failure,operation failure and the poor outcome of patients.Studies showed that peroxidation damage of liver tissue cells caused by a large number of reactive oxygen species during reperfusion was the main damage mechanism of HIRI.The member of ROS include superoxide anion(O2-.),hydrogen peroxide(H2O2)and hydroxyl radical(OH.)and so on.The chemical properties of ROS is very reactive.ROS can react with proteins,enzymes,nucleic acids,cytoskeleton and lipid in the cells,lead to the mitochondrial dysfunction and membrane structure injury,even lead to the death of cells and tissues.The lung is one of the organs which was highly sensitive to the oxygen supply in the body.Whether the remote organ lung was in oxidative stress state and was peroxidation damaged when HIRI happens.Superoxide dismutase(SOD)is one kind of important antioxidant enzyme in the body,which is mainly responsible for the removal of O2-.It can convert O2-to H2O2 and oxygen,so it plays an important role in antioxidative stress.There are three subtypes of SOD in mammals.Mn-SOD was located in mitochondria,the Cu/Zn-SOD was mainly located in cytoplasm and nucleus.Extracellular superoxide dismutase(extracellular,SOD,EC-SOD)is the only one subtype of SOD,which is located outside the cell.It can bind with the cell surface and extracellular matrix through the heparin-binding domain(HBD).The previous studies about SOD functions were mainly focused on the antioxidant effect of Mn-SOD and Cu / Zn-SOD.But,recent studies show that EC-SOD may have a stronger anti-oxidative stress and anti-inflammatory effects.EC-SOD is highly expressed in lung.How to change of EC-SOD expression in lung tissue and if EC-SOD has antioxidant effect when HIRI happens,which will need to be researched.We established hepatic Ischemia-Reperfusion injury model of rats by clipping the hepatic blood vessel released both hepatic left and middle lobes as well as bile duct pedicle with non-damage vascular clamp for 30 mins.Then observed the MDA and H2O2 levels in lung tissue,mRNA and protein expression level of EC-SOD,To explore the oxidative stress level in lung tissue and the antioxidant function of EC-SOD after hepatic ischemia reperfusion injury process.Objective:To observe the MDA and H2O2 levels as well as mRNA and protein expression changes of EC-SOD in lung of liver ischemia-reperfusion injury model,to explore the oxidative stress level in lung tissue of HIRI model and the antioxidant function of EC-SOD during hepatic ischemia reperfusion injury process.Methods: 1 The preparation of HIRI model and detected samplesMale Wister rat(200±10g)were divided randomly into control group and HIRI group.The rats was anesthetized with 6% chloral hydrate,according to the method of Kohli et al,the hepatic blood vessels and bile ducts pedicle were isolated,then clipping the hepatic blood vessel released both hepatic left and middle lobes as well as bile duct pedicle with non-damage vascular clamp for 30 mins to establish the hepatic Ischemia-Reperfusion injury model of rats.The control group only isolated the hepatic blood vessels and bile ducts pedicle and not clipping.After 6 hours,the blood was collected for ALT(alanine aminotransferase)determination.The rats were killed and harvested the liver and lung.liver was fixed with 4% paraformaldehyde for HE staining to observe the morphological changes.The lungs were placed in liquid nitrogen for the determination of the mRNA expression level,protein level of EC-SOD,to detecte the MDA and H2O2 content in lung.2.The index and methods 2.1 The morphological change of the liverThe liver sample were dehydrated,transparent.embedded in paraffin,cranked out 5 micron thick common section,HE stained,then observed by light microscope.2.2 The determination of serum ALT levelsThe collected blood was centrifuged for 10 mins at 3000 rpm to isolate the serum.The serum ALT levels was determined by automatic biochemical analyzer.2.3 The determination of serum MDA contentserum MDA content was determined by Nanjing Jiancheng assay kit.2.4 The Preparation of lung homogenates and determination of MDA content.The iced lung tissue were quickly homogenized with 10mg/100 ? l homogenate buffer(50mmol/LKPB,pH7.4,1mmol/LBenzamidine,1mmol/LPMSF,0.1% Tween-20,0.5mol/L NaCl,1mmol/L EDTANa3 ?-Mercaptoethanol).The homogenate was centrifuged at 4000rpm(20min,4?),The supernatant was the 10% lung homogenate.The MDA content in 10%lung homogenate was determined by Nanjing Jiancheng assay kit.2.5 The determination of H2O2 content in lung.The iced lung tissue were quickly homogenized with 10mg/100 ? l homogenate buffer(50mmol/LKPB,pH7.4,1mmol/LBenzamidine,1mmol/LPMSF,0.1% Tween-20,0.5mol/L NaCl,1mmol/L EDTANa3 ?-Mercaptoethanol).The homogenate was centrifuged at 4000rpm(20min,4?),Then collected the supernatant to preparate the 10% lung homogenate.The H2O2 content in lung homogenate was determined by Molybdate colorimetric method and expressed in amount of hydrogen peroxide in per gram sample(mmol/g pro).2.6 The determination of mRNA level of EC-SOD in lungThe total RNA were extracted with Trizol,About 3?g total RNA was reverse transcribed into cDNA then RT-PCR.The ratio of amplification products of EC-SOD to GAPDH represents the relative mRNA expression levels 2.7The determination of protein level of EC-SOD in lungThe protein level of EC-SOD was estimated by Western Blot.The rat lung tissue was homogenized and collected the supernatant after centrifugation.The total protein was determined with the modified Lowry method.The amount of loading protein in electrophoresis was 65 ug.The EC-SOD antibody was added to the PVDF membrane after transfer film and closed process.The PVDF membrane was stood for overnight at room temperature.Then the anti-rabbit IgG antibody labeled by horseradish peroxidase was added again.The film was developed,fixed and dried according to the instructions of the kit.Then the film was scaned and analyzed with Gel imaging system.The protein content was expressed with the optical density values of the bands.Results:1 The morphology change of liver under light microscopeThe liver cells of control group were arranged in cords around central vein,The size of Hepatic sinusoid among the Hepatic cord was same,no significant dilatation and congestion.But the liver tissue of HIRI group showed serious congestion,The hepatic sinusoid had obvious dilatation and congestion.Liver cells were shrinked because of pressure.The stainning of the liver cell cytoplasm became shallow.There were a large of vacuoles in cytoplasm.A part of liver cells showed significant edema,increased volume and lighter staining.2 The levels of serum ALTThe serum ALT of control group was 20.03±5.23U/L,The serum ALT of HIRI group was 87.43±9.06 U/L.The serum ALT levels of HIRI group was significantly higher than that of control group(P<0.01).3 The MDA content of serumThe serum MDA content of control group was 12.69±2.29umol/L,The serum MDA content of HIRI group was17.54±1.96 umol/L,The serum MDA content of HIRI group was significantly higher than that of control group(P<0.01).4 The MDA content in lung homogenateThe MDA content in lung of control group was 9.81±1.89mmol/g,The MDA content in lung of HIRI group was14.09±2.47 mmol/g,The MDA content in lung of HIRI group was significantly higher than that of control group(P<0.01).5 The H2O2 content in lung homogenateThe H2O2 content in lung of control group was 13.39±1.97mmol/g,The H2O2 content in lung of HIRI group was 19.52±3.03 mmol/g,The H2O2 content in lung of HIRI group was significantly higher than that of control group(P<0.01).6 The relative expression of EC-SOD mRNA in lungThe relative expression of EC-SOD mRNA in lung were determined by RT-PCR.The ratio of amplification products of EC-SOD to GAPDH represents the relative mRNA expression levels.The expression level of EC-SOD mRNA of HIRI group(0.91±0.13)were significantly higher than that of control group(0.49±0.08,P<0.01).The result showed that the gene expression of EC-SOD in lung tissue of HIRI group were enhanced.7 The protein level of EC-SOD in lungThe protein level of EC-SOD of HIRI group(1.09±0.18)was significantly higher than that of control group(0.71±0.12)(P<0.01).Conclusion:1 The MDA and H2O2 content in lung of HIRI model were significantly enhanced,which showed that the lung tissue was also suffered oxidative damage when liver ischemia reperfusion injury happened.2 The mRNA and protein level of EC-SOD in lung were significantly enhanced when lung tissue was suffered oxidative damage induced by hepatic Ischemia-Reperfusion injury,which showed that antioxidase EC-SOD may be involved in the antioxidative stress reaction during the process. |
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