Expression Chang Of EC-SOD In Cardiac Muscle Of Hepatic Ischemia-reperfusion Injury Rats

Liver ischemia-reperfusion injury?HIRI? is a pathological physiological reaction frequently happened in liver surgery such as liver transplantation, liver resection and so on, which is also an important reason for liver transplantation failure, liver dyfunction and poor outcome of patients. Many studies have shown that oxidative stress response will be induced and a large number of reactive oxygen species?ROS? will be produced when block the liver blood flow temporarily and then restore its blood supply. The peroxide damaged of liver cells by ROS is one of the main mechanisms of HIRI. ROS include superoxide anion(O^2-.), hydrogen peroxide?H₂O₂? and hydroxyl radical?OH.? and so on. The chemical propertie of ROS is very reactive, which can react with some important cytoplasmic and membrane proteins?including structural proteins and functional proteins?, then change the structure of cell membrane, damage cell function, and even cause the death of cells and tissues.Liver is not only an important metabolic site,such as carbohydrate, lipid and protein, but also an important excretion, secretion and biological transformation organ in the body. So liver dyfunction inevitably lead to the abnormal function of other nearby or remote vital organs.The cardiac muscle is one of organ which can consume a large amount of oxygen and is sensitive to the ischemia and hypoxia reaction. When blocking the liver blood and then restore its perfusion and liver was suffered from oxidative stress damage, Whether the cardiac muscle will also suffer from oxidative damage, which need to be studied.Superoxide dismutase?SOD? is one kind of important antioxidant enzyme in the body, which is mainly responsible for the removal of O^2-. It can convert O^2- 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?EC-SOD? is the only one subtype of SOD, which is located outside the cell. EC-SOD can bind with the cell surface and extracellular matrix through the heparin-binding domain?HBD?. The previous studies about SOD were mainly focused on the antioxidant functions of Mn-SOD and Cu / Zn-SOD. But, recent studies show that EC-SOD may have a stronger anti-oxidative stress effect. studies show that the risk of hypertension and ischemic cardiac muscle disease was increased significantly in patients with decreased EC-SOD content in cardiac tissue. Decreased binding capacity of EC-SOD and extracellular matrix could lead to the increasing of the risk of cardiovascular and ischemic cardiac muscle disease. Moreover, EC-SOD expression is reduced in patients with cardiac muscle failure. This suggests that EC-SOD may play an important role in maintenance of cardiac function. How to change of EC-SOD expression in cardiac muscle of HIRI model and if EC-SOD has antioxidant effect during the process, which was not reported.We established HIRI 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 content and H₂O₂ content in myocardial tissue, LDH level in serum, mRNA and protein expression level of EC-SOD, to explore the oxidative stress status in myocardial tissues and the antioxidant function of EC-SOD after hepatic ischemia reperfusion injury.Objective:To observe the MDA content and H₂O₂ content in myocardial tissues of liver ischemia-reperfusion injury model as well as mRNA and protein expression changes of antioxidase EC-SOD, to explore the oxidative stress status in myocardial tissues and the antioxidant function of EC-SOD after hepatic ischemia reperfusion injury. Methods:1 Animals and preparation of hepatic ischemia reperfusion injury modelsMale Wister rat weighting 200±10 were divided randomly into control group?Con? and hepatic ischemia reperfusion injury?HIRI?. Anesthetized rats with 6% chloral hydrate, according to the method of Kohli et al to isolate hepatic blood vessels and bile ducts pedicle, 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? and LDH?lactate dehydrogenase? determination. The rats were killed and harvested the liver and cardiac muscle. liver was fixed with 4% paraformaldehyde for HE staining to observe the morphological changes. The cardiac muscle was putted in ice saline and gently squeezed with tweezers to remove the blood. Then the cardiac muscle 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. 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 LDH levelsLDH activity was detected with LD- L rate method according to the kit instructions. 2.4 The Preparation of cardiac muscle homogenates and determination of MDA content. The iced cardiac muscle 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 Na Cl,1mmol/L EDTANa3 ?-Mercaptoethanol?. The homogenate was centrifuged at 4000rpm?20min, 4??, The supernatant was the 10 % cardiac muscle homogenate. The MDA content in 10 %cardiac muscle homogenate was determined by Nanjing Jiancheng assay kit. 2.5 The determination of H₂O₂ content in cardiac muscle.The iced cardiac muscle 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% cardiac muscle homogenate. The H₂O₂ content in cardiac muscle 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 myocardial tissuesThe 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 2.7 The determination of protein level of EC-SOD in myocardial tissuesThe protein level of EC-SOD was estimated by Western Blot. The rat myocardial tissues 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 62 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 levels of serum LDHThe serum LDH of control group was 481.5±67.15 U/L, The serum LDH of HIRI group was 658.83±94.15 U/L. The serum LDH levels of HIRI group was significantly higher than that of control group?P<0.01?.4 The MDA content in cardiac muscle homogenateThe MDA content in cardiac muscle of control group was 9.24±1.72mmol/g, The MDA content in cardiac muscle of HIRI group was12.73±1.84 mmol/g, The MDA content in cardiac muscle of HIRI group was significantly higher than that of control group?P<0.01?.5 The H₂O₂ content in cardiac muscle homogenateThe H₂O₂ content in cardiac muscle of control group was 10.56±1.72mmol/g, The H₂O₂ content in cardiac muscle of HIRI group was 14.63±2.25 mmol/g, The H₂O₂ content in cardiac muscle of HIRI group was significantly higher than that of control group?P<0.01?.6 The relative expression of EC-SOD mRNA in cardiac muscleThe relative expression of EC-SOD mRNA in cardiac muscle 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?1.01±0.15? were significantly higher than that of control group?0.69±0.11, P<0.01?.The result showed that the gene expression of EC-SOD in cardiac muscle tissue of HIRI group were enhanced.7 The protein level of EC-SOD in cardiac muscleThe protein level of EC-SOD of HIRI group?0.69±0.09? was significantly higher than that of control group?0.48±0.07??P<0.01?.Conclusion:The hepatic Ischemia-Reperfusion injury model of rats can be established by clipping the hepatic blood vessel released both hepatic left and middle lobes as well as bile duct pedicle.The MDA content, H₂O₂ content in myocardial tissues and serum LDH activity of HIRI model were significantly increased, which showed that myocardial tissue has suffered from peroxidation damage and myocardial function is impaired.The mRNA level, protein level of EC-SOD in myocardial tissues of HIRI model were significantly enhanced, which indicated that antioxidant enzyme EC-SOD may play a role in antioxidant effect during the process.