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

Temporary blocking hepatic blood flow for a while then resuming its blood supply is needed during liver resection, liver transplantation and treatment of severe liver trauma, which lead to the hepatic ischemia reperfusion injury(HIRI). HIRI is a common pathological and physiological phenomenon during liver surgery operation. HIRI can cause severe liver cell damage and liver dysfunction. This damage may be one of main reasons that cause postoperative liver failure and bad outcome for patients. Researches have shown that one major mechanism causing the injury is oxidative stress. The Human body produce large amounts of reac-tive oxygen species(ROS) during ischemia reperfusion, for example superoxide anion(O2-), hydroxyl radical(OH), hydrogen peroxide and so on. ROS are very reactive and can damage proteins, DNA and lipids in the cell, which will lead to the death of cell.Superoxide dismutase(SOD) is the one of important antioxidant enzymes which is responsible for the removal of O2-, and plays an important role in antioxidant reaction. It can convert O2- to H2O2, oxygen. There are three subtypes of SOD in mammals. Mn-SOD is located in mitochondria, the Cu/Zn-SOD is mainly located in cytoplasm and nucleus. Extracellular superoxide dismutase(extracellular SOD, EC-SOD) is the only one subtype of SOD located outside the cell. It is binded with the cell surface and extracellular matrix by the heparin-binding domain(HBD). Secondly, EC-SOD is also present in the extracellular fluid, such as serum, cerebrospinal fluid, synovial fluid, ascites and so on. The previous studies about SOD functions were mainly focused on the antioxidant effect of Mn-SOD and Cu / Zn-SOD. But recent studies showed that EC-SOD may have a stronger anti-oxidative stress and anti-inflammatory effects. The expression change andthe antioxidant function of EC-SOD in HIRI model are not reported.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 minutes. The morphological changes of liver tissue, MDA content, m RNA and protein level of EC-SOD in liver were observed after 6 hours to explore the oxidative stress level in liver and antioxidation function of EC-SOD during ischemia and reperfusion, which will provide a new idea for prevention and treatment of hepatic ischemia-reperfusion injury.Objective: To observe the oxidative stress level and expression changes of EC-SOD in liver ischemia-reperfusion injury model of rat, and investigate their role in anti-oxidative stress.Methods:1 Animals and preparation of hepatic ischemia reperfusion injury models12 male Wister rat weighting 200±10 were divided randomly into control group(Con) and hepatic ischemia reperfusion injury group(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 minutes 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 were collected, 3000 rpm centrifugal 10 min to isolate the serum for ALT(alanine aminotransferase) determination. The rats were killed and harvested the livers, a part of livers were fixed with 4% paraformaldehyde for HE staining and the morphological changes of the liver tissue were observed. Another part of livers were placed in liquid nitrogen for the determination of the level of EC-SOD expression and MDA2 The index and methods2.1 The determination of serum ALT levelThe serum ALT level was determined by automatic biochemical analyzer.2.2 The morphological change of the liverThe liver samples were dehydrated, transparent, embedded in paraffin, cranked out 5 micron thick common sections, HE stained, then observed by light microscope.2.3 The preparation of liver homogenates and determination of MDA content.The iced liver tissues were quickly homogenized with 10mg/100μl homogenate buffer(50mmol/LKPB, p H7.4, 1mmol/LBenzamidine, 1mmol/LPMSF, 0.1% Tween-20, 0.5mol/L Na Cl, 1mmol/L EDTANa3 β-Mercaptoethanol). The homogenate was centrifuged at 4000rpm(20min, 4℃), Then collected the supernatant to prepare the 10%liver homogenate. The MDA content in 10% liver homogenate was determined by assay kit.2.4 The determination of m RNA level of EC-SOD in liverThe total RNA were extracted with Trizol. About 3μg total RNA was reverse transcribed into c DNA, and then RT-PCR was conducted. The ratio of amplification products of EC-SOD to GAPDH represents the relative m RNA expression levels.2.5 The determination of protein level of EC-SOD in liverThe protein level of EC-SOD was estimated by Western Blot. The rat liver 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 56 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 Ig G antibody labeled by Fluorescence was added again. Then the image was scanned with two-color infrared imaging systems to analyzed images value.Results:1 The morphology change of liver under light microscopeThe liver cells of control group were arranged in cords around central vein, no significant dilatation and congestion. The size of Hepatic sinusoid among the Hepatic cord was same. But the liver tissues of HIRI group showedserious congestion, The hepatic sinusoid had obvious dilatation and congestion. Liver cells were shrinked because of pressure. The staining of part of the cytoplasm became shallow. There were a large of vacuoles in cytoplasm, but had no obvious necrosis.2The 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. And the serum ALT levels of HIRI group was significantly higher than that of control group(P<0.01).3 The MDA content in liver homogenateThe MDA content in liver of control group was 8.72±1.53mmol/g. The MDA content in liver of HIRI group was 12.75±2.25 mmol/g. And the MDA content in liver of HIRI group was significantly higher than that of control group(P<0.01).4 The relative expression of EC-SOD m RNA in liverThe relative expression of EC-SOD m RNA in liver tissues was determined by RT-PCR. The expression level of EC-SOD m RNA of control group were 0.68±0.11, the expression level of EC-SOD m RNA of HIRI group were 1.13±0.18.And the expression level of EC-SOD m RNA of HIRI group was significantly higher than that of control group(P<0.01).The result showed that the gene expression of EC-SOD in liver tissue of HIRI group were enhanced.5 The protein level of EC-SOD in liverThe protein level of EC-SOD in control group was 0.74±0.14; the protein level of EC-SOD in HIRI group was 1.10±0.22. The protein level of EC-SOD of HIRI group was significantly higher than that of control group(P<0.01). The result showed that the protein level of EC-SOD in liver tissue of HIRI group was increased.Conclusion:1 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.2 The liver tissue of hepatic ischemia reperfusion injury model has been subjected to oxidative stress injury and the liver function is damaged badly. The gene and protein expression of EC-SOD are significantly enhanced, which show that it may play an important role in anti-oxidative stress during hepatic ischemia reperfusion injury and have protective function for liver cells.