| Liver ischemia-reperfusion injury is a pathological physiological phenomena frequently encountered in liver surgery such as liver transplantation, liver resection and so on. HIRI is an important reason for liver transplantation failure, liver dyfunction and the poor outcome of patients. Studies have shown that liver cells were peroxide damaged by a large number of reactive oxygen species during reperfusion, which was the main damage mechanism of HIRI. ROS including superoxide anion(O2-.), hydrogen peroxide(H2O2) and hydroxyl radical(OH.). The chemical properties of ROS is very reactive. So ROS can react with important structural proteins and functional protein in the cells and membrane, change the structure of the cell membrane, affecting cell function, and even cause the death of cells and tissues.The liver is an important metabolic organ in the body. So liver dyfunction will lead to the metabolic disorders and dyfunction of other nearby or remote vital organs.The heart has a great need for oxygen, so it was highly sensitive to the ischemia and hypoxia reaction in the body. Whether the remote organs was in oxidative stress state and was peroxidation damaged when the liver blood supply was restored after blocking and the liver was in highly oxidative stress state which was unclear.Prx III is one member of the peroxidases peroxiredoxin family which was found in recent years. Studies have shown that Prx III was located in the mitochondria, and it is the only one specific protein enzymes of the family which was located in mitochondria. Mitochondrial respiratory chain is the important source of ROS in the cell. In recent years, the research also further confirmed that Prx III may be the key enzyme for clearing H2O2 in the mitochondria. The oxygen consumption of the heart is very strong in the body. Energy required of myocardial cell was provided mainly through mitochondrial oxidative phosphorylation, a large number of ROS was produced during the process. So, the heart is the most vulnerable to ROS peroxide damage in the body. How to change of the Prx III expression in myocardial tissue and whether Prx III participate in oxidative stress reaction when HIRI happens. It is not reported.We established hepatic Ischemia-Reperfusion injury model of rats by clipping the hepatic blood vessel released both hepatic right and middle lobes as well as bile duct pedicle with non-damage vascular clamp for 30 mins. Then observed the LDH level in serum, MDA levels in Myocardial tissue, m RNA and protein expression changes of Prx III in heart, To investigate the oxidative stress status in heart during hepatic ischemia reperfusion injury process and the antioxidant function of Prx III.Objective: To observe the oxidative stress levels in heart of liver ischemia-reperfusion injury model as well as m RNA and protein expression changes of Prx III. To explore the role of Prx III in oxidative stress reaction.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 Kohlietal to isolate hepatic blood vessels and bile ducts pedicle, then clipping the hepatic blood vessel released both hepatic right 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 heart. liver was fixed with 4% paraformaldehyde for HE staining to observe the morphological changes. The heart was putted in ice saline and gently squeezed with tweezers to remove the blood. Then the heart were placed in liquid nitrogen for the determination of the m RNA expression level, protein level of Prx III, to detecte the MDA content in heart. 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 ALT levelsLDH activity was detected with LD- L rate method according to the kit instructions. 2.4 The Preparation of heart homogenates and determination of MDA content.The iced heart tissue 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℃), The supernatant was the 10%heart homogenate. The MDA content in 10%heart homogenate was determined by Nanjing Jiancheng assay kit. 2.5 The determination of m RNA level of Prx III in heartThe total RNA were extracted with Trizol, About 3μg total RNA was reverse transcribed into c DNA then RT-PCR. The ratio of amplification products of Prx III to GAPDH represents the relative m RNA expression levels 2.6The determination of protein level of Prx III in heartThe protein level of Prx III was estimated by Western Blot. The rat heart 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 68 ug. The Prx III 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 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 481.50±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 heart homogenateThe MDA content in heart of control group was 9.24±1.72mmol/g, The MDA content in heart of HIRI group was12.73±1.84 mmol/g, The MDA content in heart of HIRI group was significantly higher than that of control group(P<0.01).5 The relative expression of Prx III m RNA in heartThe relative expression of Prx III m RNA in heart were determined by RT-PCR. The ratio of amplification products of Prx III to GAPDH represents the relative m RNA expression levels. The expression level of Prx III m RNA of HIRI group were significantly higher than that of control group(P<0.01).The result showed that the gene expression of Prx III in heart tissue of HIRI group were enhanced.6 The protein level of Prx III in heartThe protein level of Prx III of HIRI group(0.84±0.18) was significantly higher than that of control group(0.58±0.13)(P<0.05).Conclusions: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 LDH levels of serum and the MDA content of myocardial tissue in HIRI model were significantly increased which showed that myocardial tissue has suffered from peroxidation damage. The m RNA level, protein level of Prx III were significantly enhanced which indicated that Prx III was involved in oxidative stress induced by ischemia-reperfusion... |
PDF Full Text Request