| The liver is an important metabolic organ in the body. It not only take part in important material metabolism such as protein and fat, but also has the important function of excretion, secretion, detoxification. So liver dyfunction will lead to the metabolic disorders and dyfunction of other vital organs. Liver ischemia-reperfusion injury is a pathological physiological phenomena frequently encountered in liver surgery such as liver transplantation, liver resection and so on. Studies have shown that HIRI is an important reason for liver failure, operation failure and the poor outcome of patients.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 lung is one of the organs which was highly sensitive to the oxygen content in the body. Whether the remote organs was in oxidative stress state and was peroxidation damaged when HIRI happens.Prx VI is one member of the peroxidases Peroxiredoxin family which was found in recent years. in mammalian, Prx VI highly express in alveolar type II epithelial cells. Studies have shown that Prx VI has the biological activity of glutathione peroxidase. So its main function is responsible for the reduction of H2O2 and phospholipid peroxides. The m RNA and protein levels of Prx VI was significantly enhanced in lung epithelial cells after treating with high concentration of oxygen, paraquat and H2O2 to cause oxidative stress reaction. The ability of removing H2O2 of cell was significantly enhanced and inhibit the peroxidation reaction when the expression of Prx VI in lung epithelial cell line was induced. It show that Prx VI may play an important role in removal of ROS and preventing peroxidation damage in lung tissue., How to change of the expression level of Prx VI when HIRI happens, The question were 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 mins. Then observed the MDA levels, m RNA and protein expression changes of Prx VI in lung, To investigate the oxidative stress status in lung during hepatic ischemia reperfusion injury process and the antioxidant function of Prx VI.Objective: To observe the oxidative stress levels in lung of liver ischemia-reperfusion injury model as well as m RNA and protein expression changes of Prx VIMethods: 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) 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 m RNA expression level, protein level of Prx VI, to detecte the MDA 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 Preparation of lung homogenates and determination of MDA content.The iced lung tissue were quickly homogenized with 10mg/100 μ l 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% lung homogenate. The MDA content in 10%lung homogenate was determined by Nanjing Jiancheng assay kit. 2.4 The determination of m RNA level of Prx VI in lungThe 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 VI to GAPDH represents the relative m RNA expression levels 2.5The determination of protein level of Prx VI in lungThe protein level of Prx VI 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 61 ug. The Prx VI 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 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).4 The relative expression of Prx VI m RNA in lungThe relative expression of Prx VI m RNA in lung were determined by RT-PCR. The ratio of amplification products of Prx VI to GAPDH represents the relative m RNA expression levels. The expression level of Prx VI 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 VI in lung tissue of HIRI group were enhanced.5 The protein level of Prx VI in lungThe protein level of Prx VI of HIRI group(1.02±0.19) was significantly higher than that of control group(0.65±0.13)(P<0.01).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 m RNA level, protein level of Prx VI and MDA content in lung were significantly enhanced in hepatic ischemia reperfusion injury model, which showed that the lung tissue has been hurt by peroxidation reaction and Prx VI was involved in oxidative stress. |
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