| The kidneys can not only excrete metabolic waste in the body, maintain sodium, potassium, calcium and other electrolyte stability and acid-base balance, but also has the function of endocrine. So the kidney is very important to maintain the internal environment balance of body. Studies have shown that neighbors and the remote organs of kidney such as heart, lung, liver, intestine and brain function will be severely damaged when kidney function is damaged.Renal Ischemia-Reperfusion Injury(RIRI) is a common pathophysiological process in the clinical treatment such as acute renal artery occlusion or kidney transplantation and so on. RIRI is also an important factor that leads to delayed function recovery after kidney transplantation and acute renal failure. The kidney will produce large amounts of reactive oxygen species(ROS) during ischemia-reperfusion process and the kidney is in a high degree of oxidative stress status which is one of mechanisms of ischemia-reperfusion injury. ROS include 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 can affect cell function, even cause cell and tissue death.Prx VI is one member of the peroxidases Peroxiredoxin family which was found in recent years. In mammalian, Prx VI is highly expressed 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. The content of H2O2 and MDA in the lung tissues of Prx VI knockout mouse were significantly higher than those of control group. These results show that Prx VI has strong anti-oxidative stress function in lung tissue and may play an important role in removal of ROS and preventing peroxidation damage in lung tissue. The lung is one of the organs which is very sensitive to the oxygen content in the body. whether the lung will be in a state of oxidative stress and suffered oxidative damage, how to change of the expression level of Prx VI when RIRI happens, The questions were not reported.We first established Renal Ischemia-Reperfusion Injury model of rats by clipping the renal artery with non-damage vascular clamp. After 24 h of reperfusion, we observed the MDA and H2O2 content in lung, m RNA and protein expression of Prx VI, to explore peroxide damage degree of lung and the antioxidant effect of Prx VI during Kidney ischemia-reperfusion injury, Which will provide a new way for prevention and treatmemt of lung damage induced by Renal Ischemia-Reperfusion Injury.Objective: Observing the oxidative stress state and expression changes of Prx VI m RNA and protein in lung of renal ischemia-reperfusion injury model, to investigate the role of Prx VI in peroxidation damage induced by RIRI.Methods: 1 Animals and preparation of Renal Ischemia-Reperfusion Injury models Male Wistar rat weighting 200±10g were purchased from the Experimental Animal Center of Hebei Medical University. The rats were divided randomly into control group(Con) and Renal Ischemia-Reperfusion Injury(RIRI) group. There are 6 rats in each group. First the rats were anesthetized with 6% chloral hydrate, then we established Renal Ischemia-Reperfusion Injury model of rats according to the method of YU Xiao-Dong et al. We exposed the kidneys of RIRI group rats, first removed the right kidney, then separated the left renal artery and cliped the left renal arterywith non-damage vascular clamp near the renal hilum. We could see that the colour of kidney became gradually dark red from bright red. Removed the vascular clamp after 45 mins and restored the blood supply. The colour of kidney quickly became bright red from dark red again which showed that the reperfusion was successful. The control group rats were only removed the right kidney and separated the left renal artery, but not cliped the left renal artery. After 24 hours, the blood was collected and centrifuged for 10 mins at 3000 rpm to isolate the serum for determination of Serum Creatinine(SCr) and Blood Urea Nitrogen(BUN). The rats were killed and harvested the kidney and lung. the kidney was fixed with 4% paraformaldehyde for HE staining and observing the morphological changes. The lung was placed in liquid nitrogen for the determination of m RNA expression level, protein level of Prx VI, to detecte the MDA and H2O2 content in lung. 2 The index and methods 2.1 The determination of serum SCr and BUN The SCr level in serum was measured using the picric acid method. The BUN level in serum was measured by enzyme-coupled rate method. 2.2 The morphological observation of the kidney by HE staining The kidney sample were dehydrated, transparent. embedded in paraffin, cranked out 5 micron thick common section, HE stained, then observed the morphological change of kidney by light microscope. 2.3 The determination of MDA content in lung. 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℃), Then collected the supernatant to preparate the 10% lung homogenate. The MDA content in 10 % lung homogenate was determined by Nanjing Jiancheng assay kit. 2.4 The determination of H2O2 content in lung. The iced lung tissue were quickly homogenized with 10mg/100 μ lhomogenate 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 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). 2.5 The determination of m RNA level of Prx VI in lung The total RNA were extracted with Trizol, About 3μg total RNA was reverse transcribed into c DNA then RT-PCR, using GAPDH as internal control. The ratio of amplification products of Prx VI to GAPDH represents the relative m RNA expression levels. 2.6The determination of protein level of Prx VI in lung The 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 64 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 Fluorescence was added again. Then the image was scaned with two-color infrared imaging systems to analyzed images value.Results: 1 The morphology change of kidney under light microscope The structure and shape of glomerulus, renal capsule, proximal tubule, distal convoluted tubule and collecting duct of control group were neat under light microscope. But the glomerulus of RIRI group was shrinking. The size of glomerular became smaller. Renal capsule cavity was expanded. Lumen of tubular was also expanded obviously. Some epithelial cells of proximal tubule showed edema change and their cytoplasm became loose. Renal stroma also showed edema change. The gap between tubular was expanded. Lumen of collecting duct was expanded and their epithelial cell showed edema change.2 The level of SCr in serum The serum SCr of control group was 103.444±8.465μmol/L, The serum SCr of RIRI group was 131.153±17.814μmol/L. The serum SCr levels of RIRI group was significantly higher than that of control group(P<0.05). 3 The level of BUN in serum The serum BUN of control group was 4.462±0.541 mmol/L, The serum BUN of RIRI group was 13.685±4.397 mmol/L. The serum BUN levels of RIRI group was significantly higher than that of control group(P<0.05). 4 The MDA content in lung homogenate The MDA content in lung of control group was 7.63±1.68mmol/g, The MDA content in lung of RIRI group was 10.89±1.74 mmol/g, The MDA content in lung of RIRI group was significantly higher than that of control group(P<0.01). 5 The H2O2 content in lung homogenate The H2O2 content in lung of control group was 16.51±2.39mmol/g, The H2O2 content in lung of RIRI group was 21.85±4.16 mmol/g, The H2O2 content in lung of RIRI group was significantly higher than that of control group(P<0.05). 6 The relative expression of Prx VI m RNA in lung The relative expression of Prx VI m RNA in lung were determined by RT-PCR. The expression level of Prx VI m RNA of control group were 0.72±0.17, the expression level of Prx VI m RNA of RIRI group were 1.09±0.23, The expression level of Prx VI m RNA of RIRI group was significantly higher than that of control group(P<0.01).The result showed that the gene expression of Prx VI in lung tissue of RIRI group were enhanced. 7 The protein level of Prx VI in lung The protein level of Prx VI in control group was 0.62±0.13, the protein level of Prx VI in RIRI group was 0.88±0.16. The protein level of Prx VI of RIRI group was significantly higher than that of control group(P<0.01). The result showed that the protein level of Prx VI in lung tissue of RIRI group were increased.Conclusion: 1 The Renal Ischemia-Reperfusion Injury model of rats can be successfully established by clipping left renal artery with non-damage vascular clamp near the renal hilum. 2 Renal ischemia-reperfusion could lead to high oxidative stress state of lung and oxidative damage. 3 The gene and protein expression of Prx VI in lung of renal ischemia reperfusion injury modle were significantly enhanced, which showed that Prx VI may be involved in the oxidative stress response induced by ischemia-reperfusion and have protective function for lung tissue. |
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