Oxidative Stress Level And Expression Chang Of EC-SOD In Heart Of RIRI Model

Renal ischemia reperfusion injury（RIRI）is a kind of frequent pathophysiological reaction in clinic,RIRI is more common in clinical surgery,shuh as nephron sparing surgery,renal crush injury and so on.The occurrence of RIRI can delay the recovery of renal function after operation,which even can lead to the failure of surgery and renal failure.A large number of animal experiments and clinical studies showed that when the blood of the kidneys is blocked,the cells in different parts of the kidney will be damaged due to ischemia and hypoxia.When the renal blood supply is restored again,there will be a large number of reactive oxygen species（ROS）released from renal tissue cells,which further aggravate the existing cell injury and lead to the occurrence of renal ischemia-reperfusion injury.So,oxidative stress is thought to be one of the main mechanisms of RIRI.Superoxide dismutase（SOD）is the major ROS scavenging enzyme.There are three SOD subtypes: Manganese superoxide dismutase（Mn-SOD）、Copper zinc superoxide dismutase（Cu/Zn-SOD）Extracellular superoxide dismutase（EC-SOD）.EC-SOD is the only SOD isoform that can remove extracellular ROS.In the past,there were many studies on the antioxidant function of Mn-SOD and Cu/Zn-SOD.But recent studies have found: compared with Cu/Zn-SOD and Mn-SOD,EC-SOD may play a more important role in anti oxidative stress and anti inflammation.Research showed that: The decrease of EC-SOD expression in myocardial tissue could significantly increase the incidence of hypertension and ischemic myocardial disease for patients;The decreasing of binding force for EC-SOD and extracellular matrix increased significantly the risk of cardiovascular disease and ischemic heart disease.In addition,the expression level of EC-SOD was significantly decreased in heart failure patients.These studies indicate that EC-SOD may play an important role in maintaining the normal function of myocardium.The heart is the power organ of the blood circulation.It takes a lot of oxygen and nutrients to maintain the normal function of the heart.So,The heart is very sensitive to ischemia and hypoxia.When renal tissue appeared ischemia reperfusion injury and was oxidative stress state,Whether myocardial tissue is also subject to oxidative damage due to oxidative stress response Whether the expression of EC-SOD in myocardial tissue was changed.Objective: To explore whether the expression of EC-SOD gene and protein was changed,and whether the change was related to the content of H₂O₂ and MDA after RIRI.Methods: 1 Establishment of RIRI animal model and treatment of test specimenWistar rats（weighting 200±10g）were provided by the experimental animal center of Hebei Medical University.rats were divided randomly into Renal Ischemia-Reperfusion Injury（RIRI）group and control group.The experimental animal model of RIRI was established according to the method of Yu Xiaodong et al.The rats were anesthetized by intraperitoneal injection of 6% chloral hydrate（5ml / kg）.The RIRI group rats were fixed on operating table and fully exposed bilateral kidneys after alcohol disinfection.After the right kidney was excised,the left renal artery of the rat was isolated and clipped by a non traumatic artery clamp near the renal hilum to block renal blood supply,At this time,we can see that the color of the kidney changes from bright red to light red.After 45 minutes of blocking blood perfusion,the artery clamp was removed to restore blood perfusion of the left kidney.At this time,the left renal artery was rapidly filling,and the color of the kidney changed from light red to bright red.The results showed that the blood reperfusion of the left kidney was successful.The disinfection and laparotomy process of control group was consistent with the RIRI model group.However,only the right kidney was removed after exposing bilateral kidneys.The left renal artery was isolated,but the blood perfusion was not blocked.Rats were anesthetized again after 24 hours of renal perfusion,blood was collected from the right carotid artery and was centrifuged at 3000 rpm for 10 mins in order to isolate the serum,for detection of the Urea Nitrogen（BUN）and the Creatinine（SCr）.The kidneys of rats were removed and fixed with 4% paraformaldehyde fixative to observed the morphological changes by HE staining.Then hearts were harvested and quickly placed in liquid nitrogen,and then transferred to-80 refrigerator after freezing for determination of the EC-SOD gene and protein expression level,malondialdehyde（MDA）and hydrogen peroxide（H₂O₂）content in myocardial tissue.2 The detection index and determination method 2.1 The determination of SCr and BUN in serum of ratsThe SCr content and the BUN content in serum of rats were measured by picric acid method and enzyme-coupled rate method.The determination process of SCr and BUN are carried out strictly according to the operating instructions of the kit.2.2 The observation of the kidney morphological changeThe morphological change of kidney was observed by HE staining.First the kidney specimens fixed by 4% paraformaldehyde were dehydrated by gradient alcohol,xylene transparent and paraffin embedded,then kidney specimens were cranked out 5 micron thick common section and carried out hematoxylin eosin staining treatment.The Olympus optical microscope was used to observe the morphological changes of the kidney tissue and photographed.2.3 The myocardial tissue homogenate and determination of MDA contentThe cooled homogenate buffer（KPB 50mmol/L,Benzamidine 1mmol/L,EDTANa3 1mmol/L,PMSF 1mmol/L,Tween-20 0.1%,NaCl 0.5mol/L,β-Mercaptoethanol 5mmol/L,pH7.4）.was added to the frozen myocardial tissue according to the ratio of 10mg/100μl,the tissue was homogenized in ice water bath.The homogenate was centrifuged at 4℃with 4000 rpm,for 20 mins,the supernatant was named 10% myocardial tissue homogenate.The MDA content of the homogenate was determined by thiobarbituric acid colorimetry method.The whole detection process was carried out in accordance with the operational guidelines of MDA assay kit from Nanjing jiancheng.2.4 The myocardial tissue homogenate and determination of H₂O₂ contentThe cooled homogenate buffer（KPB 50mmol/L,Benzamidine 1mmol/L,EDTANa3 1mmol/L,PMSF 1mmol/L,Tween-20 0.1%,Na Cl 0.5mol/L,β-Mercaptoethanol 5mmol/L,pH7.4）.was added to the frozen myocardial tissue according to the ratio of 10mg/100μl,the tissue was homogenized in ice water bath.The homogenate was centrifuged at 4 with 4000 rpm,for 20 mins,the ℃supernatant was named 10% myocardial tissue homogenate.The H₂O₂ content of the homogenate was determined by the Molybdate colorimetric method,The H₂O₂ content of myocardial tissue was expressed by the amount of H₂O₂ per gram of protein（mmol/g Pro）.2.5 The detection of EC-SOD gene expression in myocardial tissueThe total RNA in rats myocardial tissue were extracted with the Trizol reagent,and 3μg total RNA were reverse transcribed into template cDNA.glyceraldehyde-3-phosphate dehydrogenase（GAPDH）was used as internal control,then PCR amplification.The gray value of amplified products were analysed.The relative expression of EC-SOD gene was represented by the ratio of EC-SOD gray value to GAPDH gray value.2.6 The detection of EC-SOD protein expression in myocardial tissueThe relative expression level of EC-SOD protein in rat myocardial tissue was determined by Western blot,Rat myocardial tissue was prepared into homogenate,the supernatant was collected after centrifugation,protein denaturation was made by boiling method and modified Lowry method for protein quantitation.The loading protein amount was 65 ug in SDS-PAGE gel electrophoresis,The EC-SOD antibody diluted with 5% skimmed milk was added to the PVDF membrane after transfering membrane and 5% skimmed milk closed treatment,the PVDF membrane was at shaker for Shocking overnight at 4℃.The IgG antibody labeled by Fluorescence was added to PVDF membrane after rinsing membrane with Tween-TBS rinse solution.The strip was scaned with dual color infrared imaging system and analyzed the gray value.2.7 The correlation analysis between EC-SOD mRNA,protein expression and MDA content,H₂O₂ content in rat myocardial tissueThe correlation between EC-SOD mRNA,protein expression and MDA content,H₂O₂ content in rat myocardial tissue were analyzed with pearson correlation statistical method.Results:1 The morphology observation of rats renal tissue with HE stainingMorphological changes of renal tissue in rats were observed by HE staining method,under light microscope: the morphology of glomerulus and renal capsule was normal with no glomus atrophy and cystic expansion,the morphology of proximal、distal convoluted tubules and collecting ducts was normal,the renal interstitial structure was no abnormal;But some glomerular of RIRI group was shrinking.The size also became smaller.Follicular cysts,renal tubular lumen,collecting duct lumen showed obvious expansion;In addition,the RIRI group also showed renal interstitial edema and enlargement of renal tubular space.There was no obvious morphological changes between the two groups under the light microscope.2 The change of SCr content in serumCompared with the SCr content of control group in serum（103.444± 8.465μmol/L）,the SCr content of RIRI group in serum（131.153±17.814μmol/ L）was significantly increased（P<0.05）.3 The change of BUN content in serumThe BUN content of RIRI group in serum（13.685±4.397 mmol/L）was significantly increased than the BUN content of control group in serum（4.462±0.541 mmol/L）,（P<0.05）.4 The change of MDA content in myocardial tissueCompared with the MDA content of control group in myocardial tissue（10.18±1.77mmol/g）,the MDA content of RIRI group in myocardial tissue（14.01±2.29 mmol/g）was significantly increased（P<0.01）.5 The change of H₂O₂ content in myocardial tissueCompared with the H₂O₂ content of control group in myocardial tissue（13.93±1.88 mmol/g）,the H₂O₂ content of RIRI group in myocardial tissue（18.56±2.56 mmol/g）was significantly increased（P<0.01）.6 The expression change of EC-SOD gene in rats myocardial tissue.The relative expression of EC-SOD gene in rats myocardial tissue was determined by RT-PCR.GAPDH was used as internal control.Compared with the EC-SOD mRNA expression of control group in myocardial tissue（0.73±0.11）,the EC-SOD mRNA expression of RIRI group in myocardial tissue（1.15±0.17）was significantly increased（P<0.01）.The result showed that the EC-SOD mRNA expression in rats myocardial tissue of RIRI group was significantly increased.7 The expression change of EC-SOD protein in rats myocardial tissueThe relative expression level of EC-SOD protein was detected by Western blot.Compared with EC-SOD protein expression level of control group in rats myocardial tissue（0.65±0.1）,the EC-SOD protein expression level of RIRI group in rats myocardial tissue（1.07±0.12）was significantly increased（P<0.01）.The result showed that EC-SOD protein expression level in rats myocardial tissue of RIRI group was significantly increased after renal ischemia reperfusion injury.8 The correlation between EC-SOD mRNA,protein expression and MDA content,H₂O₂ content in rat myocardial tissueThe correlation between EC-SOD mRNA,protein expression and MDA content,H₂O₂ content in rat myocardial tissue were analyzed with pearson statistical method.There were positive correlation between EC-SOD mRNA level and MDA content（r=0.70,P<0.05）,between EC-SOD mRNA level and H₂O₂ content（r=0.632,P<0.05）.There were also positive correlation between EC-SOD protein level and MDA content（r=0.616,P<0.05）,between EC-SOD protein level and H₂O₂ content（r=0.641,P<0.05）.Conclusion: The expression of EC-SOD gene and protein in myocardial tissue in RIRI rats was increased,the change was positive correlated with H₂O₂ and MDA content.which indicated that EC-SOD may play an important role in antioxidation during the RIRI process.