The Effect Of Sulforaphane On Oxidative Stress Of Renal Ischemia Reperfusion Injury 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,renal artery occlusion and renal resection or renal ransplantation and so on.A large number of animal experiments and clinical studies showed that a large number of reactive oxygen species?ROS?were produced during temporary occlusion of the renal blood perfusion and subsequent recovery of the blood supply,which led to severe injury of renal tissue cells.Therefore,oxidative stress is considered to be one of the main mechanisms of ischemia-reperfusion injury.nuclear factor erythroid 2-related factor 2?Nrf2?is the most potent transcriptional regulator of antioxidant stress.Nrf2 regulates the expression of antioxidant protein by interacting with antioxidant responsive element?ARE?.It is an important endogenous antioxidant stress pathway.Superoxide dismutase?SOD?is the major ROS scavenging enzyme in the body.It can convert hydroxyl radical?OH-?into oxygen by disproportionation reaction.At the same time,SOD is one of the downstream target genes of Nrf2 transcription regulation.Sulforaphane?SFN?was widely presenced in cruciferous vegetables such as broccoli and blong to isothiocyanate.A large number of studies show that SFN is an inducer of Nrf2 pathway.It can play a biological role in antioxidation and anti-tumor by regulating the expression of Nrf2 and its downstream genes.In this study,we first established renal ischemia-reperfusion injury model of rat by clipping the renal artery with non-damage vascular clamp.At the same time,the Nrf2 inducer sulforaphane was given to RIRI model.The morphological and functional changes,the H₂O₂ content and MDA content,activity and expression level of antioxidant protein SOD of RIRI modeltreated with sulforaphane were observed,to investigate the antioxidant role of sulforaphane in renal ischemia reperfusion injury.Objective:observing the morphological and functional changes,the H₂O₂ content and MDA content,activity and expression level of antioxidant protein SOD of RIRI model treated with sulforaphane,to explore whether sulforaphane can enhanced ROS scavenging effect and reduce renal tissue peroxidation injury by upregulating expression of Nrf2 downstream gene SOD,which will provide data basis for the research about prevention and cure of sulforaphane on renal ischemia reperfusion injury.Methods:1 Establishment of RIRI animal model and treatment of test specimen24 male Wistar rats?weighting 200±10g?purchased from the experimental animal center of Hebei Medical University were divided randomly into control group,Renal Ischemia-Reperfusion Injury?RIRI?group,Ischemia +sulforaphane group?SFN1 group?,Reperfusion + sulforaphane group?SFN2group?.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,it can be observed that the color of the kidney changes from bright red to dark red.After 45 minutes of blocking blood perfusion,the artery clamp was removed to restoring blood perfusion of the left kidney.At this time,the left renal artery was rapidly filling,and the color of the kidney changed from dark red to bright red.The results showed that the blood reperfusion of the left kidney was successful.The disinfection and laparotomy process 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.Sulforaphane diluted with DMSO was evenly applied to the surface of the small intestine ofSFN1 group immediately after clamping the left renal artery,the remaining steps are the same as the RIRI group.Sulforaphane diluted with DMSO was evenly applied to the surface of the small intestine of SFN2 group immediately after removing blood vessels to restore the renal blood perfusion,the remaining steps are the same as the RIRI group,only equal DMSO was evenly applied to the surface of the small intestine of control group and RIRI group.Rats were anesthetized again after 24 hours of renal perfusion,the right carotid artery blood was collected and centrifuged for 10 mins at 3000 rpm to isolate serum,for detection of Creatinine?SCr?and Urea Nitrogen?BUN?.The kidneys were harvested after killing the rats.The suitable size of kidney specimens were fixed by 4% paraformaldehyde fixative,morphological changes of renal tissue were observed by HE staining.The kidney was quickly placed in liquid nitrogen,and then transferred to-80 refrigerator after freezing,for determination of the SOD activity and gene expression level,malondialdehyde content?MDA?and hydrogen peroxide?H₂O₂?content in kidney.2 The detection index and determination method2.1 The determination of SCr and BUN in serum of ratsThe SCr content in serum of rats was measured by picric acid method.The BUN content in serum of rats was measured by 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 detection of MDA content in renal tissue of ratsThe content of MDA in renal tissue was determined by thiobarbituric acid colorimetric method.The testing process was carried out according to the operating instructions of the kit.2.4 The detection of H₂O₂ content in renal tissue of ratsThe content of H₂O₂ in renal tissue was determined by Molybdate colorimetric method.The testing process was carried out according to the operating instructions of the kit.2.5 The detection of SOD activity in renal tissue of ratsThe SOD activity in renal tissue was determined by xanthine oxidase method.The testing process was carried out according to the operating instructions of the kit.2.6 The detection of SOD gene expression in kidneyThe total RNA in kidney were extracted with RNA extraction kit,2?g quantified RNA was reverse transcribed into template c DNA.glyceraldehyde-3-phosphate dehydrogenase?GAPDH?was used as internal control,then Real-Time PCR relative quantitative analysis.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: for rats of control group,the morphology and structure of renal proximal convoluted tubule and distal convoluted tubule were clear and complete,the morphology of glomerulus was regular,the size of Renal capsule is normal.for rats of RIRI group,glomerular obviously shrinked and size also became smaller.The renal capsule gap was widened significantly,the cell of proximal convoluted tubule and distal convoluted tubule were shrinked,bubble could be seen in cytoplasm,duct lumen showed obvious expansion.for rats of SFN2 group,glomerular lightly shrinked and size became smaller.Eosinophilic staining was enhanced.The renal capsule was expanded significantly,the cell of Proximal convoluted tubule was light swelling,the gap in renal interstitium became narrow,the lumen of distal tubules were widened.For rats of SFN1 group,eosinophilic staining of glomerular was enhanced,the renal capsule gap was widened slightly,renal interstitium gap became narrowrenal,the lumen of distal tubules showed slight swelling and are lighter than SFN2 group.2 The change of SCr content in serumCompared with the SCr content of control group in serum?59.87±6.76?mol/L?,the SCr content of RIRI group?358.08±22.20?mol/L?,SFN2group?294.14±18.76?mol/L?,SFN1 group?93.60±13.16?mol/L?in serum was significantly increased?P<0.05?.but the SCr content of SFN2 group and SFN1 group in serum was significantly lower than that of RIRI group?P<0.05?,the SCr content of SFN1 group in serum was significantly lower than that of SFN2 group?P<0.05?.3 The change of BUN content in serumCompared with the BUN content of control group in serum?6.35±1.15mmol/L?,the BUN content of RIRI group?24.80±2.53 mmol/L?,SFN2 group?20.01±1.78 mmol/L?,SFN1 group?12.32±1.69 mmol/L?in serum was significantly increased?P<0.05?.but the BUN content of SFN2 group and SFN1 group in serum was lower than that of RIRI group?P<0.05?,the BUN content of SFN1 group in serum was lower than that of SFN2 group?P<0.05?.4 The change of MDA content in kidneyCompared with the MDA content of control group in kidney?72.29±14.75 mmol/g?,the MDA content of RIRI group?284.32±17.48mmol/g?,SFN2 group?186.52±17.50 mmol/g?,SFN1 group?161.31±14.03mmol/g?in kidney was significantly increased?P<0.05?.but the MDA content of SFN2 group and SFN1 group in kidney was significantly lower than that of RIRI group?P<0.05?,the MDA content of SFN1 group in kidney was lower than that of SFN2 group?P<0.05?.5 The change of H₂O₂ content in kidneyCompared with the H₂O₂ content of control group in kidney?85.70±10.66mmol/g?,the H₂O₂ content of RIRI group?260.62±48.50 mmol/g?,SFN2group?160.84±23.34 mmol/g?,SFN1 group?121.22±18.49 mmol/g?in kidney was significantly increased?P<0.05?.but the H₂O₂ content of SFN2 group andSFN1 group in kidney was significantly lower than that of RIRI group?P<0.05?,the H₂O₂ content of SFN1 group in kidney was lower than that of SFN2 group?P<0.05?.6 The change of SOD activity in kidneyCompared with the SOD activity of control group in kidney?112.47±9.09U/mg pro?,the SOD activity of RIRI group?28.56±6.11 U/mg pro?,SFN2group?43.85±5.29 U/mg pro?,SFN1 group?65.44±8.91 U/mg pro?in kidney was significantly decreased?P<0.05?.but the SOD activity of SFN2 group and SFN1 group in kidney was higher than that of RIRI group?P<0.05?,the SOD activity of SFN1 group in kidney was higher than that of SFN2 group?P<0.05?.7 The expression change of SOD gene in kidney.The relative expression of SOD m RNA in kidney was determined by Real-Time PCR.GAPDH was used as internal control.Compared with the SOD m RNA expression of control group in kidney?0.93±0.13?,the SOD m RNA expression of RIRI group?1.15±0.15?,SFN2 group?1.36±0.18?,SFN1group?1.39±0.18?in kidney was increased?P<0.05?.but the SOD m RNA expression of SFN2 group and SFN1 group in kidney was higher than that of RIRI group?P<0.05?,However,there was no significant difference for m RNA expression between SFN2 group and SFN1 group?P>0.05?.The result showed that: SFN upregulated the expression of SOD during renal ischemiareperfusion injury.Conclusion:1 After renal ischemia reperfusion injury,renal tissue was in oxidative stress state and suffered from peroxidative damage.2 After renal ischemia reperfusion injury,sulforaphane enhanced scavenging effect for ROS and decrease the peroxidative damage degree of kidney tissue by upregulating the expression of Nrf2 and downstream gene SOD.3 Compared with the sulforaphane administration after reperfusion,giving sulforaphane immediately after ischemia can effectively reduce theoxidative stress level and peroxidative damage degree of renal tissue and improve the morphological and functional changes of the kidney.