| Renal ischemia reperfusion injury(RIRI)is a common pathophysiological phenomenon during the surgical treatment of renal diseases in Department of Urology,It occurs during the process of clinical treatment such as renal crush injury,nephron retention,renal transplantation and so on.A large number of reactive oxygen species(ROS)were produced during the process of renal ischemia and reperfusion,which can lead to kidney damage.Therefore,oxidative stress is considered to be one of the main mechanisms of ischemia-reperfusion injury.The liver is also an important digestion,secretion,detoxification and excretion organ in the human body.Our previous research showed that liver was also exposed to oxidative stress and subjected peroxidative damage after Renal ischemia reperfusion injury.Nuclear factor erythroid 2-related factor 2(Nrf2)is a potent transcriptional regulator of endogenous antioxidant stress.Nrf2 regulates the expression of antioxidant proteins by interacting with antioxidant response elements(ARE).Superoxide dismutase(SOD)is the major ROS scavenging enzyme in the body,SOD is also one of the downstream target genes of Nrf2 transcription regulation.Sulforaphane(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.SFN are rich in broccoli and other cruciferous vegetables.Whether SFN can improve RIRI induced-liver peroxidative damage by antioxidant pathways? It is not reported.In this study,the rat model of RIRI was established by clamping the left renal artery and the SFN was given to RIRI model.The morphological and functional changes,the H2O2 content and MDA content,activity and expression level of antioxidant protein SOD in liver of RIRI model treated with SFN were observed,To investigate the antioxidant role of SFN inprevention of liver peroxidative damage induced by RIRI.Objective: Observing the morphological and functional changes,the H2O2 content and MDA content,activity and expression level of antioxidant protein SOD in liver of RIRI model treated with sulforaphane,to explore whether sulforaphane can enhanced ROS scavenging effect in liver and reduce liver peroxidative damage degree by upregulating expression of Nrf2 downstream gene SOD,which will provide data basis for the research about prevention and cure of sulforaphane on liver tissue injury induced by 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).Establishing the experimental animal model of RIRI was.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 of SFN1 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 Alanine aminotransferase(ALT).The liver were harvested after killing the rats.The suitable size of liver specimens were fixed by 4%paraformaldehyde fixative,morphological changes of liver were observed by HE staining.The liver 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 liver.2 The detection index and determination method2.1 The determination of ALT in serum of rats The content of ALT in serum was detected by microplate assay.The determination process of ALT are carried out strictly according to the operating instructions of the kit.2.2 The observation of liver morphological change The morphological change of liver was observed by HE staining.First the liver specimens fixed by 4% paraformaldehyde were dehydrated by gradient alcohol,xylene transparent and paraffin embedded,then liver 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 liver,then photographed.2.3 The detection of MDA content in liver of rats The content of MDA in liver 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 liver of rats The content of H2O2 in liver 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 liver of rats The SOD activity in liver 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 liver The total RNA in liver were extracted with RNA extraction kit,2μg 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 liver tissue with HE staining The morphological changes of liver tissue in rats were observed by HE staining method,under light microscope: for rats of control group,the size of hepatocytes were uniform and arranged in cords around central vein,the size of hepatic sinusoid were uniform,no abnormality were observered.for rats of RIRI group,the size of hepatocyte became smaller due to shrinking,hepatic sinusoid was expanded.for rats of SFN1 group,the size of Hepatocyte was increased due to swelling,hepatic sinusoids were compressed and became narrow.Vacuolar degeneration could be seen occasionally.for rats of SFN2 group,the partial liver cells are atrophic,the hepatic sinusoid is slightly dilated.A large number of vacuoles could be seen in liver cells and are more than SFN1 group.2 The change of ALT content in serum Compared with the ALT content of control group in serum(15.02±2.43U/L),the ALT content of RIRI group(210.27±29.94 U/L),SFN2group(161.71±21.76 U/L),SFN1 group(111.25±17.94 U/L)in serum was significantly increased(P<0.05).but the ALT content of SFN2 group and SFN1 group in serum was lower than that of RIRI group(P<0.05),the ALT content of SFN1 group in serum was lower than that of SFN2 group(P<0.05).3 The change of MDA content in liver Compared with the MDA content of control group in liver(45.08±6.35mmol/g),the MDA content of RIRI group(96.49±12.38 mmol/g),SFN2group(68.86±9.15 mmol/g),SFN1 group(54.62±9.57 mmol/g)in liver was increased(P<0.05).but the MDA content of SFN2 group and SFN1 group in liver was lower than that of RIRI group(P<0.05),the MDA content of SFN1 group in liver was lower than that of SFN2 group(P<0.05).4 The change of H2O2 content in liver Compared with the H2O2 content of control group in liver(55.49±8.75mmol/g),the H2O2 content of RIRI group(124.15±18.52 mmol/g),SFN2group(96.09±12.14 mmol/g),SFN1 group(75.00±11.14 mmol/g)in liver was increased(P<0.05).but the H2O2 content of SFN2 group and SFN1 group in liver was lower than that of RIRI group(P<0.05),the H2O2 content of SFN1 group in liver was lower than that of SFN2 group(P<0.05).5 The change of SOD activity in liver Compared with the SOD activity of control group in liver(133.33±14.21U/mg pro),the SOD activity of RIRI group(53.36±7.28 U/mg pro),SFN2group(73.46±8.96 U/mg pro),SFN1 group(86.75±10.16 U/mg pro)in liver was decreased(P<0.05).but the SOD activity of SFN2 group and SFN1 group in liver was higher than that of RIRI group(P<0.05),the SOD activity of SFN1 group in liver was higher than that of SFN2 group(P<0.05).6 The expression change of SOD gene in liver.The relative expression of SOD m RNA in liver was determined by Real-Time PCR.GAPDH was used as internal control.Compared with the SOD m RNA expression of control group in liver(0.79±0.09),the SOD m RNA expression of RIRI group(0.99±0.15),SFN2 group(1.22±0.15),SFN1group(1.31±0.13)in liver was increased(P<0.05).but the SOD m RNA expression of SFN2 group and SFN1 group in liver 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 in liver during renal ischemia-reperfusion injury.Conclusion:1 After renal ischemia reperfusion injury,sulforaphane enhanced scavenging effect for ROS in liver and decrease the peroxidative damage degree of liver tissue by upregulating the expression of Nrf2 and downstream gene SOD of hepatic tissue.2 Compared with the sulforaphane administration after reperfusion,giving sulforaphane immediately after ischemia can effectively reduce the oxidative stress level and peroxidative damage degree of hepatic tissue and improve the morphological and functional changes of the liver. |
