The Effect Of Sulforaphane On Intestinal Injury Induced By Renal Ischemia-Reperfusion Injury Rats

Objective:.The kidney is an important organ in the human body,as part of the urinary system,plays a key role in maintaining the stability of the human body environment,mainly for the excretion of harmful substances,reabsorption of useful substances,such as potassium ions,sodium ions,amino acids and other substances,so as to maintain acid-base balance,prevent the occurrence of water,electrolyte disorder.In addition,the kidneys are known to regulate blood pressure,promote the formation of red blood cells,and promote the activation of vitamin D.Therefore,once the kidney is damaged,it will lead to the instability of the internal environment,water and electrolyte disturbances,and even life endanger.It is reported that when the kidney is damaged,it can cause other organs such as liver,brain,lung and other organs to have corresponding damage.Renal ischemia reperfusion injury?RIRI?is a complex pathophysiological process,along with the deepening of research in recent years,compared to the pathophysiology of RIRI exact involved is a reactive oxygen species?ROS?generated a large number of reactive oxygen species,is a kind of single electron containing oxygen free radical substances.Common including superoxide anion?O₂^-?,hydroxyl radical??OH?and hydrogen peroxide?H₂O₂?etc.Under normal circumstances,ROS plays an important physiological function in cells,and can be the body antioxidant system removed,but when the production of ROS,then the effect will show up,can cause the nucleic acid,protein,mitochondria and microsome of material damage,at the same time,ROS can activate a variety of transcription factors,can directly or indirectly involved in ischemia reperfusion injury.Sulforaphane is an isothiocyanate,extracted from cruciferous vegetables,is a non-toxic,complex natural plant ingredients,and high concentrations of sulforaphane from broccoli and broccoli sprouts.The former form of glucosinolates in plants can be hydrolyzed through the hydrolysis of black mustard glucosinase,with the most content in broccoli.Sulforaphane?SFN?antioxidant and anticancer properties have been confirmed by many studies,and sulforaphane activates the Nrf2 pathway.Studies have found thatNrf2?Nuclear factor erythroid-derived 2-like 2?is nuclear transcription factor.According to research,Nrf2 is the strongest antioxidant reaction found.Under normal circumstances,Nrf2 is located in the cytoplasm.When the cells undergo oxidative stress,Nrf2 is phosphorylated under the action of ERK and other kinases,and it enters into the nucleus and starts the transcription of SOD.The application of antioxidant properties of sulforaphane in renal ischemia reperfusion injury,should be able to reduce ischemia-reperfusion injury.Does renal ischemia-reperfusion injury cause intestinal damage?The application of sulforaphane could play against oxidative damage,and even reduce intestinal injury?With these doubts,we first prepare the rat model of renal ischemia-reperfusion injury,then observe the renal function changes and pathological sections to observe whether the model is successfully prepared.The pathological changes of intestinal tissue,H₂O₂ content,MDA content,SOD activity,superoxide dismutase?SOD?protein expression and mRNA in the rat RIRI model were further observed.Discussion on renal ischemia reperfusion rat model whether caused by intestinal injury,application of sulforaphane might play a role in reducing the stress injury of intestinal antioxidant oxidation,in order to provide some ideas and experimental basis for clinical treatment.Methods:1 The preparation of RIRI models and animals groupA new practical model of renal ischemia-reperfusion model was prepared with reference to Yu Xiaodong and other reports.The rats were randomly divided into control group?Con?and renal ischemia reperfusion model group?RIRI?,ischemia+sulforaphane group?M group?,reperfusion+sulforaphane group?N group?.2 The pathological examination of renal tissue and intestinal tissue in rats was examined by HE staining.3 The observation index and its methodThe serum levels of SCr and BUN were detected,the content of MDA in the intestinal tissue homogenate,the content of H₂O₂ in the intestinal tissue homogenate,and the activity of SOD in the intestinal tissue were measured.Operate strictly in accordance with the operating procedures given by the purchased kit instructions.4 Detection of SOD gene expression in rat intestinal tissue by real-time quantitative RT-PCRThe total RNA kit was used to extract the total organization RNA according to the experimental procedure given..2 g total RNA reverse transcription.Using GAPDH as a reference,the relative expression amount of SOD was measured by quantitative real-time PCR.5 Determination of SOD protein in intestinal tissue of rats by Western blotThe total protein of rat intestinal tissue was extracted and the protein expression level of superoxide dismutase?SOD?in intestinal tissue of rats was determined by Western blot method.6 Data processingAll data were analyzed by SPSS 19 software.The data were expressed by?x?SD.Groups were compared with multiple mean variance analysis,and P<0.05 was statistically significant.Results:1.The morphology change of kidney and intestineKidney tissue microscope display that in the Con group,the renal tubular morphological structure was complete and clear,the size of the renal capsule was normal,and the glomerular morphology was regular.In the RIRI group,atrophy of the renal vasculature was observed;dilation of the renal cysts appeared;occasionally,cytoplasmic cytoplasm of the tubular epithelial cells was loosened;edema of the renal interstitium developed,and the gap expanded;the collecting ducts also showed changes in lumen expansion.In group M,the renal vasculature slightly atrophied,the space between the renal cysts was slightly widened,and the proximal tubules were slightly swollen.In group N,there was atrophy of the renal vascular globules,significant dilation of renal tubules,enlargement of the gap between renal cysts,and widening of distal tubules.Intestinal tissue optical microscope:Con group rats showed complete intestinal villi,goblet cell morphology is normal.The morphological structure of small intestine tissue in RIRI group,M group and N group showed no significant difference compared with the control group.2.The level of serum SCrCompared with the SCr content of control group in serum60.07±6.46?mol/L,the SCr content of RIRI group 360.15±22.814?mol/L,M group 92.9±13.06?mol/L,N group 295.04±18.43?mol/L in serum was significantly increased,P<0.05.But the SCr content of M group and N group in serum was significantly lower than RIRI group,P<0.05.But the SCr content of M group and N group in serum was significantly lower than RIRI group,P<0.05.The SCr content of M group in serum was significantly lower than N group,P<0.05.3.The BUN level in the serumCompared with the BUN content of control group in serum6.563±1.44mmol/L,the BUN content of RIRI group 25.07±2.39mmol/L,M group 11.36±1.44mmol/L,N group 21.06±1.51mmol/L in serum was significantly increased,P<0.05.But the BUN content of M group and N group in serum was significantly lower than RIRI group,P<0.05.The BUN content of M group in serum was significantly lower than N group,P<0.05.4.The MDA content in the intestine homogenateCompared with the MDA content of control group in intestine72.67±13.77mmol/g,the MDA content of RIRI group 285.07±17.33mmol/g,M group 162.67±14.57mmol/g,N group 185.67±17.77mmol/g in intestine was significantly increased,P<0.05.But the MDA content of M group and N group in intestine was significantly lower than RIRI group,P<0.05.The MDA content of M group in intestine was significantly lower than N group,P<0.05.5.The H₂O₂ content in the intestine homogenateCompared with the H₂O₂ content of control group in intestine87.67±10.77mmol/g,the H₂O₂ content of RIRI group 261.67±48.37mmol/g,M group 121.67±18.87mmol/g,N group 161.67±22.43mmol/g in intestine was significantly increased,P<0.05.But the H₂O₂ content of M group and N group in intestine was significantly lower than RIRI group,P<0.05.The H₂O₂ content of M group in intestine was significantly lower than N group,P<0.05.6.The change of SOD activity in intestineCompared with the SOD activity of control group in intestine112.67±9.33U/mg pro,the SOD activity of RIRI group 28.67±6.33U/mg pro,M group 65.67±8.39U/mg pro,N group 43.67±5.33U/mg pro in intestine was significantly decreased,P<0.05.But the SOD activity of M group and N group in intestine was significantly higher than RIRI group,P<0.05.The SOD activity of M group in intestine was significantly higher than N group,P<0.05.7.The expression level of the SOD mRNA in intestineThe expression level of the SOD mRNA in intestine were determined by RT-PCR.GAPDH was used as internal control.Compared with the SOD mRNA expression of control group in intestine 0.92±0.11,the SOD mRNA expression of RIRI group 1.14±0.13,M group 1.34±0.17,N group 1.64±0.17 in intestine was increased,P<0.05.But the SOD mRNA expression of M group and N group in intestine was higher than RIRI group,P<0.05.However there was no significant difference for mRNA expression between M group and N group,P>0.05.8.The protein level of the SOD in intestineThe protein level of the SOD was estimated by Western Blot.Compared with the protein level of the SOD of control group in intestine 0.97±0.12,the protein level of the SOD of RIRI group 1.12±0.17,M group 1.38±0.16,N group 1.34±0.19 in intestine was increased,P<0.05.But the protein level of the SOD of M group and N group in intestine was higher than RIRI group,P<0.05.However there was no significant difference for protein level of the SOD between M group and N group,P>0.05.Conclusion:1.Renal ischemia reperfusion injury causes the intestine to be in a high degree of oxidative stress and suffer from oxidative damage.2.After Renal ischemia reperfusion injury,SFN enhanced scavenging effect for ROS and decrease the peroxidative damage degree of intestine 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 the oxidative stress level and peroxidative damage degree of intestine tissue.