莱菔硫烷对大鼠肾缺血再灌注损伤模型氧化应激的影响

发布时间：2018-03-13 13:51

  本文选题：肾缺血再灌注损伤　切入点：莱菔硫烷　出处：《河北医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：肾缺血再灌注损伤(Renal ischemia reperfusion injury,RIRI)是临床上一种常见的病理生理现象,多见于肾单位保留术、肾挤压伤、肾动脉阻断术以及肾脏切除或移植等临床治疗过程中。大量动物实验和临床研究均发现:在暂时性阻断肾脏的血液灌注,随后又重新恢复其血液供应的过程中,会有大量活性氧族(reac-tive oxygen species,ROS)产生,导致肾组织细胞的严重损伤。因此,氧化应激被认为是引发缺血再灌注损伤的主要机制之一。核因子E2相关因子2(nuclear factor erythroid 2-related factor 2,Nrf2)是目前发现的最强抗氧化应激转录调控因子。Nrf2通过与抗氧化反应元件(antioxidant responsive element,ARE)相互作用调节抗氧化蛋白的表达,是机体内重要的内源性抗氧化应激通路。超氧化物歧化酶(Superoxide Dismutases,SOD)是机体内主要的ROS清除酶系,能通过歧化反应将羟自由基(OH-)转化成氧。同时,SOD也是Nrf2转录调控的下游靶基因之一。莱菔硫烷(sulforaphane,SFN)在西兰花等十字花科蔬菜中大量存在,属于异硫氰酸盐。大量研究表明:SFN是Nrf2通路的诱导剂,可通过上调Nrf2及其下游基因的表达发挥抗氧化、抗肿瘤等生物学作用。本研究采用无损伤动脉夹钳夹肾动脉法建立大鼠RIRI模型,同时给予RIRI模型组大鼠Nrf2诱导剂莱菔硫烷,观察莱菔硫烷处理的大鼠RIRI模型肾组织形态学及功能变化、H_2O_2含量、MDA含量及抗氧化蛋白SOD活性及表达变化,探讨莱菔硫烷在RIRI中发挥的抗氧化作用。目的:通过观察莱菔硫烷处理的大鼠RIRI模型肾组织形态学和功能变化、H_2O_2含量、MDA含量及抗氧化蛋白SOD活性及表达变化,探讨莱菔硫烷是否可通过上调Nrf2下游基因SOD的表达,增强机体对ROS的清除作用,降低肾组织过氧化损伤程度,为莱菔硫烷对RIRI的防治研究提供数据依据。方法:1动物模型建立及检测标本的处理Wistar大鼠来源于河北医科大学实验动物中心,雄性(体重200±10g)24只,随机分为对照组(Control组)、肾脏缺血再灌注损伤模型组(RIRI组)、缺血+莱菔硫烷组(SFN1组),灌注+莱菔硫烷组(SFN2组)。按照余晓东等人的模型制备方法建立RIRI实验动物模型:腹腔注射6%水合氯醛(5ml/kg)麻醉大鼠。将RIRI组大鼠固定于手术台上,酒精消毒后开腹充分暴露双侧肾脏,先将大鼠的右侧肾脏切除后,再钝性地分离其左侧肾动脉,并采用无创伤性动脉夹在接近肾门部位夹闭左侧肾动脉,阻断肾脏血液供应,此时可以观察到肾脏颜色由鲜红色迅速转变为暗红色。血液灌注阻断45分钟后弃去动脉夹,重新恢复左侧肾脏的血液灌注,此时可见左侧肾动脉迅速充盈,肾脏颜色也随即由暗红色转变为鲜红色,提示左侧肾脏的血液重新灌注成功。Control组大鼠的消毒开腹过程与RIRI模型组一致,但该组大鼠双侧肾脏暴露后只切除其右侧肾脏,钝性地分离左侧肾动脉,但并不夹闭阻断血液灌注。SFN1组大鼠在夹闭左侧肾动脉后立即将二甲基亚砜稀释的莱菔硫烷均匀涂抹于小肠表面,其余操作步骤同RIRI组。SFN2组大鼠在弃去血管夹恢复肾脏血液灌注后立即将二甲基亚砜稀释的莱菔硫烷均匀涂抹于小肠表面,其余操作步骤同RIRI组。Control组和RIRI组只在小肠表面涂抹等量的二甲基亚砜。恢复大鼠肾脏血流灌注24小时后重新麻醉大鼠,收集右颈总动脉血液,3000转/分,离心10分钟,分离并收集血清,用于血清肌酐(Serum Creatinine,SCr)、血清尿素氮(Blood Urea Nitrogen,BUN)含量检测;大鼠处死后切取其肾脏,取适宜大小的肾脏标本置于4%多聚甲醛固定液中固定,用于HE染色法观察大鼠肾脏组织的形态学改变。将其余肾脏组织迅速置于液氮中,待冷冻后转移至-80℃冰箱保存备用,用于大鼠肾组织SOD活性、基因表达水平测定,丙二醛(malondialdehyde,MDA)含量和过氧化氢(hydrogen peroxide,H_2O_2)含量测定。2检测指标及测定方法2.1大鼠血清内SCr和BUN含量检测采用苦味酸法检测大鼠血清SCr含量;采用酶偶联速率法测定大鼠血清BUN含量。SCr和BUN含量的测定过程严格按照试剂盒操作说明进行。2.2大鼠肾脏组织形态结构改变的观察采用HE染色法观察肾脏组织形态结构的改变。将4%多聚甲醛固定的肾脏标本按梯度酒精脱水、二甲苯透明,石蜡包埋处理后,将肾脏标本切片厚度约5μm,然后进行苏木精伊红染色处理,采用奥林帕斯光学显微镜观察肾组织形态学改变并拍照。2.3大鼠肾组织MDA含量检测肾组织MDA含量采用硫代巴比妥酸比色法测定,检测过程按试剂盒操作说明进行。2.4大鼠肾组织H_2O_2含量检测肾组织H_2O_2含量采用钼酸比色法测定,检测过程按试剂盒操作说明进行。2.5大鼠肾组织SOD活性检测肾组织SOD活性采用黄嘌呤氧化酶法测定,检测过程按试剂盒操作说明进行2.6大鼠肾组织SOD基因表达水平检测采用RNA提取试剂盒裂解提取大鼠肾组织内的总RNA。RNA定量后将2μg RNA反转录成模板c DNA。以甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)作为内参照,进行Real-Time PCR相对定量分析。结果:1大鼠肾组织的HE观察结果HE法观察大鼠肾组织形态结构改变。光镜下可见:Control组大鼠肾近曲小管、远曲小管形态结构清晰完整。肾小球形态规则、肾小囊大小正常。RIRI组大鼠:肾小球明显萎缩体积变小,肾小囊间隙显著增宽,近曲小管、远曲小管细胞萎缩,胞质内可见空泡,管腔扩张明显。SFN2组大鼠:肾小球略有萎缩体积变小嗜伊红染色增强,肾小囊有所显扩张,近曲小管细胞略显肿胀,小管之间间隙变窄,远曲小管管腔增宽。SFN1组大鼠:肾小球嗜伊红染色有所增强,肾小囊间隙略显增宽,近曲小管略有肿胀,小管之间间隙变窄,远曲小管管腔略增宽程度轻于SFN2组。2血清内SCr含量的改变与Control组大鼠血清内SCr的含量(59.87±6.76μmol/L)相比,RIRI组(358.08±22.20μmol/L)、SFN2组(294.14±18.76μmol/L)、SFN1组(93.60±13.16μmol/L)大鼠血清SCr含量均明显升高(P0.05),但SFN2组和SFN1组大鼠血清SCr含量低于RIRI组(P0.05),SFN1组大鼠血清SCr含量又低于SFN2组(P0.05)。3血清内BUN含量的改变与Control组大鼠血清内BUN的含量(6.35±1.15 mmol/L)相比,RIRI组(24.80±2.53 mmol/L)、SFN2组(20.01±1.78 mmol/L)、SFN1组(12.32±1.69mmol/L)大鼠血清BUN含量均明显升高(P0.05),但SFN2组和SFN1组大鼠血清BUN含量低于RIRI组(P0.05),SFN1组大鼠血清BUN含量又低于SFN2组(P0.05)。4肾组织MDA含量的改变与Control组大鼠肾组织MDA含量(72.29±14.75 mmol/g)相比,RIRI组(284.32±17.48 mmol/g)、SFN2组(186.52±17.50 mmol/g)、SFN1组(161.31±14.03 mmol/g)大鼠肾组织MDA含量均明显升高(P0.05),但SFN2组和SFN1组大鼠肾组织MDA含量明显低于RIRI组(P0.05),SFN1组大鼠肾组织MDA含量又低于SFN2组(P0.05)。5肾组织H_2O_2含量的改变与Control组大鼠肾组织H_2O_2含量(85.70±10.66 mmol/g)相比,RIRI组(260.62±48.50 mmol/g)、SFN2组(160.84±23.34 mmol/g)、SFN1组(121.22±18.49 mmol/g)大鼠肾组织H_2O_2含量均明显升高(P0.05),但SFN2组和SFN1组大鼠肾组织H_2O_2含量明显低于RIRI组(P0.05),SFN1组大鼠肾组织H_2O_2含量又低于SFN2组(P0.05)。6大鼠肾组织SOD活性改变与Control组大鼠肾组织SOD活性(112.47±9.09 U/mg pro)相比,RIRI组(28.56±6.11 U/mg pro)、SFN2组(43.85±5.29 U/mg pro)、SFN1组(65.44±8.91 U/mg pro)大鼠肾组织SOD活性均明显降低(P0.05),但SFN2组和SFN1组大鼠肾组织SOD活性高于RIRI组(P0.05),SFN1组大鼠肾组织SOD活性又高于SFN2组(P0.05)。7肾组织SOD基因的表达改变Real-Time PCR法测定大鼠肾组织SOD的m RNA相对表达水平,GAPDH作为扩增内对照。与Control组大鼠肾组织SOD m RNA表达量(0.93±0.13)相比,RIRI组(1.15±0.15)、SFN2组(1.36±0.18)、SFN1组(1.39±0.18)大鼠肾组织SOD m RNA表达水平均升高(P0.05)。SFN2组和SFN1组大鼠肾组织SOD的m RNA相对表达水平又高于RIRI组(P0.05),但SFN2组和SFN1组SOD的m RNA相对表达水平无明显差异(P�e0.05)。此结果表明:在RIRI发生过程中,SFN上调了SOD基因表达水平。结论:1肾脏缺血再灌注损伤发生后,肾组织处于氧化应激状态并遭受过氧化损伤。2肾脏缺血再灌注损伤发生后,莱菔硫烷通过上调Nrf2下游基因SOD的表达,增强机体对ROS的清除作用,降低了肾组织的过氧化损伤程度。3与再灌注后给药相比,缺血后即刻给予莱菔硫烷更能有效降低肾组织氧化应激水平和过氧化损伤程度,改善肾脏的形态结构和功能改变。
[Abstract]:Renal ischemia reperfusion injury (Renal ischemia reperfusion injury, RIRI) is a common clinical pathological and physiological phenomenon in nephron sparing surgery, renal crush injury, renal artery occlusion and renal resection or transplantation in the clinical treatment process. A large number of animal experiments and clinical studies have found that in the temporary occlusion of blood renal perfusion, then returned to the blood supply, there will be a large number of reactive oxygen species (reac-tive oxygen, species, ROS), causing serious damage of renal tissue cells. Therefore, oxidative stress is considered to be one of the main causes of ischemia-reperfusion injury. Nuclear factor E2 related factor 2 (nuclear factor erythroid 2-related factor 2, Nrf2.Nrf2) is currently the strongest antioxidant stress transcription factor found with antioxidant response element (antioxidant, responsive element, ARE) interaction adjustment Section expression of antioxidant proteins is an important endogenous antioxidant stress pathway. Superoxide dismutase (Superoxide Dismutases SOD) is the main body of the ROS scavenging enzyme system, the hydroxyl radical by disproportionation (OH-) into oxygen. At the same time, one of the downstream target gene SOD and Nrf2 transcription. Sulforaphane (sulforaphane, SFN) in the presence of a large number of broccoli and other cruciferous vegetables, which belongs to the isothiocyanates. Many studies show that SFN is the inducer of the Nrf2 pathway, through the upregulation of the expression of Nrf2 and its downstream genes play anti oxidation, anti tumor biological effects. This study uses non-invasive artery clamp renal artery clamping method to establish a rat model of RIRI, given the RIRI model rats induced by Nrf2 agent sulforaphane, observe the changes of morphology and function in renal tissue of RIRI rat model of sulforaphane treated H_2O_2 content, MDA content and The changes and expression of antioxidant protein SOD activity, to explore the antioxidant effects of sulforaphane play in RIRI. Objective: To observe the changes of morphology and function in renal tissue of RIRI rat model of sulforaphane treatment H_2O_2 content and expression of MDA SOD protein content and antioxidant activity of sulforaphane, whether through upregulation of the expression of Nrf2 the downstream gene of SOD, enhanced the scavenging effect of ROS, reduce the renal oxidative damage degree, provide data basis for the study of sulforaphane on prevention and treatment of RIRI. Methods: 1 animal models and specimens and Wistar rats from the experimental animal center of Hebei Medical University, male (weight 200 + 10g) 24 were randomly divided into control group (Control group), model group renal ischemia reperfusion injury (RIRI group), ischemia + sulforaphane group (SFN1 group), reperfusion + sulforaphane group (SFN2 group) according to Yu Xiaodong et al. The preparation method of the RIRI model to establish an experimental animal model: intraperitoneal injection of 6% chloral hydrate (5ml/kg) in anesthetized rats. The rats in RIRI group were fixed on the operating table, alcohol disinfection after laparotomy fully exposed bilateral kidneys, the right kidney of rats after resection, and blunt separation of the left renal artery, and the clip near the hilum part clamping left renal artery without traumatic occlusion of renal artery, blood supply, this can be observed in the kidney color quickly changed from bright red to dark red. The blood perfusion after occlusion for 45 minutes and discard the artery clamp, restoring blood perfusion of the left kidney, this is visible on the left renal artery rapidly filling kidney the color was dark red shift by bright red, suggesting that the left kidney blood reperfusion success in rats of.Control group and RIRI model group disinfectionand operation process, but the rats exposed only after resection of bilateral kidneys The right kidney, blunt separation of left renal artery occlusion, but not blocking the blood perfusion of rats in the.SFN1 group in the clamping of the left renal artery immediately after sulforaphane two DMSO diluted evenly on the surface of the small intestine, the remaining steps with the RIRI group.SFN2 group of rats in the blood discarding recovery pipe clamp renal blood perfusion immediately after sulforaphane two DMSO diluted evenly on the surface of the small intestine, two DMSO remaining steps with the RIRI group.Control group and RIRI group only apply in the small intestine. The equivalent surface recovery of rat kidney perfusion again after 24 hours of anesthesia in rats, the right carotid artery blood collection 3000, RPM, centrifugal separation for 10 minutes, and serum was collected for serum creatinine (Serum Creatinine, SCr), blood urea nitrogen (Blood Urea, Nitrogen, BUN) were detected; the rats were killed after excision of the kidney, and suitable for small samples of kidney structure In 4% paraformaldehyde fixative, used to observe the morphological changes of renal tissue in rats by HE staining. The kidney tissue rapidly in liquid nitrogen, to be frozen after transfer to -80 deg.c refrigerator spare, for the activity of SOD in rat renal tissue, to determine the level of gene expression, C two aldehydes (malondialdehyde, MDA) and the content of hydrogen peroxide (hydrogen peroxide, H_2O_2) determination of.2 detection index and the method for the determination of 2.1 rat serum SCr and BUN were detected by picric acid method to detect serum SCr content in rats; process of determination of rat serum BUN content of.SCr and BUN was determined by enzyme coupling rate method in strict accordance with the kit instructions were observed. The change of.2.2 in rat kidney tissue morphology observed by HE morphology of kidney tissue staining. The change of poly 4% kidneys were fixed in formaldehyde dehydrated by gradient alcohol, toluene two transparent, Paraffin embedded renal tissue sections after the treatment, the thickness of about 5 m, and then by hematoxylin eosin staining, using Olympus optical microscope to observe the morphological changes of the kidney tissue was determined by colorimetric method of thiobarbituric acid photo MDA content of renal tissue to detect the content of MDA in renal tissue of.2.3 rats, the detection process according to the kit instructions than the molybdate colorimetric method for determination of H_2O_2 content of renal tissue to detect the content of H_2O_2 in renal tissue of.2.4 rats, the detection process according to the kit instructions for the SOD activity of renal tissue to detect the activity of SOD in renal tissue of.2.5 rats was determined by xanthine oxidase method, the detection process according to the kit instructions for renal SOD gene expression of rats was detected by 2.6 RNA extraction kit to extract total RNA.RNA cracking quantitative renal tissue in rats after 2 g RNA reverse transcription template C DNA. with glyceraldehyde dehydrogenase (Gly -3- Ceraldehyde-3-phosphate dehydrogenase, GAPDH) as internal reference, Real-Time PCR relative quantitative analysis. Results: 1 rat renal tissue HE results HE method was used to observe the morphology of rat kidney. Under light microscope: in Control group of renal proximal tubules, distal convoluted tubule structure is clear and complete. Glomerular morphology rules. The renal capsule size of normal rats in the.RIRI group: glomerular atrophy volume, renal capsule gap widened significantly, proximal tubules, distal convoluted tubule atrophy, intracytoplasmic vacuoles, dilated obviously in.SFN2 rats: glomerular shrink slightly smaller eosinophilic staining enhancement, renal capsule has significant expansion the proximal tubule cells, slightly swollen, narrow tubules between the distal tubule lumen widened.SFN1 rats glomerular eosinophilic staining increased, renal capsule clearance slightly widened, proximal tubules slightly swollen, small 绠′箣闂撮棿闅欏彉绐，

本文编号：1606655