Nesfatin-1预处理在大鼠肾缺血再灌注损伤模型中的保护作用及相关机制的研究

发布时间：2018-06-08 20:06

本文选题：肾缺血再灌注损伤 + Nesfatin-1　；参考：《武汉大学》2016年博士论文

【摘要】：背景：肾脏缺血再灌注损伤(Renal ischemia reperfusion injury, RIRI)在泌尿外科领域的临床工作中是一种常见的病理生理过程,是肾移植术、肾部分切除术、肾组织楔形切除术、肾盂肾实质联合切开取石术、失血性休克及主动脉修补术等疾病所共同遭受的一个多种因素和环节参与的病理过程。肾脏组织的血流灌注非常丰富,且对缺血再灌注损伤很敏感,如果手术中肾脏组织缺血时间过长,它不仅可引起急性肾小管坏死,造成急性肾功能衰竭的重要因素之一,同时也是肾移植术后肾功能异常及影响患者肾功能的恢复的重要原因。相关的研究表明,肾IRI是影响肾移植术后肾组织存活时间的危险因素。由肾缺血再灌注损伤所引发的相关疾病的发病率和死亡率持续增高,因此,如何有效的干预肾IRI引起的肾功能损害及肾功能的保护越来越多的受到关注,在泌尿外科领域中仍然是一个值得探究的重要问题。肾IRI的发病机理错综复杂,目前尚未完全阐明,目前的研究资料表明,肾缺血再灌注损伤的病理生理机制可能与氧自由基的大量产生、炎症介质反应、Ca2+超载及细胞凋亡等相关。Nesfatin-1是一种下丘脑分泌的神经多肽类物质,在能量平衡和代谢过程中发挥着十分重要的作用。研究证实,nesfatin-1具有多种生化和生理功能,在不同的人体疾病和动物模型中参与了各种病理生理过程。现有的研究表明,nesfatin-1现已应用于心肌缺血再灌注损伤、肠缺血再灌注损伤、胃粘膜损伤、蛛网膜下腔出血性损伤及创伤性脑损伤的动物模型中,具有一定的保护作用。但目前尚未见nesfatin-1防治肾IRI的相关报道。因此,我们推测nesfatin-1可能在大鼠的肾缺血再灌注损伤中具有保护作用。本课题拟探究nesfatin-1预处理对大鼠肾缺血再灌注损伤有无保护作用和其相关的可能机制进行初步的研究。目的：以nesfatin-1为研究对象,评估n esfatin-1对大鼠肾缺血再灌注损伤的影响并探索其可能的作用机制。方法：体重为200-250g的雄性Wistar大鼠24只,随机分成三组,即伪手术组(Sham组或正常对照组)、肾缺血再灌注组(I/R组)、肾缺血再灌注组+nesfatin-1预处理组。每组的大鼠各为8只。肾I/R组及肾I/R+ nesfatin-1预处理组的大鼠通过采用右肾切除加微型无创伤性动脉夹夹闭左侧肾蒂的方法建立大鼠肾缺血再灌注模型。第一部分：检测nesfatin-1预处理后对肾功能(BUN、Scr)的影响,通过HE和PAS染色观察肾脏组织的病理形态学变化；第二部分：通过检测超氧化物歧化酶(SOD)、丙二醛(MDA)、过氧化氢酶(CAT)及还原型谷胱甘肽(GSH)量的变化评估iesfatin-1预处理对肾缺血再灌注损伤在氧化应激方面的作用；第三部分：通过检测TUNEL、Caspase-3、Bcl2 及 Bax的表达量的变化评价nesfatin-1预处理对肾缺血再灌注损伤在凋亡方面的影响；第四部分：通过检测髓过氧化物酶(MPO)、TNF-α、IL-1β、IL-6 及 NF-κB的表达情况,以评价nesfatin-1预处理对肾缺血再灌注损伤在炎症方面的影响。结果：1、肾功能检测结果显示,与肾I/R组相比较,I/R+nesfatin-1组的尿素氮(BUN)和肌酐(Scr)水平显著低于肾I/R组(P0.05),表明nesfatin-1预处理具有一定保护肾功能的作用。2、光镜下检测发现,与肾I/R组相比较,I/R+nesfatin-1组的组织形态学变化明显减轻,病理学评分明显降低(P0.01),表明nesfatin-1预处理可以减轻肾IRI所导致的肾脏组织的形态学损伤。3、氧化应激途径的影响：结果显示,与肾I/R组相比较,I/R+nesftin-1组的SOD、CAT、GSH的活性明显增加(P0.01),而MDA的水平显著降低(P0.01),表明了经nesftin-1预处理后氧自由基清除酶SOD、CAT、GSH的活性增加,使肾IRI时增多的氧自由基清除增加,从而使增加的氧自由基水平降低,最终使其介导生成的MDA表达水平减少。4、凋亡方面的影响：与肾I/R组相比较,I/R+nesftin-1组的TUNEL凋亡表达数目显著降低(P0.05),进一步通过比较Caspase-3、Bcl2、Bax的表达量及Bcl2/Bax的比值发现,：/R+nesftin-1组的Caspase-3和Bax的表达显著降低(P0.01),Bcl2的表达量显著增高(P0.01),Bcl2/Bax之比显著增加(P0.05),说明I/R+nesftin-1组的细胞凋亡降低,表明nesftin-1预处理可以在肾缺血再灌注损伤中减少凋亡细胞的发生。5、炎症方面的影响：结果显示,I/R+nesftin-1组的MPO、TNF-α、IL-1β、IL-6及NF-κB的表达量显著降低,表明nesfatin-1预处理能够在肾缺血再灌注损伤中减少炎症反应的发生。结论：1、在大鼠的肾缺血再灌注损伤中,Nesfatin-1预处理具有肾功能的保护作用,并能减轻肾脏组织的形态学损伤。2、Nesfatin-1预处理对肾缺血再灌注损伤的肾脏组织具有显著的抗氧化作用。3、Nesfatin-1预处理对肾缺血再灌注损伤具有明显的抗凋亡效应,其作用可能是通过抑制细胞凋亡及上调抗凋亡基因表达的路径而实现的。4、Nesfatin-1预处理在肾缺血再灌注损伤中抑制了炎症反应的中性粒细胞的活化,并抑制炎性因子的释放,从而减轻了炎症反应,对大鼠的肾IRI发挥肾脏保护作用。
[Abstract]:Background: Renal ischemia reperfusion injury (RIRI) is a common pathophysiological process in the clinical work in the field of Department of urology. It is a renal transplantation, partial nephrectomy, renal tissue wedge resection, renal pelvis renal parenchyma incision and stone removal, hemorrhagic shock and aortic repair. The renal tissue is very rich in blood flow and is very sensitive to ischemia reperfusion injury. If the time of renal tissue ischemia is too long during the operation, it can not only cause acute renal tubular necrosis, but also one of the important factors of acute renal failure, and it is also a kidney transplant. Abnormality of postoperative renal function and important reasons for the recovery of renal function in patients. Related studies have shown that renal IRI is a risk factor affecting the survival time of renal tissue after renal transplantation. The incidence and mortality of related diseases caused by renal ischemia-reperfusion injury continue to increase, and how to effectively interfere with renal function caused by renal IRI More and more attention has been paid to the protection of damage and renal function, which is still an important issue in the field of Department of urology. The pathogenesis of renal IRI is complicated and is not fully elucidated. The present research data show that the pathogenesis of renal ischemia reperfusion injury may be associated with a large number of oxygen free radicals and inflammation. .Nesfatin-1 is a neuropolypeptide secreted by the hypothalamus, which is a neuropeptide secreted by the hypothalamus. It plays a very important role in the process of energy balance and metabolism. It has been proved that nesfatin-1 has many biochemical and physiological functions and has been involved in various pathophysiology in different human diseases and animal models. Process. Existing studies have shown that nesfatin-1 has been applied to myocardial ischemia reperfusion injury, intestinal ischemia-reperfusion injury, gastric mucosal injury, subarachnoid hemorrhage injury and traumatic brain injury in animal models, but there is no related report on the prevention and treatment of renal IRI by nesfatin-1. Therefore, we speculate that NES Fatin-1 may play a protective role in renal ischemia-reperfusion injury in rats. This subject is to explore the protective effect of nesfatin-1 preconditioning on renal ischemia reperfusion injury in rats and its possible mechanism. Objective: To evaluate the effect of n esfatin-1 on renal ischemia-reperfusion injury in rats by using nesfatin-1 as the research object. Influence and explore its possible mechanism of action. Methods: 24 male Wistar rats weighing 200-250g were randomly divided into three groups, namely, pseudo operation group (Sham group or normal control group), renal ischemia reperfusion group (group I/R) and +nesfatin-1 preconditioning group of renal ischemia reperfusion group. Each group was 8 rats each. Kidney I/R group and kidney I/R+ nesfatin-1 preconditioning group. Rat model of renal ischemia reperfusion was established by using right nephrectomy and mini traumatic artery clamp to clamp the left renal pedicle. Part 1: the effects of nesfatin-1 preconditioning on renal function (BUN, Scr) were detected, and the morphological changes of renal tissue were observed by HE and PAS staining. The second part: by detecting SUPEROXYGEN Changes in SOD, MDA, catalase (CAT) and reduced glutathione (GSH); the role of iesfatin-1 preconditioning on oxidative stress in renal ischemia reperfusion injury; third part: evaluation of the expression of TUNEL, Caspase-3, Bcl2 and Bax by the evaluation of nesfatin-1 preconditioning for renal ischemia and reperfusion The effect of perfusion injury on apoptosis; Fourth: by detecting the expression of myeloperoxidase (MPO), TNF- alpha, IL-1 beta, IL-6 and NF- kappa B in order to evaluate the effect of nesfatin-1 preconditioning on renal ischemia reperfusion injury in inflammation. Results: 1, renal function test results showed that the urine of the renal I/R group was compared with the kidney I/R group and the urine of the I/R+nesfatin-1 group. The level of BUN and creatinine (Scr) was significantly lower than that of the renal I/R group (P0.05), which showed that nesfatin-1 preconditioning had a certain role in protecting the renal function.2. Under the light microscope, the histologic changes of the I/R+nesfatin-1 group were obviously reduced and the pathological score was significantly reduced (P0.01), indicating that nesfatin-1 preconditioning could reduce the IR renal function. The morphological damage of renal tissue caused by I was.3, the effect of oxidative stress pathway. The results showed that the activity of SOD, CAT, GSH in the group I/R+nesftin-1 was significantly increased (P0.01) compared with the I/R group of the kidney (P0.01), and the level of MDA decreased significantly (P0.01). The increase of oxygen free radical scavenging increased the increased oxygen free radical level, and finally reduced the level of MDA expression by.4 and apoptosis. Compared with group I/R, the number of TUNEL apoptotic expression in group I/R+nesftin-1 decreased significantly (P0.05), and the expression of Caspase-3, Bcl2, Bax and Bcl2/Bax were compared. The expression of Caspase-3 and Bax in the:/R+nesftin-1 group was significantly decreased (P0.01), the expression of Bcl2 increased significantly (P0.01), and the ratio of Bcl2/Bax increased significantly (P0.05), indicating that the apoptosis in the I/R+nesftin-1 group decreased, indicating that nesftin-1 pretreatment could reduce the incidence of apoptotic cells in the renal hemorrhage reperfusion injury. Results: the results showed that the expression of MPO, TNF- a, IL-1 beta, IL-6 and NF- kappa B in the I/R+nesftin-1 group decreased significantly, indicating that nesfatin-1 preconditioning could reduce the occurrence of inflammatory reaction in renal ischemia reperfusion injury. Conclusion: 1, in rats with renal ischemia reperfusion injury, Nesfatin-1 preconditioning has the protective effect of renal function and can be reduced. The morphological damage of renal tissue is.2, Nesfatin-1 preconditioning has a significant antioxidant effect on renal ischemia reperfusion injury.3. Nesfatin-1 preconditioning has obvious anti apoptosis effect on renal ischemia-reperfusion injury, and its effect may be achieved by inhibiting apoptosis and increasing the path of anti apoptotic gene expression. .4, Nesfatin-1 preconditioning inhibits the activation of neutrophils in the inflammatory response and inhibits the release of inflammatory factors in the renal ischemia-reperfusion injury, thus alleviates the inflammatory response and plays a renal protective role in the renal IRI of rats.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R692;R-332

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