Necrostatin-1对大鼠肾缺血再灌注损伤的保护作用

发布时间：2018-02-24 21:50

本文关键词： Necrostatin-1受体相互作用蛋白1 受体相互作用蛋白3 缺血再灌注损伤程序性坏死　出处：《天津医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的：通过建立大鼠肾缺血再灌注损伤(Ischemia-Reperfusion Injury, IRI)模型,观察其对大鼠肾病理、生化指标、炎症因子及细胞程序性坏死的影响,并观察Necrostatin-1(Nec-1)对肾的保护作用,探讨其可能的机制,为临床治疗肾缺血再灌注损伤提供理论依据。方法：将90只雄性SD大鼠按随机数字表法分为3组：假手术组、DMSO对照组、Nec-1组,每组30只；每组按2h、12h、24h再分为3组,每组10只。DMSO对照组和Nec-1组采用夹闭双侧大鼠肾动脉45min再恢复血流的方法制成肾缺血再灌注模型,于模型完成前15min尾静脉给药。假手术组不夹闭双侧肾动脉。Nec-1组于再灌15min前尾静脉注射Nec-11mg/kg(由DMSO容解),DMSO对照组注射等体积DMSO,假手术组只游离双肾,不作任何处理。模型成功后收集血清及肾脏组织；检测血清肌酐(serum creatinine, Scr).血尿素氮(blood urea nitrogen, BUN);HE染色观察肾组织病理改变并计算肾小管坏死评分；酶联免疫吸附试验(enzyme linked immunosorbent assay, ELISA)方法检测肿瘤坏死因子-α (TNF-α)白细胞介素-1p(IL-1p)浓度；Western blot方法检测受体相互作用蛋白1和3(RIP1,RIP3)的表达。结果：1.与假手术组相比,DMSO对照组及Nec-1组大鼠各时间点Scr、BUN水平均升高(P0.01),以DMSO组升高更为明显。随着时间推移,两组Scr、BUN水平均升高,Nec-1组大鼠各时间点Scr、BUN水平比DMSO对照组均降低(P0.05)。 2.假手术组大鼠肾组织结构清晰,肾小管上皮细胞完整,排列整齐。DMSO对照组肾小管结构异常,细胞脱落坏死、肿胀、管型形成、炎细胞浸润明显。与DMSO对照组相比,Nec-1组细胞脱落坏死、肿胀、管型形成均减轻,炎细胞相对减少。与假手术组相比,DMSO组与Nec-1组大鼠各时间点肾小管坏死死评分均明显升高(P0.01)以DMSO组升高更为显著。随着时间推移,两组Scr. BUN水平均升高,Nec-1组大鼠各时间点肾小管坏死评分水平比DMSO对照组均降低(P0.05)。 3.DMSO对照组及Nec-1组大鼠各时间点TNF-α、IL-1β表达比假手术组明显升高(P0.01),以DMSO组升高更为明显。随着时间推移,两组TNF-α、IL-1β水平均升高,Nec-1组大鼠各时间点TNF-α、IL-1β比DMSO对照组均降低(P0.01)。 4.假手术组RIP1和RIP3蛋白的表达量很低。与假手术组相比,DMSO对照组及Nec-1组RIP1和RIP3蛋白从2h开始就有较明显表且的表达,且随时间升高(P0.01)。随着时间推移,两组RIP1和RIP3蛋白水平均升高,与DMSO对照组相比,Nec-1组RIP1和RIP3蛋白的表达差异无统计学意义(P0.05)。结论：1.肾缺血再灌注损伤可导致大鼠肾组织结构破坏,细胞脱落坏死,肾小管坏死评分明显升高,反应肾功能水平的Scr、BUN水平均升高。Nec-1能有效减轻肾缺血再灌注损伤,改善肾功能。 2.肾缺血再灌注损伤可激活大鼠免疫反应,引起TNF-α、IL-1β等炎症因子明显升高。Nec-1能有效抑制免疫炎症反应,减轻损伤。 3.肾缺血再灌注损伤可引起严重的程序性坏死,RIP1和RIP3蛋白升高明显。Nec-1能抑制RIP1和RIP3蛋白活性,减轻程序性坏死对肾组织损伤程度。 4.Nec-1能明显改善大鼠肾缺血再灌注损伤组织破坏、炎症反应,减少程序性坏死的发生,其机制可能与抑制细胞程序性坏死有关。
[Abstract]:Objective: to establish the rat renal ischemia reperfusion injury (Ischemia-Reperfusion, Injury, IRI) model, observe the biochemical indicators of renal pathology, rats, inflammatory factors and programmed cell necrosis Necrostatin-1 (Nec-1), and to observe the protective effect on the kidney, to explore the possible mechanism for the clinical treatment of renal ischemia reperfusion injury and provide a theoretical basis.
Methods: 90 male SD rats were randomly divided into 3 groups: sham operation group, DMSO control group, Nec-1 group, 30 rats in each group; each group according to the 2H, 12h, 24h was subdivided into 3 groups, 10 rats in each group.DMSO control group and Nec-1 group by clamping bilateral renal artery of 45min rats to restore blood flow by renal ischemia reperfusion model, before the completion of 15min intravenous injection in the model. The sham operation group without occlusion of bilateral renal artery in.Nec-1 group after 15min reperfusion before intravenous injection of Nec-11mg/kg (DMSO, DMSO solution containing) control group was injected with equal volume of DMSO, the sham operation group only free double kidney, without any treatment. Serum and kidney tissue after successful model; detection of serum creatinine (serum creatinine, Scr). Blood urea nitrogen (blood urea, nitrogen, BUN); HE staining was used to observe the pathological change of kidney tissue and renal tubular necrosis score; enzyme linked immunosorbent assay (enzyme linked immunosorbent as Say (ELISA) method was used to detect the concentration of tumor necrosis factor alpha (TNF- alpha) interleukin -1p (IL-1p). Western blot method was used to detect the expression of receptor interacting protein 1 and 3 (RIP1, RIP3).
Results: 1. compared with sham operation group, DMSO control group and Nec-1 group at different time points in rats Scr, BUN levels were significantly increased (P0.01), in DMSO group increased more significantly. With the passage of time, two groups of Scr, BUN levels were elevated, Nec-1 rats at each time point Scr, BUN level than DMSO the control group were lower (P0.05).
2. sham operation group rats renal tissue structure is clear, renal tubular epithelial cells, arranged in.DMSO group of renal tubular abnormalities, exfoliative cell necrosis, swelling, tube formation, inflammatory cell infiltration. Compared with DMSO control group, Nec-1 group of exfoliated cells necrosis, swelling and tube formation were reduced, inflammatory cells relatively reduced. Compared with sham operation group, DMSO group and Nec-1 group at different time points in rat renal tubular and bad scores were significantly increased (P0.01) in DMSO group increased more significantly. With the passage of time, Scr. BUN levels were increased in all two groups, each time point of renal tubular necrosis in rats in group Nec-1 were better than DMSO the control group were lower (P0.05).
Each time point 3.DMSO control group and Nec-1 group rats TNF- alpha, IL-1 beta expression was significantly higher than that of sham operation group (P0.01), in DMSO group increased more significantly. With the passage of time, two groups of TNF- alpha, IL-1 beta levels were elevated, each time point TNF- alpha Nec-1 rats, IL-1 beta DMSO the control group were lower (P0.01).
The expression of RIP1 4. in sham operation group and RIP3 protein is very low. Compared with sham operation group, DMSO group and Nec-1 group of RIP1 and RIP3 protein 2H from the start and has obvious expression, and increased with time (P0.01). With the passage of time, two groups of RIP1 and RIP3 protein levels were elevated. Compared with DMSO control group, no statistically significant difference between group RIP1 and the expression of Nec-1 RIP3 protein (P0.05).
Conclusion: 1., renal ischemia-reperfusion injury can lead to the destruction of renal structure, cell loss and necrosis, and the increase of renal tubular necrosis. The level of Scr and BUN in response to renal function is increased..Nec-1 can effectively alleviate renal ischemia-reperfusion injury and improve renal function.
2., renal ischemia-reperfusion injury can activate the immune response in rats, induce TNF-, IL-1 and other inflammatory factors to increase significantly,.Nec-1 can effectively inhibit immune inflammatory response and reduce injury.
3., renal ischemia-reperfusion injury can cause severe procedural necrosis. RIP1 and RIP3 protein increase..Nec-1 can inhibit the activity of RIP1 and RIP3 protein, and reduce the degree of renal damage in programmed necrosis.
4.Nec-1 can obviously improve the tissue destruction, inflammatory reaction and reduce the occurrence of programmed necrosis in rats with renal ischemia-reperfusion injury. The mechanism may be related to the inhibition of programmed cell death.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R699.2

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