褪黑素对脑缺血再灌注后脑组织中Nox2和Nox4表达影响的实验研究

发布时间：2018-06-13 10:40

  本文选题：缺血性脑卒中 + 缺血再灌注　； 参考：《苏州大学》2014年硕士论文

【摘要】：目的：迅速溶栓复流治疗是缺血性脑卒中急性期治疗成功的前提和基础，但是该过程引起的氧化应激是导致脑组织二次伤害即缺血/再灌注(I/R)损伤的重要因素。而NADPH氧化酶，尤其是其中的Nox2和Nox4在I/R引起的氧化应激中发挥着重要作用。褪黑素是迄今为止最强的内源性自由基清除剂，且有文献报道表明褪黑素可以抑制脑缺血再灌注引起的脑损伤，但是其作用机制尚不明确。我们拟利用本研究探讨在缺血再灌注条件下，，褪黑素发挥脑保护作用的具体机制，明确褪黑素是否通过调节Nox2和Nox4发挥作用。 方法：利用30只雄性Sprague-Dawley (SD)大鼠，将其随机分为三组：假手术组，缺血再灌注组，褪黑素预处理+缺血再灌注组。利用线栓法制备大鼠大脑中动脉栓塞(MCAO)模型（通过大鼠大脑中动脉栓塞后相应神经功能障碍表现验证模型制备成功）。 然后利用荧光分光光度计检测脑组织ROS水平、TUNEL染色检测脑组织细胞凋亡情况、Western blot检测Nox2和Nox4蛋白水平、同时检测TUNEL与Nox2、Nox4的免疫荧光共染的细胞数目。相应实验数据结果应用SPSS18.0统计分析软件，对数据进行统计学分析。 结果： 1）与假手术组相比，缺血再灌注组ROS水平显著提高，尤其是在缺血半球尤为显著，褪黑素预处理可以显著抑制缺血再灌注引起的缺血半球以及对侧半球中ROS水平的上升。 2）与假手术组相比，缺血再灌注组凋亡细胞显著增多，尤其是在缺血半球尤为显著；而褪黑素预处理可以显著抑制缺血再灌注引起的缺血半球以及对侧半球中细胞的凋亡。 3)与假手术组相比，缺血再灌注组Nox2和Nox4的蛋白水平显著上升；而褪黑素预处理可以显著抑制缺血再灌注引起的Nox2和Nox4的蛋白水平上升。提示：褪黑素可能通过降低Nox2和Nox4的表达，抑制ROS的产生，进而抑制细胞凋亡。 4）免疫荧光共染表明：TUNEL阳性细胞伴随着Nox2或者Nox4的高表达；褪黑素预处理显著降低了Nox2/TUNEL双阳性细胞和Nox4/TUNEL双阳性细胞的数目。 结论： 在脑缺血再灌注过程中，褪黑素可能通过抑制Nox2和Nox4的表达发挥抗氧化和抗凋亡的作用。
[Abstract]:Objective: rapid thrombolytic reflow therapy is the premise and foundation of successful treatment in acute phase of ischemic stroke, but oxidative stress induced by this process is an important factor leading to secondary injury of brain tissue, I. E. ischemia / reperfusion injury. NADPH oxidase, especially Nox2 and Nox4, plays an important role in I / R induced oxidative stress. Melatonin is the strongest endogenous free radical scavenger so far, and it has been reported that melatonin can inhibit brain injury induced by cerebral ischemia and reperfusion, but its mechanism is not clear. We intend to use this study to explore the specific mechanism of melatonin in cerebral protection under ischemia-reperfusion conditions, and to determine whether melatonin plays a role by regulating Nox2 and Nox4. Methods: thirty male Sprague-Dawley SD rats were randomly divided into three groups: sham operation group, ischemia reperfusion group and melatonin preconditioning ischemia reperfusion group. The MCAO model of middle cerebral artery embolization (MCAO) was established by the method of thread embolization. Then the Ros level of brain tissue was detected by fluorescence spectrophotometer and Tunel staining was used to detect the apoptosis of brain tissue. Western blot was used to detect the levels of Nox2 and Nox4 proteins, and the number of Tunel and Nox2Nox4 immunofluorescence co-stained cells were also detected. The corresponding experimental data were analyzed by SPSS 18.0 statistical analysis software. Results: 1) compared with sham operation group, Ros level in ischemia reperfusion group was significantly higher than that in sham operation group, especially in ischemic hemisphere. Melatonin pretreatment significantly inhibited the increase of Ros levels in the ischemic and contralateral hemispheres induced by ischemia and reperfusion. Melatonin preconditioning significantly inhibited apoptosis in ischemic and contralateral hemispheres induced by ischemia and reperfusion. 3) compared with sham operation group, melatonin preconditioning significantly inhibited apoptosis in ischemic and contralateral hemispheres. The protein levels of Nox2 and Nox4 increased significantly in ischemia-reperfusion group, while melatonin preconditioning significantly inhibited the protein levels of Nox2 and Nox4 induced by ischemia-reperfusion. These results suggest that melatonin inhibits the production of Ros by decreasing the expression of Nox2 and Nox4, and thus inhibits apoptosis. 4) Immunofluorescence co-staining indicates that the high expression of Nox2 or Nox4 is associated with the expression of Nox2 or Nox4 in the cells with positive cells. Melatonin pretreatment significantly decreased the number of Nox2 / Tunel double positive cells and Nox4 / Tunel double positive cells. Conclusion: melatonin may inhibit the expression of Nox2 and Nox4 during cerebral ischemia-reperfusion.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743.31

【参考文献】

相关期刊论文 前1条

1 王威;唐圣松;;吞噬细胞NADPH氧化酶活性的调控[J];国际病理科学与临床杂志;2009年04期

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