Sestrin2在大鼠蛛网膜下腔出血后早期脑损伤中的作用及机制

发布时间：2018-03-05 14:56

本文选题：Sestrin2　切入点：蛛网膜下腔出血　出处：《重庆医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景Sestrin2(Sesn2)是sestrin家族的重要成员,它是一组高度保守的蛋白质,起初是作为抗氧化体系的重要成员被发现,在机体受到应激包括遗传物质损伤、氧化应激和缺氧等均可以诱导其产生。Sesn2已被公认为是一种强有力的自由基清道夫。有研究表明sestrin2在神经退行性病变(MPTP帕金森模型)中发挥神经保护作用。亦有实验表明sestrin2在肝脏通过加剧Keap1的降解从而起到抗氧化保护作用,减轻了肝细胞的氧化应激损伤。但其在蛛网膜下腔出血(Subarachnoid hemorrhage,SAH)后早期脑损伤(Early brain injury,EBI)中的作用尚未见报道。目的探讨Sestrin2在大鼠蛛网膜下腔出血后早期脑损伤中的作用及机制。方法(1)通过将自体动脉血注入大鼠视交叉前池,建立大鼠SAH模型。(2)建立Sesn2干扰的大鼠SAH模型,于SAH建模前24小时脑室注射Sesn2 Si RNA.(3)观察干扰Sesn2基因对大鼠SAH早期脑损伤后脑组织组织形态学变化(尼氏染色)的影响。(4)通过检测脑组织匀浆中SOD活性和MDA含量,观察Sesn2沉默后对大鼠SAH早期脑损伤后神经细胞氧化应激水平的影响。(5)观察干扰Sesn2基因在大鼠SAH早期脑损伤中对Keap1/Nrf2通路及下游抗氧化酶HO-1、NQO1(Real-time q PCR和Western blot)的表达的影响。结果(1)与SAH组相比,干扰Sesn2后,出血灶脑组织中神经元数量减少、萎缩,尼氏小体消失。(2)同Sham组相比,SAH组中MDA的含量明显增高,而SOD的活性降低。与SAH组相比,干扰Sesn2后,MDA的含量进一步增高,而SOD的活性则降低更加明显,氧化应激水平明显增强。(3)与Sham组相比,SAH组Sesn2、Nrf2、HO-1、NQO1的表达显著增加,Keap1表达降低。与SAH组相比,干扰Sesn2后,Sesn2、Nrf2及抗氧化酶中HO-1、NQO1的表达均明显降低,Keap1的表达明显升高;结论(1)干扰Sesn2可加重SAH后神经细胞组织学损伤及氧化应激水平,提示Sesn2对神经细胞具有一定的抗氧化保护作用。(2)蛛网膜下腔出血(SAH)之后初期Keap1/Nrf2传导通路会被激活,从而也使这一传导通路下游的各种产物的表达被激活,于是使此通路下游的表达产物增加,由此发挥一定的保护神经细胞免受氧化损伤的作用,Sesn2沉默后加重脑氧化应激损伤的机制可能是通过降低Keap1的降解从而抑制Keap1/Nrf2通路,减少其下游一系列抗氧化酶、解毒酶的合成,使氧化应激损伤加剧。
[Abstract]:Background Sestrin2 (Sesn2) is an important member of the sestrin family. It is a highly conserved group of proteins, initially found as an important member of the antioxidant system. Stress in the body includes genetic material damage. Oxidative stress and hypoxia have been recognized as a powerful scavenger of free radicals induced by oxidative stress and hypoxia. Some studies have shown that sestrin2 plays a neuroprotective role in neurodegenerative lesions (MPTP Parkinson model). Tests have shown that sestrin2 acts as an antioxidant protection in the liver by increasing the degradation of Keap1. The role of Sestrin2 in early brain injury after subarachnoid hemorrhage was not reported. Objective to explore the role of Sestrin2 in early brain injury after subarachnoid hemorrhage in rats. Methods autologous arterial blood was injected into the anterior cistern of optic chiasma in rats. Establish rat SAH model. 2) establish rat SAH model with Sesn2 interference. The effects of interfering Sesn2 gene on the morphological changes of brain tissue (Nissl staining) after early brain injury in rats with SAH were observed 24 hours before SAH modeling. The activity of SOD and the content of MDA in the homogenate of brain tissue were detected. To observe the effect of Sesn2 silencing on the oxidative stress level of nerve cells after early brain injury in rats with SAH. (5) to observe the effect of interfering Sesn2 gene on the expression of Keap1/Nrf2 pathway and its downstream antioxidant enzyme HO-1NQO1Real-time Q PCR and Western blot in early brain injury of rats with SAH. Results 1) compared with SAH group, After interfering with Sesn2, the number of neurons in the brain tissue of hemorrhagic focus decreased, atrophy, and the Nissl corpuscles disappeared.) compared with Sham group, the content of MDA and the activity of SOD in Sham group increased significantly. Compared with SAH group, the content of SOD increased further after interfering with Sesn2. The activity of SOD was significantly decreased, and the level of oxidative stress was significantly increased. (3) compared with Sham group, the expression of Sesn2Nrf2Nrf2HO-1NQO1 in Sham group was significantly increased, and compared with SAH group, the expression of NQO1 in Sesn2Nrf2HO-1 group decreased significantly. After interfering with Sesn2, the expression of HO-1NQO1 in both Sesn2Nrf2 and antioxidase was significantly decreased, conclusion the interference of Sesn2 could aggravate the histological injury and oxidative stress level of nerve cells after SAH, and the expression of HO-1NQO1 in HO-1NQO1.Conclusion interference with Sesn2 can aggravate the histological injury and oxidative stress of nerve cells after SAH. It is suggested that Sesn2 has a certain antioxidant protective effect on nerve cells. (2) the Keap1/Nrf2 conduction pathway is activated in the early stage after subarachnoid hemorrhage (SAH), which also activates the expression of various products downstream of this pathway. Thus, the downstream expression products of this pathway were increased, which may play a role in protecting neurons from oxidative damage. The mechanism of Sesn2 silencing exacerbating brain oxidative stress may be to inhibit the Keap1/Nrf2 pathway by reducing the degradation of Keap1. It reduces the synthesis of a series of antioxidant enzymes and detoxifying enzymes, and exacerbates oxidative stress damage.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R743.35

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