槲皮素通过Nrf2通路对创伤性脑损伤后线粒体损伤的保护作用研究
发布时间：2018-12-18 02:28
【摘要】：研究背景随着经济、社会的发展,创伤性脑损伤(traumatic brain injury,TBI)成为生活中致死率和致残率较高的疾病之一[1,2]。其对患者的身心健康造成了一定程度的不利影响,也给社会带来了严重负担。TBI后脑组织代谢加速而此时受伤脑组织中的线粒体的形态、功能发生改变致其无法进行正常的能量代谢从而使细胞的正常生理功能发生故障,最终导致后期更为严重的继发性损伤[3]。而在此过程中,大量线粒体形态和结构遭到破坏,导致线粒体无法正常供应能量,引起神经元细胞的死亡。所以,增强线粒体自身的生物合成以改善线粒体的能量代谢,可大大减少细胞的损伤,起到保护神经元的作用。第一部分槲皮素可在TBi后激活Nrf2通路并保护线粒体损伤目的探讨槲皮素通过Nrf2途径对TBI后线粒体损伤的保护作用。方法选取雄性成年ICR小鼠,采用自由落体模型,分为四组:假手术组(sham组)、外伤组(TBI组)、外伤+溶剂组(TBI+vehicle组)、外伤+槲皮素组(TBI+quercetin组)。在伤后24h取伤灶周围大脑皮层组织,使用Western blot检测细胞核及胞浆的中Nrf2蛋白的表达情况;检测线粒体及胞浆中Bax及CytC的浓度。使用免疫组化检测Nrf2蛋白由胞浆向胞核转移情况;用酶标仪检测线粒体SOD及MDA浓度。结果与假手术组相比,外伤组和溶剂组Nrf2表达增强且向核内转移明显增多。线粒体Cyt C及SOD含量减少,而Bax与MDA含量增多。槲皮素干预后不但促进了 Nrf2的入核和表达,而且使创伤性脑损伤后线粒体的氧化应激相关蛋白表达减少。。结论槲皮素可进一步激活TBI后Nrf2信号通路,减轻TBI后线粒体的破坏。第二部分Nrf2通路在槲皮素减轻TBI后线粒体破坏中的作用目的应用Nrf2基因敲除小鼠来验证Nrf2通路在TBI后槲皮素对线粒体保护过程中的作用。方法选取野生型[Nrf(+/+)]小鼠与Nrf2基因敲除型[Nrf(-/-)]小鼠,采用相同的自由落体颅脑损伤模型。并在伤后30min给予腹腔注射槲皮素。在伤后24h取伤灶周围大脑皮层组织使用Western blot检测线粒体及胞浆中Bax及Cyt C的浓度。用酶标仪检测线粒体SOD及MDA浓度。结果与野生型小鼠相比,Nrf2基因敲除小鼠TBI后线粒体保护作用明显减弱。结论Nrf2通路在槲皮素在TBI后线粒体保护中起重要作用。
[Abstract]:Background with the development of economy and society, traumatic brain injury (traumatic brain injury,TBI) has become one of the diseases with high mortality and disability rate. It has a certain degree of adverse effect on patients' physical and mental health, and also brings a serious burden to society. After TBI, the metabolism of brain tissue accelerates and the mitochondria in the brain tissue are injured. The change of function leads to the failure of normal energy metabolism, which leads to the breakdown of normal physiological function of cells, and finally leads to more serious secondary injury in the later stage [3]. During this process, a large number of mitochondria were destroyed in morphology and structure, resulting in the mitochondria unable to supply energy normally and resulting in the death of neuronal cells. Therefore, enhancing the biosynthesis of mitochondria to improve the energy metabolism of mitochondria can greatly reduce cell damage and protect neurons. Part one: quercetin could activate Nrf2 pathway after TBi and protect mitochondria from mitochondrial damage. Objective to investigate the protective effect of quercetin on mitochondrial damage induced by TBI through Nrf2 pathway. Methods male adult ICR mice were divided into four groups: sham operation group (sham group), trauma group (TBI group), trauma solvent group (TBI vehicle group) and traumatic quercetin group (TBI quercetin group). The expression of Nrf2 protein in nucleus and cytoplasm and the concentration of Bax and CytC in mitochondria and cytoplasm were detected by Western blot. The transfer of Nrf2 protein from cytoplasm to nucleus was detected by immunohistochemistry, and the concentration of SOD and MDA in mitochondria was detected by enzyme labeling instrument. Results compared with sham operation group, the expression of Nrf2 in trauma group and solvent group increased significantly. The contents of Cyt C and SOD in mitochondria decreased, while the contents of Bax and MDA increased. Quercetin not only promoted the entry and expression of Nrf2, but also decreased the expression of oxidative stress related proteins in mitochondria after traumatic brain injury. Conclusion Quercetin can further activate the Nrf2 signaling pathway after TBI and alleviate the mitochondrial damage after TBI. The second part of the role of Nrf2 pathway in reducing mitochondrial damage after TBI by quercetin objective to investigate the role of Nrf2 pathway in mitochondrial protection after TBI by using Nrf2 gene knockout mice. Methods Wild-type [Nrf (/)] mice and Nrf2 knockout type [Nrf (- / -) mice] were selected and the same free-fall brain injury model was used. Quercetin was injected intraperitoneally into 30min after injury. The concentrations of Bax and Cyt C in mitochondria and cytoplasm were detected by Western blot in the cortical tissue around the lesion 24 hours after injury. The concentrations of mitochondrial SOD and MDA were detected by enzyme labeling instrument. Results compared with wild-type mice, the mitochondrial protection of Nrf2 knockout mice was significantly weakened after TBI was removed. Conclusion Nrf2 pathway plays an important role in the mitochondrial protection of quercetin after TBI.
【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R651.15
，
本文编号：2385168
[Abstract]:Background with the development of economy and society, traumatic brain injury (traumatic brain injury,TBI) has become one of the diseases with high mortality and disability rate. It has a certain degree of adverse effect on patients' physical and mental health, and also brings a serious burden to society. After TBI, the metabolism of brain tissue accelerates and the mitochondria in the brain tissue are injured. The change of function leads to the failure of normal energy metabolism, which leads to the breakdown of normal physiological function of cells, and finally leads to more serious secondary injury in the later stage [3]. During this process, a large number of mitochondria were destroyed in morphology and structure, resulting in the mitochondria unable to supply energy normally and resulting in the death of neuronal cells. Therefore, enhancing the biosynthesis of mitochondria to improve the energy metabolism of mitochondria can greatly reduce cell damage and protect neurons. Part one: quercetin could activate Nrf2 pathway after TBi and protect mitochondria from mitochondrial damage. Objective to investigate the protective effect of quercetin on mitochondrial damage induced by TBI through Nrf2 pathway. Methods male adult ICR mice were divided into four groups: sham operation group (sham group), trauma group (TBI group), trauma solvent group (TBI vehicle group) and traumatic quercetin group (TBI quercetin group). The expression of Nrf2 protein in nucleus and cytoplasm and the concentration of Bax and CytC in mitochondria and cytoplasm were detected by Western blot. The transfer of Nrf2 protein from cytoplasm to nucleus was detected by immunohistochemistry, and the concentration of SOD and MDA in mitochondria was detected by enzyme labeling instrument. Results compared with sham operation group, the expression of Nrf2 in trauma group and solvent group increased significantly. The contents of Cyt C and SOD in mitochondria decreased, while the contents of Bax and MDA increased. Quercetin not only promoted the entry and expression of Nrf2, but also decreased the expression of oxidative stress related proteins in mitochondria after traumatic brain injury. Conclusion Quercetin can further activate the Nrf2 signaling pathway after TBI and alleviate the mitochondrial damage after TBI. The second part of the role of Nrf2 pathway in reducing mitochondrial damage after TBI by quercetin objective to investigate the role of Nrf2 pathway in mitochondrial protection after TBI by using Nrf2 gene knockout mice. Methods Wild-type [Nrf (/)] mice and Nrf2 knockout type [Nrf (- / -) mice] were selected and the same free-fall brain injury model was used. Quercetin was injected intraperitoneally into 30min after injury. The concentrations of Bax and Cyt C in mitochondria and cytoplasm were detected by Western blot in the cortical tissue around the lesion 24 hours after injury. The concentrations of mitochondrial SOD and MDA were detected by enzyme labeling instrument. Results compared with wild-type mice, the mitochondrial protection of Nrf2 knockout mice was significantly weakened after TBI was removed. Conclusion Nrf2 pathway plays an important role in the mitochondrial protection of quercetin after TBI.
【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R651.15
，
本文编号：2385168
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