硫氧还蛋白-1参与雌激素在过氧化氢诱导的心肌细胞凋亡中的保护作用

发布时间：2018-03-14 05:53

本文选题：硫氧还蛋白　切入点：氧化应激　出处：《广西医科大学》2013年硕士论文　论文类型：学位论文

【摘要】：国内外大量实验研究表明心血管疾病与氧化应激损伤密切相关,而氧化应激被认为是在众多心血管疾病中诱发凋亡的主要因素,那么干预氧化应激可能成为心血管疾病治疗的新靶点。既往研究发现雌激素具有一定的抗氧化作用,且对心脏有保护作用,但具体通过何种信号通路来实现保护作用研究得不太清楚；而硫氧还蛋白(Trx)作为维持细胞内蛋白质还原状态的重要的二硫键还原酶,在对抗细胞的氧化应激中也起着重要的作用。另外,硫氧还蛋白与雌激素在保护心肌细胞作用中的相互关系鲜见报道。硫氧还蛋白(thioredoxin,Trx)系统是一个控制细胞还原/氧化状态和细胞增殖/生存的广泛表达的氧化还原系统,尤其在多种心血管病变的组织中有较高的表达,对心血管疾病的发生发展具有广泛的影响。目的本研究旨在探索硫氧还蛋白-1(Trx-1)是否参与了雌激素(E2)对过氧化氢(H2O2)诱导的凋亡心肌细胞的保护效应及相关的细胞信号转导通路。方法(1)用MTT法观察E2和H2O2对H9c2细胞活性的影响；(2)采用反转录PCR及Western blot分别检测不同浓度E2处理H9c2细胞后,胞内Trx-1基因mRNA和蛋白的表达；(3)采用DHE染色及Hochest33342染色来观察单独H2O2刺激和E2预处理对H9c2细胞的胞内活性氧(ROS)和凋亡水平的影响；(4)用Western blot法检测在E2对凋亡心肌细胞的保护作用中Trx-1的蛋白表达以及相关的促凋亡和抗凋亡信号通路中主要的信号分子Akt、P-Akt、P38、P-P38、JNK、P-JNK、Bcl-2、Bax、caspase-3的表达水平。结果(1)MTT法分析显示H202可呈浓度依赖性降低H9c2细胞的活性(P0.05),选择100uM H2O2处理12小时作为后续实验的诱导条件；仅用不同浓度的E2(1nM、10nM、100nM)处理H9c2细胞,对其活性均无明显影响(P0.05)；用不同浓度的E2预处理H9c2细胞24小时后,E2呈浓度依赖性明显减轻H202对H9c2细胞活性的损伤,10nM、100nM处理时无差异,所以选择10nM E2预处理24小时作为后续实验的干预条件。(2)雌激素呈剂量依赖的提高Trx-1mRNA和蛋白的表达,在浓度达10n M时最为明显。(3)外源性H202引起氧化应激能够增加细胞内ROS的产生,损伤H9c2细胞活性,引发凋亡,而雌激素预处理能减轻H202造成的心肌细胞的氧化应激和凋亡。(4)雌激素能减少p38磷酸化、caspase3活化以及减少Bax激活,促进Akt活化,从而达到保护心肌的效应。结论(1)外源性H202能模拟自然界氧化应激,刺激细胞内ROS的产生。(2)雌激素能呈剂量依赖的提高Trx-1的表达从而通过促进抗凋亡信号通路PI3K/Akt的激活,抑制促凋亡信号通路JNK/P38的激活,以及抑制依赖线粒体的caspase级联反应,来实现对H202诱导的凋亡心肌细胞的保护作用。
[Abstract]:A large number of experimental studies at home and abroad show that cardiovascular disease is closely related to oxidative stress injury, and oxidative stress is considered to be the main factor that induces apoptosis in many cardiovascular diseases. Intervention in oxidative stress may become a new target for the treatment of cardiovascular disease. Previous studies have found that estrogen has a certain antioxidant effect and has a protective effect on the heart. However, it is not clear what kind of signaling pathway is used to protect the cells. Thioredoxin (TRX) is an important disulfide bond reductase to maintain the reduction state of protein in cells. It also plays an important role in combating oxidative stress in cells. The relationship between thioredoxin and estrogen in the protection of cardiomyocytes is rarely reported. Thioredoxin Trx) system is a widely expressed redox system that controls cell reduction / oxidation state and cell proliferation / survival. Especially in a variety of cardiovascular disease tissue has a high expression, cardiovascular disease has a wide range of influence on the development. Objective to investigate whether thioredoxin-1 (TRX-1) is involved in the protective effect of estrogen E2) on the apoptosis of cardiomyocytes induced by H 2O 2 (H 2O 2) and its related signal transduction pathway. Methods 1) the effect of E _ 2 and H _ 2O _ 2 on the activity of H9c2 cells was observed by MTT assay. Reverse transcription PCR and Western blot were used to detect the effects of E _ 2 and H _ 2O _ 2 on the activity of H9c2 cells. Expression of mRNA and protein of intracellular Trx-1 gene: DHE staining and Hochest33342 staining were used to observe the effects of H2O2 alone and E2 pretreatment on intracellular reactive oxygen species (Ros) and apoptosis level in H9c2 cells.) Western blot assay was used to detect the effect of E2 on apoptotic cardiomyocytes. The protein expression of Trx-1 and the expression of caspase-3 in the main signal molecule Aktnk, P-AktP38, P-P38, JNKK P-JNK-2Bcl-2Bax-2in the apoptotic and anti-apoptotic signaling pathway. Results MTT assay showed that H202 could decrease the activity of H9c2 cells in a dose-dependent manner, and treated H9c2 cells with 100UM H2O2 for 12 hours. There was no significant effect on the activity of H9c2 cells after pretreatment with different concentrations of E2 for 24 hours in a concentration-dependent manner, and no difference was found in the treatment of H9c2 cells with 10nMN 100nM treated with H202 in a concentration-dependent manner, and no significant difference was found in the treatment of H9c2 cells with different concentrations of E2 in a concentration-dependent manner. Therefore, 10nM E2 preconditioning for 24 hours was chosen as the intervention condition in the follow-up experiment. (2) estrogen increased the expression of Trx-1mRNA and protein in a dose-dependent manner. When the concentration reached 10 nm, the expression of Trx-1mRNA and protein was the most obvious, and the oxidative stress induced by exogenous H202 could increase the production of ROS in the cells. Estrogen pretreatment can reduce oxidative stress and apoptosis of cardiomyocytes induced by H202) estrogen can reduce the activation of p38 phosphorylated caspase3 and Bax, and promote Akt activation. In order to achieve the effect of myocardial protection. Conclusion exogenous H202 can mimic natural oxidative stress and stimulate the production of ROS in cells. Estrogen can increase the expression of Trx-1 in a dose-dependent manner and inhibit the activation of JNK/P38 by promoting the activation of PI3K/Akt signaling pathway. And inhibiting the mitochondrial dependent caspase cascade to protect the apoptosis cardiomyocytes induced by H 202.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R363

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