Allicin对氧化应激状态下人RPE细胞保护作用的初步研究
发布时间：2019-06-06 21:11
【摘要】：目的：研究天然物质蒜素是否能保护人视网膜色素上皮细胞(ARPE-19)免受过氧化氢(H2O2)-诱导损伤,并确定蒜素对过氧化氢诱导的氧化应激损伤的人视网膜色素上皮细胞(ARPE-19)的影响和潜在分子机制。为年龄相关性黄斑变性等与氧化应激相关的眼病防治研究方面提供实验室证据。方法：1.ARPE-19细胞暴露在250μM或500μM过氧化氢内12或24小时,后培养在预先备好的各种浓度的蒜素内(0、5、10、20或40μg／毫升)4 h。RPEs的活力使用MTT试验评估。确定蒜素对过氧化氢诱导ARPE-19细胞的影响。2.对浓度梯度的蒜素预处理的ARPE-19细胞进行250μM过氧化氢诱导后通过2、7'-二氯荧光乙酰乙酸钠(DCFH-DA)荧光探针检测细胞内活性氧(ROS)的生成,比色法检测细胞内ROS、谷胱甘肽(GSH)/二硫化谷胱甘肽(GSSG)比值和丙二醛(MDA)的水平,确定蒜素对ARPE-19细胞过氧化氢诱导损伤的影响。3.浓度梯度的蒜素预处理的ARPE-19细胞进行250μM浓度过氧化氢诱导的氧化应激状态下通过荧光定量PCR检测和蛋白质印迹检测ARPE-19细胞核蛋白质mRNA,核蛋白NOX4,NQO1及Nrf2表达水平。进一步确定蒜素保护视网膜色素上皮细胞免受氧化应激损伤的潜在机制。4.浓度梯度的蒜素预处理的ARPE-19细胞进行250μM浓度过氧化氢诱导的氧化应激状态下通过基因转染方法对Nrf2基因进行干涉,进一步应用荧光定量PCR检测法、蛋白质印迹检测法、MTT法等检测ARPE-19细胞核蛋白质和胞浆蛋白质内Nrf2相关mRNA表达水平、Nrf2蛋白质表达水平,评估Nrf2抑制效率、比较各组间生存力的差异,确定Nrf2在蒜素保护RPEs在H202诱导损伤机制中的调节效应。结果：1.ARPE-19细胞暴露于过氧化氢内处理12或24小时后导致细胞生存能力显著损失。蒜素可以逆转RPEs被过氧化氢的损伤效应,此现象是剂量依赖性的。2.在ARPE-19细胞内,蒜素通过减少细胞内的ROS、MDA的水平和增加GSH/GSSG来减弱H202-诱导的氧化应激反应。3.蒜素保护过氧化氢诱导的视网膜色素上皮细胞(ARPE-19 cells)的潜在机制可能是通过调节ROS-相关酶的表达水平,包括SOD、NOX4、NQO1以及Nrf2。4.蒜素介导的过氧化氢一诱导损伤的保护作用与Nrf2的调节有关。蒜素通过剂量-依赖的方式增强Nrf2相关的mRNA的表达水平。抑制Nrf2可显著减弱蒜素对过氧化氢-诱导的细胞的保护作用。然而,抑制Nrf2并不能完全抑制蒜素的保护作用。结论：1.外源性H202可诱导人视网膜色素上皮细胞ARPE-19细胞发生氧化应激损伤。蒜素可以保护视网膜色素上皮细胞免受氧化应激损伤,此保护作用是剂量依赖性的。2.蒜素通过调节细胞内ROS水平、GSH/GSSG、MDA及SOD等实现对视网膜色素上皮细胞的保护作用的。3.蒜素保护视网膜色素上皮细胞免受氧化应激损伤的潜在的机制可能是通过调节ROS相关酶SOD、NOX4、NQO1以及Nrf2等的表达实现的。
[Abstract]:Objective: to study whether allicin can protect human retinal pigment epithelial cells (ARPE-19) from hydrogen peroxide (H2O2)-induced injury. The effect and potential molecular mechanism of allicin on human retinal pigment epithelial cells (ARPE-19) induced by hydrogen peroxide were determined. To provide laboratory evidence for the prevention and treatment of age-related macular degeneration and other eye diseases related to oxidative stress. Methods: 1.ARPE-19 cells were exposed to 250 渭 M or 500 渭 M hydrogen peroxide for 12 or 24 hours, and then cultured in various concentrations of allicin (0, 5, 10, 20 or 40 渭 g / ml) 4 h.RPEs activity was evaluated by MTT test. To determine the effect of allicin on ARPE-19 cells induced by hydrogen peroxide. 2. After 250 渭 M hydrogen peroxide induction of ARPE-19 cells pretreated with allicin, the production of reactive oxygen species (ROS) and intracellular ROS, were detected by 2,7 and dichlorofluorescent sodium acetoacetate (DCFH-DA) fluorescence probe, and the intracellular ROS, was detected by colorimetric assay. The ratio of glutathione (GSH) / disulfide (GSSG) and the level of malondialdehyde (MDA) were determined to determine the effect of allicin on hydrogen peroxide induced damage in ARPE-19 cells. 3. The expression of nuclear protein mRNA, nuclear protein NOX4,NQO1 and Nrf2 in ARPE-19 cells pretreated with allicin was detected by fluorescence quantitative PCR and Western imprinting under 250 渭 M hydrogen peroxide induced oxidative stress. To further determine the potential mechanism of allicin in protecting retinal pigment epithelial cells from oxidative stress. 4. ARPE-19 cells pretreated with allicin were interfered with Nrf2 gene by gene transfer under 250 渭 M hydrogen peroxide induced oxidative stress. Fluorescence quantitative PCR assay and Western imprinting method were further used. MTT assay was used to detect the expression of Nrf2-related mRNA and Nrf2 protein in nuclear protein and cytoplasmic protein of ARPE-19, to evaluate the inhibitory efficiency of Nrf2, and to compare the difference of survivability among the three groups. To determine the regulatory effect of Nrf2 on the mechanism of allicin protective RPEs in H 202 induced injury. Results: the survival ability of 1.ARPE-19 cells was significantly lost after exposure to hydrogen peroxide for 12 or 24 hours. Allicin can reverse the damage effect of RPEs by hydrogen peroxide in a dose-dependent manner. 2. In ARPE-19 cells, allicin attenuated H202-induced oxidative stress by reducing the level of ROS,MDA and increasing GSH/GSSG. The potential mechanism of allicin in protecting retinal pigment epithelial cells (ARPE-19 cells) induced by hydrogen peroxide may be through the regulation of the expression of ROS- related enzymes, including SOD,NOX4,NQO1 and Nrf2.4.. The protective effect of allicin mediated hydrogen peroxide induced injury is related to the regulation of Nrf2. Allicin enhances the expression of Nrf2-related mRNA in a dose-dependent manner. Inhibition of Nrf2 significantly attenuated the protective effect of allicin on hydrogen peroxide-induced cells. However, inhibition of Nrf2 could not completely inhibit the protective effect of allicin. Conclusion: 1. Exogenous H 202 could induce oxidative stress damage in human retinal pigment epithelial cells ARPE-19 cells. Allicin can protect retinal pigment epithelial cells from oxidative stress in a dose-dependent manner. 2. Allicin can protect retinal pigment epithelial cells by regulating intracellular ROS level, GSH/GSSG,MDA and SOD. The potential mechanism of allicin to protect retinal pigment epithelial cells from oxidative stress may be achieved by regulating the expression of ROS related enzymes SOD,NOX4,NQO1 and Nrf2.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R774.5
本文编号：2494602
[Abstract]:Objective: to study whether allicin can protect human retinal pigment epithelial cells (ARPE-19) from hydrogen peroxide (H2O2)-induced injury. The effect and potential molecular mechanism of allicin on human retinal pigment epithelial cells (ARPE-19) induced by hydrogen peroxide were determined. To provide laboratory evidence for the prevention and treatment of age-related macular degeneration and other eye diseases related to oxidative stress. Methods: 1.ARPE-19 cells were exposed to 250 渭 M or 500 渭 M hydrogen peroxide for 12 or 24 hours, and then cultured in various concentrations of allicin (0, 5, 10, 20 or 40 渭 g / ml) 4 h.RPEs activity was evaluated by MTT test. To determine the effect of allicin on ARPE-19 cells induced by hydrogen peroxide. 2. After 250 渭 M hydrogen peroxide induction of ARPE-19 cells pretreated with allicin, the production of reactive oxygen species (ROS) and intracellular ROS, were detected by 2,7 and dichlorofluorescent sodium acetoacetate (DCFH-DA) fluorescence probe, and the intracellular ROS, was detected by colorimetric assay. The ratio of glutathione (GSH) / disulfide (GSSG) and the level of malondialdehyde (MDA) were determined to determine the effect of allicin on hydrogen peroxide induced damage in ARPE-19 cells. 3. The expression of nuclear protein mRNA, nuclear protein NOX4,NQO1 and Nrf2 in ARPE-19 cells pretreated with allicin was detected by fluorescence quantitative PCR and Western imprinting under 250 渭 M hydrogen peroxide induced oxidative stress. To further determine the potential mechanism of allicin in protecting retinal pigment epithelial cells from oxidative stress. 4. ARPE-19 cells pretreated with allicin were interfered with Nrf2 gene by gene transfer under 250 渭 M hydrogen peroxide induced oxidative stress. Fluorescence quantitative PCR assay and Western imprinting method were further used. MTT assay was used to detect the expression of Nrf2-related mRNA and Nrf2 protein in nuclear protein and cytoplasmic protein of ARPE-19, to evaluate the inhibitory efficiency of Nrf2, and to compare the difference of survivability among the three groups. To determine the regulatory effect of Nrf2 on the mechanism of allicin protective RPEs in H 202 induced injury. Results: the survival ability of 1.ARPE-19 cells was significantly lost after exposure to hydrogen peroxide for 12 or 24 hours. Allicin can reverse the damage effect of RPEs by hydrogen peroxide in a dose-dependent manner. 2. In ARPE-19 cells, allicin attenuated H202-induced oxidative stress by reducing the level of ROS,MDA and increasing GSH/GSSG. The potential mechanism of allicin in protecting retinal pigment epithelial cells (ARPE-19 cells) induced by hydrogen peroxide may be through the regulation of the expression of ROS- related enzymes, including SOD,NOX4,NQO1 and Nrf2.4.. The protective effect of allicin mediated hydrogen peroxide induced injury is related to the regulation of Nrf2. Allicin enhances the expression of Nrf2-related mRNA in a dose-dependent manner. Inhibition of Nrf2 significantly attenuated the protective effect of allicin on hydrogen peroxide-induced cells. However, inhibition of Nrf2 could not completely inhibit the protective effect of allicin. Conclusion: 1. Exogenous H 202 could induce oxidative stress damage in human retinal pigment epithelial cells ARPE-19 cells. Allicin can protect retinal pigment epithelial cells from oxidative stress in a dose-dependent manner. 2. Allicin can protect retinal pigment epithelial cells by regulating intracellular ROS level, GSH/GSSG,MDA and SOD. The potential mechanism of allicin to protect retinal pigment epithelial cells from oxidative stress may be achieved by regulating the expression of ROS related enzymes SOD,NOX4,NQO1 and Nrf2.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R774.5
【参考文献】
相关期刊论文 前1条
1 向艳芳;彭惠;;氧化应激及补体在年龄相关性黄斑变性发病中的作用机制[J];国际眼科杂志;2013年08期
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