【摘要】：目的：研究天然物質蒜素是否能保護人視網膜色素上皮細胞(ARPE-19)免受過氧化氫(H2O2)-誘導損傷,并確定蒜素對過氧化氫誘導的氧化應激損傷的人視網膜色素上皮細胞(ARPE-19)的影響和潛在分子機制。為年齡相關性黃斑變性等與氧化應激相關的眼病防治研究方面提供實驗室證據。方法：1.ARPE-19細胞暴露在250μM或500μM過氧化氫內12或24小時,后培養(yǎng)在預先備好的各種濃度的蒜素內(0、5、10、20或40μg／毫升)4 h。RPEs的活力使用MTT試驗評估。確定蒜素對過氧化氫誘導ARPE-19細胞的影響。2.對濃度梯度的蒜素預處理的ARPE-19細胞進行250μM過氧化氫誘導后通過2、7'-二氯熒光乙酰乙酸鈉(DCFH-DA)熒光探針檢測細胞內活性氧(ROS)的生成,比色法檢測細胞內ROS、谷胱甘肽(GSH)/二硫化谷胱甘肽(GSSG)比值和丙二醛(MDA)的水平,確定蒜素對ARPE-19細胞過氧化氫誘導損傷的影響。3.濃度梯度的蒜素預處理的ARPE-19細胞進行250μM濃度過氧化氫誘導的氧化應激狀態(tài)下通過熒光定量PCR檢測和蛋白質印跡檢測ARPE-19細胞核蛋白質mRNA,核蛋白NOX4,NQO1及Nrf2表達水平。進一步確定蒜素保護視網膜色素上皮細胞免受氧化應激損傷的潛在機制。4.濃度梯度的蒜素預處理的ARPE-19細胞進行250μM濃度過氧化氫誘導的氧化應激狀態(tài)下通過基因轉染方法對Nrf2基因進行干涉,進一步應用熒光定量PCR檢測法、蛋白質印跡檢測法、MTT法等檢測ARPE-19細胞核蛋白質和胞漿蛋白質內Nrf2相關mRNA表達水平、Nrf2蛋白質表達水平,評估Nrf2抑制效率、比較各組間生存力的差異,確定Nrf2在蒜素保護RPEs在H202誘導損傷機制中的調節(jié)效應。結果：1.ARPE-19細胞暴露于過氧化氫內處理12或24小時后導致細胞生存能力顯著損失。蒜素可以逆轉RPEs被過氧化氫的損傷效應,此現象是劑量依賴性的。2.在ARPE-19細胞內,蒜素通過減少細胞內的ROS、MDA的水平和增加GSH/GSSG來減弱H202-誘導的氧化應激反應。3.蒜素保護過氧化氫誘導的視網膜色素上皮細胞(ARPE-19 cells)的潛在機制可能是通過調節(jié)ROS-相關酶的表達水平,包括SOD、NOX4、NQO1以及Nrf2。4.蒜素介導的過氧化氫一誘導損傷的保護作用與Nrf2的調節(jié)有關。蒜素通過劑量-依賴的方式增強Nrf2相關的mRNA的表達水平。抑制Nrf2可顯著減弱蒜素對過氧化氫-誘導的細胞的保護作用。然而,抑制Nrf2并不能完全抑制蒜素的保護作用。結論：1.外源性H202可誘導人視網膜色素上皮細胞ARPE-19細胞發(fā)生氧化應激損傷。蒜素可以保護視網膜色素上皮細胞免受氧化應激損傷,此保護作用是劑量依賴性的。2.蒜素通過調節(jié)細胞內ROS水平、GSH/GSSG、MDA及SOD等實現對視網膜色素上皮細胞的保護作用的。3.蒜素保護視網膜色素上皮細胞免受氧化應激損傷的潛在的機制可能是通過調節(jié)ROS相關酶SOD、NOX4、NQO1以及Nrf2等的表達實現的。
[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.
【學位授予單位】：武漢大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R774.5

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相關期刊論文 前1條

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