尿酸调节Nrf2信号通路减轻6-OHDA对SH-SY5Y细胞损伤的实验研究

发布时间：2019-03-09 11:28

【摘要】：第一部分尿酸增加谷胱甘肽合成减轻6-OHDA对SH-SY5Y细胞损伤作用 目的：探讨尿酸（uric acid, UA）对6-羟基多巴胺(6-OHDA)诱导的SH-SY5Y细胞损伤发挥保护作用的机制。 方法：采用6-OHDA制作SH-SY5Y细胞损伤的帕金森病细胞模型，分为对照（DMEM）组、6-OHDA（50μmol/L）组、尿酸组（200μmol/L）以及6-OHDA（50μmol/L）+尿酸组（200μmol/L)。预给尿酸0.5h后6-OHDA作用6h、12h、14h、18h、24h后MTT比色法检测细胞活性；作用12h倒置显微镜下观测细胞形态；比色法检测谷胱甘肽含量；预给尿酸0.5h后6-OHDA作用12h后Western blot和RT-PCR法分别测定γ-谷氨酰半胱氨酸连接酶催化亚基（γ-GCLC）和γ-谷氨酰半胱氨酸连接酶调节亚基（γ-GCLM）蛋白和mRNA水平的表达量。 结果：200μmol/L尿酸对50μmol/L6-OHDA作用12h、14h、18h、24h的SH-SY5Y细胞活性下降有显著的改善（78.10±2.36%vs57.44±12.02%，80.52±3.16%vs50.15±2.32%，70.82±1.81%vs39.22±0.78%，42.84±4.73%vs27.99±4.23%，P0.01，P0.001， P0.001，P0.001）；作用12h后尿酸处理组细胞形态明显改善；谷胱甘肽含量下降有显著的改善（9.51±0.37μmol/L vs3.88±0.78μmol/L，，P0.01）;200μmol/L尿酸+50μmol/L6-OHDA组与50μmol/L6-OHDA组相比，γ-GCLC蛋白表达水平显著升高（139.85±11.62%vs49.20±7.09%，P0.01）；γ-GCLM.表达水平明显下降（95.36±18.98%vs160.05±8.59%，P0.05）。mRNA水平上γ-GCLC转录水平显著升高（110.99±14.41%vs49.19±6.87%，P0.001）；γ-GCLM.转录水平明显下降（102.70±10.43%vs190.05±8.59%，P0.05）。 结论：尿酸对6-OHDA诱导的SH-SY5Y细胞损伤具有保护作用，与增加谷胱甘肽合成有关。 第二部分尿酸对Nrf2信号通路的调节作用 目的：探讨尿酸增加谷胱甘肽生物合成的分子机制。 方法：采用6-OHDA损伤SH-SY5Y细胞制造帕金森病细胞模型，分为对照（DMEM）组、6-OHDA（50μmol/L）组、尿酸组（200μmol/L）以及6-OHDA（50μmol/L）+尿酸（200μmol/L)组，预先给予尿酸0.5h后，6-OHDA作用6h用共聚焦法观察Nrf2在胞浆、胞核的分布情况，运用胞浆胞核蛋白提取法后Western blot测定不同组别SH-SY5Y细胞上Nrf2表达情况；用基因转染siRNA敲减Nrf2方法后再运用比色法测定敲减组与未敲减组不同组别谷胱甘肽含量及细胞活力。 结果：与对照组相比，50μmol/L6-OHDA作用于SH-SY5Y细胞6h后Nrf2的分布无明显变化,胞浆胞核蛋白也无明显改变；与单独6-OHDA组相比，200μmol/L尿酸作用6h后共聚焦示Nrf2由胞浆明显转入胞核，提取胞浆胞核蛋白后尿酸处理组Nrf2核蛋白含量明显增加（196.33±35.21%vs93.11±29.25%，P0.05）；单独尿酸与对照组比，Nrf2核蛋白含量明显增加（231.36±55.19%vs100.00±33.01%，P0.01）；给予Nrf2-siRNA敲除Nrf2后尿酸+6-OHDA与单独6-OHDA组相比，谷胱甘肽含量无明显改变（4.16±0.12μmol/L vs3.64±0.50μmol/L, P0.05），细胞活力无明显升高（53.43±2.21%vs51.37±1.69%, P0.05）。 结论：尿酸对6-OHDA诱导的SH-SY5Y细胞损伤具有保护作用，与其调节Nrf2信号通路有关。
[Abstract]:Part I uric acid increases glutathione synthesis in SH-SY5Y cells. Objective: to investigate the effect of uric acid (uric acid, on the injury of SH-SY5Y cells induced by 6-OHDA. UA has a protective effect on 6-hydroxydopamine (6-OHDA)-induced SH-SY5Y cell injury. Methods: the model of Parkinson's disease (PD) induced by SH-SY5Y cells was induced by 6-OHDA. They were divided into three groups: control (DMEM) group, 6-OHDA (50 渭 mol / L) group, uric acid group (200 渭 mol / L) and 6-OHDA (50 渭 mol / L) group (200 渭 mol / L). After pretreated with uric acid 0.5 h, 6-OHDA for 6 h, 12 h, 14 h, 18 h, 24 h later, MTT colorimetric assay was used to detect cell activity, 12 h inverted microscope was used to observe cell morphology, and GSH content was measured by colorimetric method. Determination of 纬-glutamylcysteine ligase catalytic subunit (纬-GCLC) and 纬-glutamylcysteine ligase regulatory subunit (纬-GCLM) eggs by Western blot and RT-PCR after preadministration of uric acid for 12 h with 6-OHDA White and mRNA levels of expression. Results: 200 渭 mol / L uric acid significantly improved the decrease of SH-SY5Y cell activity (78.10 卤2.36%vs57.44 卤12.02%, 80.52 卤3.16%vs50.15 卤2.32%) at 12 h, 14 h, 18 h and 24 h after exposure to 50 渭 mol / L6-OHDA, respectively. 70.82 卤1.81%vs39.22 卤0.78%, 42.84 卤4.73%vs27.99 卤4.23%, P0.01, P0.001, P0.001); After treatment with uric acid for 12 hours, the morphology of the cells in the treatment group was obviously improved. The GSH content decreased significantly (9.51 卤0.37 渭 mol / L vs 3.88 卤0.78 渭 mol / L, P0.01). The expression of 纬-GCLC protein in 200 渭 mol / L uric acid 50 渭 mol / L6-OHDA group was significantly higher than that in 50 渭 mol / L6-OHDA group (139.85 卤11.62%vs49.20 卤7.09%, P0.01). 纬-GCLM. The expression level of 纬-GCLC was significantly decreased (95.36 卤18.98%vs160.05 卤8.59%, P0.05). The mRNA level of 纬-GCLC was significantly increased (110.99 卤14.41%vs49.19 卤6.87%, P0.001) and 纬-GCLM. was observed. The transcription level was significantly decreased (102.70 卤10.43%vs190.05 卤8.59%, P0.05). Conclusion: uric acid has protective effect on SH-SY5Y cells induced by 6-OHDA, which is related to the increase of glutathione synthesis. Part two Regulation of uric Acid on Nrf2 signaling Pathway objective: to explore the molecular mechanism of uric acid increasing glutathione biosynthesis. Methods: Parkinson's disease (PD) cells were induced by 6-OHDA injury in SH-SY5Y cells. They were divided into three groups: control (DMEM) group, 6-OHDA (50 渭 mol / L) group, uric acid group (200 渭 mol / L) and 6-OHDA (50 渭 mol / L) uric acid group (200 渭 mol / L). The distribution of Nrf2 in cytoplasm and nucleus was observed by confocal method after pretreated with uric acid 0.5 h and 6-OHDA for 6 h. The expression of Nrf2 in different groups of SH-SY5Y cells was measured by Western blot after extraction of cytoplasmic nucleoprotein. The glutathione content and cell viability in different groups of knock-down group and non-knock-down group were measured by colorimetric assay after gene transfection of siRNA knockdown Nrf2 method. Results: compared with the control group, there was no significant change in the distribution of Nrf2 and cytoplasmic nucleoprotein in SH-SY5Y cells treated with 50 渭 mol / L6-OHDA for 6 h. Compared with 6-OHDA alone group, the confocal Nrf2 was transferred from cytoplasm to nucleus after 6 h treatment with 200 渭 mol / L uric acid, and the content of Nrf2 nucleoprotein increased significantly after extraction of cytoplasmic nucleoprotein (196.33 卤35.21%vs93.11 卤29.25%, P0.05). Compared with the control group, the nuclear protein content of Nrf2 increased significantly (231.36 卤55.19%vs100.00 卤33.01%, P0.01). After Nrf2 knockout by Nrf2-siRNA, there was no significant change in glutathione content in uric acid 6-OHDA group compared with 6-OHDA alone group (4.16 卤0.12 渭 mol / L vs3.64 卤0.50 渭 mol / L, P0.05). There was no significant increase in cell viability (53.43 卤2.21%vs51.37 卤1.69%, P0.05). Conclusion: uric acid has protective effect on 6-OHDA-induced SH-SY5Y cell injury, which is related to its regulation of Nrf2 signaling pathway.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R742.5

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