基于 miR-34a / SIRT1 轴研究柚皮素对 H2O2 诱导的人视网膜色素上皮细胞氧化损伤的保护作用
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1.商丘医学高等专科学校五官科教研室,河南 商丘 476006; 2.郑州大学基础医学院人体解剖学教研室,郑州 450001

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R-33

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The protective effect of naringenin on H2O2 -induced oxidative damage of human retinal pigment epithelial cells via the miR-34a / SIRT1 axis
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1.Department of Otolaryngology, Shangqiu Medical College, Shangqiu 476006, China. 2. Department of Human Anatomy, School of Basic Medicine, Zhengzhou University, Zhengzhou 450001

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    摘要:

    目的 基于 miR-34a / 沉默信息调节因子 1(SIRT1)轴探讨柚皮素对 H2O2 诱导的人视网膜色素上皮细胞(RPE)氧化损伤的保护作用。 方法 MTT 法检测不同浓度的柚皮素对 H2O2 诱导的人视网膜色素上皮细胞 ARPE-19 存活率的影响以选取适宜柚皮素浓度为后续实验浓度。 取对数期生长的 ARPE-19 细胞分为对照组、 H2O2 组(200 μmol / L)、20 μg / mL 柚皮素组、20 μg / mL 柚皮素+mimics NC 组、20 μg / mL 柚皮素+miR-34a mimics 组。 采用 RT-qPCR 检测细胞中 miR-34a 和 SIRT1 mRNA 的表达,MTT 法及 Annexin V-FITC/ PI 双染分别检测细胞 存活与凋亡情况,荧光探针检测细胞内 ROS 水平,生化法检测 SOD 活性、MDA 和 GSH 水平,并采用 JC-1 法检测线 粒体膜电位(MMP),Western blot 检测 SIRT1 及 Bcl-2、Bax、Caspase-3 蛋白表达。 结果 与对照组相比,H2O2 组 ARPE-19 细胞中 miR-34a 表达、ARPE-19 细胞凋亡率、ROS、MDA 含量、Bax 和 Caspase-3 表达显著升高(P<0.05), SIRT1 mRNA 表达、ARPE-19 细胞存活率、SOD、GSH 含量、MMP 水平、SIRT1 和 Bcl-2 表达显著降低(P<0.05);与 H2O2 相比,柚皮素可改善 H2O2 诱导的 ARPE-19 细胞损伤,下调 miR-34a 表达,降低 ROS、MDA、Bax 和 Caspase-3 含量(P<0. 05),上调 SIRT1、MMP、Bcl-2 表达,增加 GSH、SOD 活性(P<0.05);上调 ARPE-19 细胞 miR-34a 的表达, 可逆转柚皮素对 H2O2 诱导的 RPE 细胞氧化损伤的保护作用。 结论 柚皮素可能通过下调 miR-34a,促进 SIRT1 的表达,抑制 RPE 细胞凋亡,保护 H2O2 诱导的 RPE 细胞氧化损伤。

    Abstract:

    Objective To investigate the protective effect of naringenin on H2O2 -induced oxidative damage of human retinal pigment epithelial (RPE) cells via the miR-34a / silent information regulator 1( SIRT1) axis. Methods MTT assay was used to detect the effect of naringenin at different concentrations on the survival rate of ARPE-19 cells treated with H2O2 , and the appropriate naringenin concentration for subsequent experiments was identified. ARPE-19 cells in logarithmic phase were divided into the control group, H2O2 group (200 μmol / L), 20 μg / mL naringenin group, 20 μg / mL naringenin+mimics NC group and 20 μg / mL naringenin +miR-34a mimics group. RT-qPCR was used to detect the expression of miR-34a and SIRT1 mRNA, MTT assay and Annexin V-FITC/ PI double staining were used to detect cell survival and apoptosis, respectively. A fluorescence probe was used to detect the level of ROS, and a biochemical method was used to detect the activity of SOD and levels of MDA and GSH. JC-1 method was used to detect the mitochondrial membrane potential (MMP), and Western blot was used to detect the proteins expression of SIRT1 and Bcl-2, Bax and Caspase-3. Results The expression of miR-34a, apoptosis rate, contents of ROS and MDA and expression of Bax and Caspase-3 in ARPE-19 cells were significantly increased in the H2O2 group compared with those in the control group (P< 0.05). The expression of SIRT1 mRNA, survival rate of APRE-19 cells, contents of SOD and GSH, level of MMP, expression of SIRT1 and Bcl-2 were significantly decreased in the H2O2 group compared with those in the control group (P< 0.05). Naringenin improved the damage of ARPE-19 cells induced by H2O2 , down-regulated the expression of miR-34a, decreased the contents of ROS, MDA, Bax and Caspase-3 (P<0.05), up-regulated the expression of SIRT1, MMP and Bcl-2 and increased the activities of GSH and SOD compared with effects in the H2O2 group (P<0. 05). Up-regulation of miR-34a expression in ARPE-19 cells reversed the protective effect of naringenin on H2O2 -induced oxidative damage in RPE cells. Conclusions Naringenin down-regulates miR-34a, promotes the expression of SIRT1, inhibits the apoptosis of RPE cells and protects RPE cells from oxidative damage induced by H2O2 .

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魏抗抗,董伟华,帖红艳,何章彪,赵 琳.基于 miR-34a / SIRT1 轴研究柚皮素对 H2O2 诱导的人视网膜色素上皮细胞氧化损伤的保护作用[J].中国比较医学杂志,2022,32(4):77~85.

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  • 收稿日期:2021-01-25
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  • 在线发布日期: 2022-06-20
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