维生素C保护视网膜色素上皮细胞的相关机制研究

The study of protection mechanism of vitamin c for RPE

目的研究氧化应激状态下维生素C作为抗氧化剂对视网膜色素上皮细胞的保护作用以及维生素C和SIRT1之间的调节机制。方法以人视网膜色素上皮细胞-19(ARPE-19)细胞为研究对象,分为空白对照组,无维生素C组(0μmol),低浓度维生素C组(20μmol),中等浓度维生素C组(100μmol)和高浓度维生素C组(500μmol)。培养过程中添加不同浓度维生素C之后对细胞加以H2O2(100μmol)处理12 h或24 h建立氧化应激模型。利用MTT法检测ARPE-19细胞存活率,膜联蛋白V-FITC凋亡检测试剂盒检测细胞凋亡以及活性氧(ROS)试剂盒测定细胞内活性氧的改变。使用SIRT1靶向siRNA进行SIRT1敲除,细胞使用预定浓度的维生素C进行孵育,并分为空白对照组,阴性对照组,siRNA干扰组。先用10 m M SIRT1激动剂白藜芦醇(RSV)和5 m M SIRT1抑制剂烟酰胺(NA)对细胞进行孵育,然后加入H2O2(100μmol)处理12 h或24 h,RT-PCR及Western blot方法检测SIRT1、p53和Foxo3基因表达水平。结果经过H2O212 h处理后,较高浓度的维生素C(500μmol)与较低浓度(20μmol)的维生素C无明显保护作用,而中等浓度的维生素C(100μmol)能够显著提高细胞的生存能力(P<0.05),减少ARPE-19细胞的凋亡数量(P<0.05),降低细胞内ROS水平(P<0.05),差异有统计学意义。维生素C的作用可上调H2O2作用后SIRT1转录因子和应激反应因子(p53和Foxo3)的表达。RSV及NA可分别上调及下调维生素C对H2O2刺激的ARPE-19细胞存活力,细胞凋亡和细胞内ROS水平的影响。RT-PCR及Western blot结果表明:维生素C浓度增加时,SIRT1表达增加,p53和Foxo3基因表达水平升高。敲除或上调SIRT1表达,可相应明显地增加或减少p53和Foxo3转录和蛋白水平的表达。结论维生素C可降低细胞内ROS水平,减少ARPE-19细胞凋亡,起到抗氧化损伤保护作用,有望成为年龄相关性黄斑变性的有效治疗方法。

Objective To investigate the protective effects of Vitamin C( Vit C) and the regulatory mechanism between Vit C and sirtuin1( SIRT1) in PREs during oxidative stress status. Methods Gradient concentrations of Vit C were used to interfere with the oxidative stress in ARPE- 19 and cell viability,apoptosis and intracellular ROS level were detected to evaluate the protective concentration of Vit C. Cell viability,apoptosis and intracellular ROS were also analyzed at indicated concentrations of Vit C after treatment of RSV or NA response to H2O2. The expression of p53 and Foxo3 in ARPE- 19 with supplement of moderate Vit C during oxidative stress was also examined. Results It was found that moderate Vit C( 100 μmol) prevented ARPE- 19 cells from damages induced by H2O2,including increasing viability,reducing apoptosis and attenuating intracellular ROS levels. However,higher concentration( 500 μmol) and lower concentration( 20 μmol) of Vit C had no such effects.Moreover Vit C caused dysregulation of some stress response factors( SIRT1,p53 and Foxo3) in ARPE- 19 cells responses to H2O2. SIRT1 activator RSV could promote the anti- apoptotic effects of Vit C and SIRT1 inhibitor NA relieved the protective functions of moderate Vit C. Furthermore,data also revealed the dysregulation of p53 and Foxo3 was dependent on the regulation of SIRT1 rather than Vit C. Conclusion The protective effect of Vit C against oxidative stress was involved in regulation of SIRT1. Combination of Vit C and RSV may be a promising therapeutic method for AMD.