【作者】 李芸；

【导师】 唐罗生；

【作者基本信息】 中南大学 ， 眼科学， 2010， 博士

【摘要】 [目的]观察α-硫辛酸(ALA)对外源性H2O2诱导的人视网膜色素上皮ARPE-19细胞的氧化损伤是否具有保护作用,并通过细胞凋亡和细胞自噬角度研究α-硫辛酸对氧化应激下ARPE-19细胞的保护作用途径及其相关的分子机制。[方法]1.人视网膜色素上皮细胞氧化应激损伤模型建立及ALA保护作用观察。外源性过氧化氢(H2O2)梯度浓度作用于ARPE-19细胞,MTT法检测细胞生存力,2’7’-二氯荧光乙酰乙酸钠(DCFH-DA)荧光探针检验细胞内活性氧(ROS)的生成,确定H2O2的适合作用浓度。α-硫辛酸(ALA,37.5μM)作用于氧化应激下ARPE-19细胞,检测细胞生存率和细胞内ROS的改变。2.氧化应激下RPE细胞凋亡及Akt-bcl-2/Bax-aspase-3途径的变化和ALA干预的影响。适合浓度(12.5mM)的H2O2作用于ARPE-19(氧化应激组),Hoechst33258染色定性细胞凋亡,Annexin V FITC-PI双染流式细胞术法定量检测氧化应激下的细胞凋亡,Western Blot法检测Akt磷酸化水平及凋亡相关因子Bcl-2、Bax蛋白表达,活性比色(DEVD法)检测Caspase-3活性。氧化应激下ARPE-19细胞以α-硫辛酸处理(ALA处理组,37.5μM)0、2、4、6、8、10小时后,Hoechst染色定性细胞凋亡,Annexin V FITC-PI双染流式细胞术法定量检测ALA干预后细胞凋亡变化,Western Blot法检测Akt磷酸化水平、凋亡相关因子Bax基因蛋白表达的变化,活性比色(DEVD法)检测Caspase-3活性的改变。3.氧化应激下RPE细胞溶酶体稳定性及自噬相关蛋白Beclin 1、Atg-5、LC3的变化和ALA干预的影响。适合浓度(12.5mM)的H2O2作用于ARPE-19(氧化应激组),吖啶橙(AO)染色测定氧化应激下的细胞溶酶体膜稳定性,Real-time PCR检测自噬相关基因Beclin-1和Atg-5的mRNA表达水平,Western Blot法检测Atg-5及LC3Ⅰ、Ⅱ的蛋白表达。氧化应激下ARPE-19细胞以α-硫辛酸处理(ALA处理组,37.5μM)0、2、4、6、8、10小时后,AO染色测定ALA干预后细胞溶酶体膜稳定性变化,Real-time PCR检测自噬相关因子Beclin-1和Atg-5的mRNA表达水平变化,Western Blot法检测Atg-5及LC3Ⅰ、Ⅱ的蛋白表达变化。[结果]1.外源性H2O2作用下,ARPE-19的细胞生存力下降且呈剂量相关性,同时ARPE-19细胞内的活性氧(ROS)浓度上升,提示H2O2处理可诱导ARPE-19细胞的氧化应激损伤；H2O2的适合作用浓度为12.5mM。ALA处理后,ARPE-19细胞氧化应激下的生存率增加,同时细胞内的ROS浓度降低。2.氧化应激组ARPE-19细胞凋亡明显增加,以早期凋亡为主(p<0.05),晚期凋亡变化不显著；Akt的磷酸化水平增强,凋亡相关因子Bcl-2表达无明显变化而Bax表达上升,Caspase-3活性上升。ALA处理组ARPE-19细胞2、4、6、8、10小时细胞凋亡百分率均下降,具有统计学意义(p<0.05)；Akt的磷酸化水平在4、6小时处显著增强,凋亡相关因子Bax蛋白在4、6、8小时处的表达降低,Bcl-2的表达及Caspase-3活性在各时间点无明显变化。3.氧化应激组ARPE-19细胞酸性泡样细胞器(AVO)明显增加,溶酶体的破裂增多,自噬相关基因Atg-5的mRNA及蛋白水平表达增加,Beclin-1的mRNA和LC3Ⅱ的蛋白表达上调,LC3Ⅰ改变不明显。ALA处理组ARPE-19细胞AO染色检测结果显示细胞溶酶体破裂减少,自噬相关因子Atg-5的mRNA及蛋白表达水平在4、6小时下调,LC3Ⅱ的蛋白表达在作用4、6小时下调,而Beclin-1的mRNA和LC3Ⅰ的蛋白检测未显示明显差异。[结论]1.外源性H2O2可诱导人视网膜色素上皮ARPE-19氧化应激,导致细胞损伤。α-硫辛酸可降低细胞内氧化应激水平,减少ARPE-19的细胞损伤及死亡,发挥保护作用。2.氧化应激下ARPE-19的细胞损伤可能与细胞凋亡增加有关。α-硫辛酸对氧化应激下ARPE-19细胞的保护作用可能通过减少细胞凋亡实现,这一作用可能与p-Akt的激活和Bax的表达下调相关。3.氧化应激下ARPE-19的细胞损伤可能与细胞自噬增强有关。α-硫辛酸对氧化应激下ARPE-19细胞的保护作用可能通过减少自噬性细胞死亡实现,可能与Atg-5和LC3Ⅱ的表达下调相关。更多还原

【Abstract】 Objective:To observe the protective effect of alpha lipoic acid(ALA) to the oxidative stressed human retinal pigment epithelium cell line ARPE-19, and investigate apoptosis and autophagy alterations and their relevant molecular mechanisms in human retinal pigment epithelium oxidative stress procedure and ALA protection.Methods:1. RPE oxidative stress model and ALA protection effect identification. Gradient concentrations of H2O2 were used to ARPE-19 cells to induce oxidative stress model. Cell viability was measured by MTT test, ROS concentration was quantified with DCFH-DA probe. ALA was used to interfere with the oxidative stress in ARPE-19, ROS and cell viability were also detected.2. Apoptosis and Akt-bcl-2/Bax-Caspase-3 pathway changes of Oxidative stressed RPE and ALA treatment. Oxidative stressed ARPE-19 cells (treated by 12.5mM H2O2) were detected for cell apoptosis by Hoechst 33258 staining, quantitative measurement of apoptosis by Annexin V FITC-PI double staining and flow cytometry; Caspase-3 activity detection by cpp32 colorimetric assay; Bcl-2 and Bax expression were measured by Western Blot.37.5μM ALA was administered to oxidative stressed ARPE-19 and apoptosis detections was carried out as above.3. Lysosomal stability and Autophagic factor atg-5, Beclin 1, LC3Ⅰ/Ⅱchanges of Oxidative stressed RPE and ALA treatment. Oxidative stressed ARPE-19 cells (treated by 12.5mM H2O2) were detected for autophagy:lysosomal membrane stability was detected by acridine orange(AO) staining. Autophagy was semiquantified by real-time PCR of Beclin-1 and Atg-5 mRNA, Western Blot of Atg-5 and LC3Ⅲexpression, treated by 0,37.5μM ALA,37.5μM ALA was administered to oxidative stressed ARPE-19 and autophagy detections was carried out as above.Results:1. Exogenous H2O2 significantly increases intracellular ROS concentration and inhibits ARPE-19 cell viability in a dose-dependent way, the optimal H2O2 concentration is 12.5mM; ALA administration decreases intracellular ROS concentration in oxidative stressed ARPE-19 cells and increases ARPE-19 cell viability under oxidative stress.2. Oxidative stressed ARPE-19 cells show early apoptosis increase (p<0.05) with significant Akt phosphorylation activation Bax upregulation, and Caspase-3 activity increase; ALA administration to oxidative stressed ARPE-19 shows early apoptosis decrease in2、4、6、8、10 hrs (p<0.05), with upregulation of Akt phosphorylation in 4、6 hrs and Bax downregulation in 4、6、8 hrs. Meanwhile, Caspase-3 activity and Bcl-2 expression shows no significant changes. 3. Oxidative stressed ARPE-19 cells show significantly increased acidic vesicular organelles (AVO) formation and more lysosomal rupture. Autophagy related genes Atg-5, Beclin 1, LC3Ⅱexpression increased; ALA administration to oxidative stressed ARPE-19 shows increased lysosomal stability and downregulated Atg-5 and LC3Ⅱexpression. Beclin 1 and LC3Ⅰshows no significant expression changes.Conclusion:1. Exogenous H2O2 induces ARPE-19 oxidative stress and cell injury; Alpha lipoic acid(ALA) shows protection effect to oxidative stressed ARPE-19 cells by reducing oxidative stress and cell death.2. Oxidative stressed ARPE-19 cell injury may be related to increased apoptosis. Alpha lipoic acid decreases ARPE-19 cell apoptosis, possibly by p-Akt activation and Bax expression downregulation.3. Oxidative stressed ARPE-19 cell injury may be related to increased autophagy. Alpha lipoic acid attenuates ARPE-19 autophagic cell death, possibly by Atg-5 and LC3Ⅱexpression downregulation.更多还原