雌激素对蓝光及过氧化氢诱导的ARPE-19细胞内活性氧生成的影响

目的:观察17-β雌二醇(E2)对蓝光及过氧化氢(H2O2)诱导的人视网膜色素上皮ARPE-19细胞内活性氧(reactive oxygen species,ROS)生成的影响方法:对培养的ARPE-19细胞予以浓度为10nmol/L,100nmol/L,1μmol/L的E2预处理2h后接受蓝光照射(470±20nm,2000±500lx)或200μmol/LH2O2处理,在处理前、处理后30min;1,2,4h行2′,7′-二氢二氯荧光黄双乙酸钠(DCFH-DA)细胞内ROS荧光染色,以490nmol/L荧光显微镜观察照相,收集细胞后以流式细胞仪检测荧光强度。结果:蓝光照射和过氧化氢均可显著增强ARPE-19细胞内的ROS荧光,提示蓝光照射和过氧化氢处理均成功诱导氧化应激,10nmol/L、100nmol/L、1μmol/L17β-雌二醇剂量依赖性地减弱蓝光照射和过氧化氢处理后ARPE-19细胞内的ROS荧光增强的幅度。结论:蓝光照射及过氧化氢处理可诱导ARPE-19细胞内活性氧水平增高;17β-雌二醇可剂量依赖性地抑制蓝光照射诱导的ARPE-19细胞内活性氧水平增高。     

视网膜色素上皮细胞线粒体DNA的氧化损伤研究

目的研究体外氧化应激模型中视网膜色素上皮(RPE)细胞线粒体DNA(mtDNA)的损伤规律。方法以过氧化氢(H2O2)诱导建立体外RPE细胞氧化应激模型,对模型中细胞mtDNA损伤进行检测,并与细胞存活率和细胞内活性氧(ROS)浓度的变化相比较。结果mtDNA损伤程度随H2O2浓度升高和作用时间延长而增加;较之细胞存活率和ROS浓度改变更为敏感。结论在RPE细胞的氧化应激中存在mtDNA的损伤改变,它与氧化剂之间具有明显的浓度与时间的依赖性。     

过氧化氢诱导的氧化应激对ARPE-19细胞中泛素化途径的激活

目的探讨H2O2诱导的氧化应激对人视网膜色素上皮（RPE）细胞系ARPE-19细胞中泛素及泛素化蛋白表达的影响。方法MTT法建立H2O2诱导ARPE-19细胞模型。不同浓度H2O2作用于ARPE-19细胞后,RT-PCR检测细胞内泛素基因转录水平,细胞免疫荧光方法检测细胞内泛素化蛋白的表达。用有（或无）蛋白酶体抑制剂MG-132预处理后再用H2O2处理ARPE-19,Western blot方法检测细胞内泛素化蛋白。结果氧化应激后UBA、UBB的转录水平明显增加（P〈0．05）,UBC无明显变化。细胞内相对分子质量为50000～250000的泛素化蛋白均增加,MG-132预处理后再用H2O2处理,细胞内增加的泛素化蛋白则多集中在相对分子质量75000以上。细胞内泛素化蛋白表达增加,核内增加明显。结论H2O2诱导的ARPE-19氧化应激过程激活细胞内泛素化途径。     

黄酮对视网膜色素上皮细胞氧化损伤的保护作用

目的:观察黄酮对氧化剂所诱导的视网膜色素上皮细胞(RPE)的保护作用。方法:在体研究中,预先给予5g/L黄酮滴眼液(3次/d),1wk后舌下静脉注射NaIO3诱导大鼠RPE变性,在2和4wk末,采用视网膜电图(ERG)测量C波。离体研究中,采用缺氧、H2O2、NaN3和t-BHP诱导RPE细胞损伤,并用MTT法检测细胞的存活率。结果:ERG的C波结果表明,第4wk末,黄酮抑制了由NaIO3诱导的大鼠RPE变性。离体研究结果表明,黄酮对多种氧化剂所诱导的RPE细胞损伤具有保护作用。结论:黄酮对氧化诱导的在体和离体视网膜色素上皮细胞均具有保护作用。     

过氧化氢对人视网膜色素上皮细胞内年龄相关性黄斑病变易感因子2表达的影响

目的 研究过氧化氢（H2O2）诱导的氧化应激对人视网膜色素上皮（ARPE-19）细胞内年龄相关性黄斑病变易感因子2（age-related maculopathy susceptibility 2,ARMS2）转录和蛋白水平的影响,初步探讨ARMS2基因在氧化应激中对视网膜色素上皮细胞的作用.方法 实验研究.选用ARPE-19细胞,以浓度为0、100、300、500、700μmol/L的H2O2作用一定时间后,应用四甲基偶氮唑盐比色法（MTT）检测各浓度组H2O2作用2 h和4 h后ARPE-19细胞生长情况;采用Realtime-PCR（SYBR Green法）和细胞免疫荧光法检测ARMS2的转录及蛋白水平变化.组间比较均采用单因素方差分析,两两比较采用最小显著性差异法,H2O2浓度与细胞活性的关系采用曲线估计分析.结果 五个浓度的H2O2作用ARPE-19细胞4 h后,吸光度值分别为0.531±0.037、0.370±0.017、0.371±0.016、0.330±0.006和0.297±0.012,差异有统计学意义（F=6.782,P=0.007）.曲线估计分析显示H2O2浓度与细胞活性呈高度负相关（r=-0.99.P=0.036）.100～700 μmol/L H2O2作用ARPE-19细胞后,Realtime-PCR结果 Ct值分别为1.154±0.007、1.324±0.022、1.350±0.011、1.280±0.031,差异有统计学意义（F=33.409.P=0.000）;H2O2浓度在300～500 μmol/L范围时,ARMS2基因mRNA表达量达到高峰,之后下降.细胞免疫荧光检测灰度值结果分别为7320±2493、14 300±848、22400±1596、23400±2405、19 200±561,差异有统计学意义（F=22.843,P=0.000）;H2O2浓度在300～500 μmol/L范围时,ARMS2蛋白表达量达到高峰,之后下降.转录和蛋白水平变化趋势一致.结论 H2O2在一定浓度范围内时,可诱导氧化应激,使ARPE-19细胞内ARMS2转录和蛋白水平增加,如果H2O2的浓度超过ARPE-19细胞耐受范围,ARMS2基因表达量下降.     

柚皮素对ARPE-19和HUVEC细胞的抗氧化作用

目的：研究柚皮素对人视网膜色素上皮细胞（ARPE-19）和人脐静脉内皮细胞（HUVEC）的抗氧化作用。 方法：采用MTT的方法检测ARPE-19和HUVEC细胞的生存率及增殖率。 结果：3，10mg／L柚皮素能显著增加ARPE-19细胞的增殖率达10．8％和11．4％。10mg／L柚皮素能提高ARPE-19细胞在缺氧，0．3mmol／LNaN，及200μmol／L，H2O2条件下的生存率分别为55．2％，69．2％及50．3％。1mg／L柚皮素能提高ARPE-19细胞在50μmol／L t-BHP和30mg／LNaIO3条件下的生存率达20．2％和30．4％。30mg／L柚皮素能够促进ARPE-19细胞在50μmol／L t-BHP条件下的增殖率达32．2％，而1mg／L柚皮素可以提高30，100，300mg／LNaIO3处理的ARPE-19细胞的增殖率达30．3％，10．3％及18．5％。3，10及30mg／L柚皮素抑制HUVEC的增殖率分别为23．9％，70．4％及77．9％。1，3mg／L柚皮素能提高HUVEC细胞在缺氧条件下的生存率达10．7％和13．1％，以及提高在300mg／LNaIO3条件下的生存率达41．2％和37．7％。3mg／L柚皮素能提高HUVEC细胞在200，400μmol／L H2O2条件下的生存率达20．1％和21．5％． 结论：柚皮素能够促进ARPE-19细胞的增殖率，抑制HU-VEC生长，同时对这两种细胞均有抗氧化作用。因此，柚臾素是治疗老年黄斑变性的很有前景的候选药物。     

黄芪含药血清对氯化钴诱导ARPE-19细胞缺氧损伤的保护作用

目的：研究黄芪含药血清对氯化钴(CoCl₂)诱导人视网膜色素上皮(ARPE-19)细胞缺氧损伤的保护作用,从而探讨黄芪是否通过抗氧化应激来改善糖尿病视网膜病变(DR)。

方法：建立CoCl₂诱导ARPE-19细胞缺氧模型,并分为以下5组：正常组(细胞正常培养,不加任何处理)、缺氧模型组(200μmol/L CoCl₂)、空白血清组(200μmol/L CoCl₂+空白血清)、低剂量含药血清组(200μmol/L CoCl₂+10%含药血清)、高剂量含药血清组(200μmol/L CoCl₂+20%含药血清)。CCK-8检测细胞活性; 试剂盒检测细胞上清液中还原型谷胱甘肽(GSH)、丙二醛(MDA)水平; ELISA检测细胞培养基中缺氧诱导因子-1α(HIF-1α)、血管内皮生长因子(VEGF)的含量; 实时荧光定量PCR(qPCR)检测VEGF、HIF-1α和脯氨酰羟化酶-2(PHD-2)的mRNA水平; Western Blot法检测VEGF、HIF-1α和PHD-2的表达情况。

结果：采用200μmol/L的CoCl₂浓度可成功建立ARPE-19细胞缺氧模型。低剂量和高剂量黄芪含药血清可抑制缺氧诱导的ARPE-19细胞增殖(P<0.05),升高ARPE-19细胞缺氧损伤中GSH水平,降低MDA含量(P<0.05)。低剂量和高剂量黄芪含药血清可抑制ARPE-19细胞缺氧损伤上清液中HIF-1α和VEGF的表达(P<0.05),以及ARPE-19细胞中VEGF、HIF-1α、PHD-2的mRNA表达和蛋白表达(P<0.05)。

结论：低剂量和高剂量黄芪含药血清通过抗氧化作用减轻CoCl₂诱导ARPE-19细胞缺氧损伤。     

骨形态发生蛋白-6对过氧化氢造成的视网膜色素上皮细胞损伤的保护作用

目的：：观察骨形态发生蛋白-6( bone morphogenetic protein,BMP-6)对过氧化氢( hydrogen peroxide,H2 O2)作用的人视网膜色素上皮株ARPE-19细胞的细胞形态、增殖以及凋亡的影响。方法：常规培养 ARPE-19细胞,分为正常对照组、75μmol/L H2 O2及150ng/mL BMP-6、75μmol/L H2 O2+150 ng/mL BMP-6环境中培养3、6、9、12 h后, MTT比色法检测细胞的活性;流式细胞仪检测细胞的周期及凋亡变化。结果：H2 O2作用组的细胞活性随着作用时间的延长逐步降低;而BMP-6+H2 O2组的细胞活性则较H2 O2组高,在3h及6h时两组相比差异有统计学意义(P<0.05);细胞形态观察发现：H2 O2培养6h后,细胞数量减少,细胞发生脱落,而添加 BMP-6后细胞脱落及凋亡均要明显减少。结论：BMP-6可以一定程度上保护视网膜色素上皮受到氧化应激的损伤。     

三氧化二砷对表皮生长因子诱导的视网膜色素上皮细胞增生和迁移的影响

背景 细胞因子失衡所导致的视网膜色素上皮(RPE)细胞的异常增生和迁移是增生性玻璃体视网膜病变( PVR)的主要病理变化之一.三氧化二砷(As2O3)是中国传统中药中的有效成分,可有效抑制肿瘤细胞的增生和迁移.但As2O3对细胞生长因子引起的RPE细胞增生和迁移的影响尚未明确. 目的 探讨As2O3对表皮生长因子(EGF)诱导的ARPE-19细胞增生和迁移的影响.方法 用无血清培养基对RPE细胞系ARPE-19细胞进行培养,将终浓度为0、0.5、1.0、2.0、5.0、10.0和20.0 μmol/L的As2O3分别加入到无血清培养基和含10 mg/L EGF的ARPE-19细胞培养液中作用24 h和48 h,通过噻唑蓝(MTT)比色法检测各培养组ARPE-19细胞活性的吸光度(A)值,以探讨As2O3对细胞的药物毒性作用,并筛选安全、有效的As2O3作用浓度.用10 mg/L EGF加入培养基诱导ARPE-19细胞迁移,分别在培养板中加入0、0.5、1.0、2.0μmol/L As2O3 作用24 h和48 h,并通过划痕试验和Transwell试验检测As2O3对EGF诱导的ARPE-19细胞迁移的影响.结果 MTT法检测发现不同浓度As2O3组作用24 h和48 h后,无血清培养组细胞A值随As2O3浓度的升高而逐渐下降,总体差异有统计学意义(F浓度=38.269,P=0.000;F时间=0.874,P=0.358).与空白对照组(0μmol/LAs2O3组)比较,0.5～ 5.0 μmol/L As2O3组ARPE-19细胞A值的差异均无统计学意义(P＞0.05).对含10 mg/LEGF组的ARPE-19细胞,药物对细胞A值的影响呈现浓度和时间依赖性(F浓度=152.155,P=0.000;F时间=51.649,P=0.000).与对照组比较,0.5～2.0μmol/L As2O3加入24 h和48 h后,A值的变化差异均无统计学意义(P＞0.05),而0.5、1.0、2.0μmol/L As2O3加入10 mg/L EGF诱导的ARPE-19细胞中作用24h和48 h后,A值的变化差异均无统计学意义(F浓度=2.215,P=0.126;F时间 =2.230,P=0.155).5.0 ～20.0 μmol/L As2O3作用于EGF诱导的ARPE-19细胞中作用后,细胞A值明显下降,与空白对照组比较差异均有统计学意义(P＜0.05),5.0～20.0 μmol/L As2O3作用24 h后,对EGF诱导的ARPE-19细胞增生抑制率分别为12％、32％、37％;作用48 h后细胞抑制率分别为39％、44％和53％.划痕试验结果显示,0.5～2.0 μmol/L As2O3对EGF诱导的ARPE-19细胞的横向迁移具有抑制作用.Transwell试验结果表明,0.5～2.0 μmol/L As2O3对10 mg/L EGF诱导的ARPE-19细胞纵向迁移有明显的抑制作用,0.5、1.0、2.0μmol/L As2O3作用12h对ARPE-19细胞的抑制率分别为22％、33％和46％. 结论 As2O3在一定浓度范围内对ARPE-19细胞无毒性作用,2.0 μmol/L以下浓度的As2O3对EGF诱导的ARPE-19细胞增生无明显影响,但可影响细胞的迁移能力,5.0 μmol/L以上浓度的As2O3可明显抑制ARPE-19细胞的增生.     

基于Nrf2通道研究柚皮素磷脂复合物对氧化损伤ARPE-19细胞的保护作用

目的：研究柚皮素(Nar)及其磷脂复合物(NPC)对叔丁基氢过氧化物(t-BHP)诱导产生的氧化损伤人视网膜色素上皮细胞(ARPE-19细胞)的保护作用及其作用机制。

方法：根据文献最佳制作工艺制备NPC。将体外培养的ARPE-19细胞分为溶媒组(用DMSO培养)、模型组(用200μmol/L t-BHP干预)、Nrf2-siRNA组(针对Nrf2基因进行细胞转染)、柚皮素组(200μmol/L柚皮素培养基预处理后加入200μmol/L t-BHP)、NPC组(200μmol/L NPC培养基预处理后加入200μmol/L t-BHP)、Nrf2-siRNA+柚皮素组(200μmol/L柚皮素预处理后Nrf2基因干扰,再加入200μmol/L t-BHP)、Nrf2-siRNA+NPC组(200μmol/L NPC预处理后Nrf2基因干扰,再加入200μmol/L t-BHP)。检测各组细胞内活性氧、丙二醛、超氧化物歧化酶、总抗氧化能力水平,分别采用RT-PCR和Western blot法检测各组细胞内血红素加氧酶-1(HO-1)、醌氧化还原酶-1(NQO-1)、谷氨酸半胱氨酸连接酶(GCL)和核因子E2相关因子2(Nrf2)的表达水平。

结果：NPC较柚皮素能明显降低ARPE-19细胞内丙二醛、活性氧的含量,提高细胞内超氧化物歧化酶、总抗氧化能力水平。柚皮素和NPC预保护ARPE-19 细胞后,Nrf2、HO-1、NQO-1和GCL mRNA的相对表达量和蛋白表达水平均较模型组和Nrf2-siRNA组升高。柚皮素组与NPC组,Nrf2-siRNA+柚皮素组与Nrf2-siRNA+NPC组细胞内4种基因和蛋白相对表达水平均有差异。Nrf2-siRNA+柚皮素组与Nrf2-siRNA组Nrf2、HO-1、NQO-1蛋白表达无显著差异。Nrf2-siRNA+NPC组与Nrf2-siRNA组4种蛋白表达均有差异,NPC作用明显强于柚皮素。

结论：柚皮素磷脂复合物能明显提高细胞内抗氧化能力,降低氧化水平,其通过激活Nrf2/ARE抗氧化应激通路上调Nrf2及其下游抗氧化酶和Ⅱ相解毒酶的表达对氧化损伤的ARPE-19细胞起到更好的保护作用。     

Antioxidant effect of hydralazine on retinal pigment epithelial cells and its potential use in the therapy of age-related macular degeneration

AIM: To investigate the antioxidant effect of hydralazine under hypoxia-induced damage on retinal pigment epithelial (ARPE-19) cells and the role of reactive oxygen species (ROS) in this effect. ·METHODS: Human retinal pigment epithelial (hRPE) cells were used to investigate the effect of hydralazine on oxidative stress, including tert-butyl hydroxyperoxide (t-BHP), H2O2, sodium azide (NaN3), and hypoxia induced cell damage. Cell viability was determined by MTT assay. RESULTS: When ARPE-19 cells were treated with oxidative stress induced by ROS, hydralazine showed concentration-dependent protection against t-BHP, H2O2 and hypoxia induced cell damage but not NaN3. Nitric oxide (NO) was not involved in this effect. ·CONCLUSION: Hydralazine showed antioxidant potential against oxidative stress induced damage in ARPE-19 cells. These effects might be caused through scavenger of ROS. Thus, hydralazine could be used for the treatment of age-related macular degeneration (AMD).     

Effects of flavone on the oxidation-induced injury of retinal pigment epithelium cells

AIM: To investigate the effect of flavone on oxidation- induced injury in retinal pigment epithelium cells. METHODS: In in vivo studies, NaIO3-induced RPE degene- ration in rat eyes was treated with 0.5% flavone eye drops 3 times a day for 1 week before and 4 weeks after NaIO3 injection. At the end of 2 and 4 weeks, all rats were measured c-wave by electroretinogram (ERG). In in vitro studies, ARPE-19 cells were treated with hypoxia, H2O2, NaN3 and t-BHP to induce cell damages. MTT assay was used to measure the viable cells. RESULTS: The ERG c-wave results showed that flavone reversed NaIO3-induced injury at the end of 4 weeks. In vitro results showed flavone reversed the various oxidants-induced injuries in RPE cells. CONCLUSION: Flavone could prevent the RPE from oxidation- induced injury both in vivo and in vitro.     

Zn2+-induced cell death is mediated by the induction of intracellular ROS in ARPE-19 cells

PURPOSE: Recent studies have shown that Zn2+ induced cell death in retinal pigment epithelial cells. Here we sought to investigate the mode of Zn2+-induced cell death and the role of reactive oxygen species (ROS) in human retinal pigment epithelial cell line, ARPE-19 cells. METHODS: Cell viability was measured by MTT assay. Cell death of ARPE-19 cells was measured by annexin V-fluorescein isothiocyanate (FITC) binding assay, TUNEL assay. The formation of intracellular ROS was measured using 2',7'-dichlorofluorescein diacetate (DCFH-DA). The activation of mitogen-activated protein kinase (MAPK) was examined by Western blot analysis. RESULTS: This study demonstrated that Zn2+ treatment induced both necrosis and apoptosis in ARPE-19 cells. Exposure of ARPE-19 cells to Zn2+ led to the activation of ERK1/2, JNK1/2/3, and p38 MAPKs. The activation of these MAPKs was blocked by treatment with the antioxidant, N-acetylcystein (NAC). More importantly, inhibition of ROS production by NAC completely prevented Zn2+-induced cell death in RPE cells. CONCLUSIONS: This study suggests that Zn2+ induces both apoptosis and necrosis in ARPE-19 cells and that its cytotoxicity may depend on the induction of intracellular ROS.     

Demonstration by redox fluorometry that sulforaphane protects retinal pigment epithelial cells against oxidative stress

PURPOSE: To quantify the effects of oxidant challenge on the redox state of adult human retinal pigment epithelial cells using microscopic autofluorescence spectroscopy and to determine whether treatment with the isothiocyanate sulforaphane protects these cells against oxidative stress. METHODS: Oxidative stress was evoked in ARPE-19 cells by H2O2 and tert-butyl hydroperoxide. Reduced nicotinamide nucleotides NAD(P)H were assessed by excitation at 366 nm with measurement of fluorescence at 450 nm. Oxidized flavoproteins were assessed by excitation at 460 nm with measurement of fluorescence at 540 nm. The ratio of these measurements served as the index of cellular redox status. RESULTS: Redox ratio and cell viability decreased in a dose-dependent manner after oxidant exposure. ARPE-19 cells treated with sulforaphane maintained significantly higher redox ratio and cell viability. The ratio for sulforaphane-treated cells after exposure to 0.64 mM H2O2 was 2.64 +/- 0.19 compared with 1.77 +/- 0.16 in untreated cells (P = 0.001). At 1.2 mM H2O2, the redox ratio of sulforaphane-treated cells was 2.30 +/- 0.18 compared with 1.76 +/- 0.13 in untreated cells (P = 0.02). Similar results were observed after insult with tert-butyl hydroperoxide. CONCLUSIONS: Redox fluorometry provides quantitative information on the redox status of living cells. Sulforaphane protects ARPE-19 cells from oxidative injury by induction of antioxidant phase 2 genes. The findings in this study describe a useful method for assessing antioxidant effects in live cells and support phase 2 gene induction as a potential treatment strategy for macular degeneration and diseases in which oxidative injury plays a causative role.     

Oxidative stress affects retinal pigment epithelial cell survival through epidermal growth factor receptor/AKT signaling pathway

AIM: To investigate the cross-talk between oxidative stress and the epidermal growth factor receptor (EGFR)/AKT signaling pathway in retinal pigment epithelial (RPE) cells. METHODS: Human RPE cell lines (ARPE-19 cell) were treated with different doses of epidermal growth factor (EGF) and hydrogen peroxide (H2O2). Cell viability was determined by a methyl thiazolyl tetrazolium assay. Cell proliferation was examined by a bromodeoxyuridine (BrdU) incorporation assay. EGFR/AKT signaling was detected by Western blot. EGFR localization was also detected by immunofluorescence. In addition, EGFR/AKT signaling was intervened upon by EGFR inhibitor (erlotinib), PI3K inhibitor (A66) and AKT inhibitor (MK-2206), respectively. H2O2-induced oxidative stress was blocked by antioxidant N-acetylcysteine (NAC). RESULTS: EGF treatment increased ARPE-19 cell viability and proliferation through inducing phosphorylation of EGFR and AKT. H2O2 inhibited ARPE-19 cell viability and proliferation and also suppressed EGF-stimulated increase of RPE cell viability and proliferation by affecting the EGFR/AKT signaling pathway. EGFR inhibitor erlotinib blocked EGF-induced phosphorylation of EGFR and AKT, while A66 and MK-2206 only blocked EGF-induced phosphorylation of AKT. EGF-induced phosphorylation and endocytosis of EGFR were also affected by H2O2 treatment. In addition, antioxidant NAC attenuated H2O2-induced inhibition of ARPE-19 cell viability through alleviating reduction of EGFR, and phosphorylated and total AKT proteins. CONCLUSION: Oxidative stress affects RPE cell viability and proliferation through interfering with the EGFR/AKT signaling pathway. The EGFR/AKT signaling pathway may be an important target in oxidative stress-induced RPE cell dysfunction.     

Hydrogen peroxide-induced cell death in a human retinal pigment epithelial cell line,ARPE-19

The loss of retinal pigment epithelium (RPE) with aging is related to age-related macular degeneration (AMD). This study was conducted to investigate the mechanism of hydrogen peroxide (H2O2) induced cell death in a human retinal pigment epithelial cell line, ARPE-19. Hydrogen peroxide was added at different concentrations to ARPE-19 cells and cultured. The cytotoxicity was assayed by mitochondrial function using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) testing. The patterns of cell damage were assessed using an acridine orange-ethidium bromide differential staining method, in situ end labeling (ISEL) assay and transmission electron microscopy (TEM). Catalase, a major antioxidant, was used to prevent cell death. The cleavage of procaspase 3 and poly (ADP-ribose) polymerase (PARP) was determined by western blot analysis. Hydrogen peroxide significantly induced cell death in ARPE-19 cells, whereas pretreatment of the cells with catalase prevented cell death. Application of the ISEL assay and acridine orange/ethidium bromide staining demonstrated that the H2O2-induced cell death occurred by an apoptotic mechanism at lower concentrations of H2O2 (400, 500, 600 microM), whereas higher concentrations of H2O2 induced necrosis rather than apoptosis. Caspase 3 was associated with the apoptotic pathway in human RPE cell death. Western blot analysis confirmed caspase 3 activation and cleavage of substrate proteins in ARPE-19 cells treated with an H2O2 concentration of 600 microM. These results indicate that treatment with H2O2 induces apoptotic and necrotic cell death in ARPE-19, and that caspase 3 is associated with apoptotic cell death. Therefore, H2O2 may induce the destruction of RPE cells in AMD by the combined effects of apoptosis and necrosis.     

信号转导/转录激活因子3抗ARPE-19细胞氧化应激研究

目的　本研究旨在观察信号转导／转录激活因子３（ｓｉｇｎａｌｔｒａｎｓｄｕｃｔｉｏｎ／ａｃｔｉｖａｔｉｏｎｏｆｔｒａｎｓｃｒｉｐｔｉｏｎｆａｃｔｏｒ３,ＳＴＡＴ３）对视网膜色素上皮细胞（ｒｅｔｉｎａｌｐｉｇｍｅｎｔｅｐｉｔｈｅｌｉｕｍｃｅｌｌｓ,ＲＰＥ）氧化应激损伤的保护作用,并探讨ＳＴＡＴ３在年龄相关性黄斑变性（ａｇｅ-ｒｅ-ｌａｔｅｄｍａｃｕｌａｒｄｅｇｅｎｅｒａｔｉｏｎ,ＡＭＤ）发病机制中的意义。方法　体外培养ＡＲＰＥ-１９细胞系,氧化低密度脂蛋白及Ｈ２Ｏ２干预培养细胞诱导氧化应激损伤,通过对细胞增殖、凋亡、活性氧族（ｒｅａｃｔｉｖｅｏｘｙｇｅｎｓｐｅｃｉｅｓ,ＲＯＳ）水平及细胞衰老分析研究,评估氧化应激损伤对ＲＰＥ的影响;实时荧光定量技术分析氧化应激过程中ＳＴＡＴ３-ｍＲＮＡ表达状况;ＳＴＡＴ３过表达载体转染ＡＲＰＥ-１９细胞,烟酰胺预处理细胞再经Ｈ２Ｏ２及氧化低密度脂蛋白干预后通过对增殖、凋亡、ＲＯＳ及细胞衰老状态的分析,了解ＳＴＡＴ３抗ＲＰＥ氧化应激效果。结果　与对照组相比,Ｈ２Ｏ２和氧化低密度脂蛋白能显著增加ＲＯＳ水平,促使细胞衰老增加,细胞的增殖显著下降而细胞凋亡显著上升（均为Ｐ＜０．０５）。氧化应激状况下ＳＴＡＴ３上游产物表达上升,氧化低密度脂蛋白及Ｈ２Ｏ２组荧光表达强度分别是３．３±１．２及３．５±１．１,与对照组相比差异均有统计学意义（均为Ｐ＜０．０５）;ＳＴＡＴ３能保护ＡＲＰＥ-１９细胞抗氧化应激损伤,使细胞增殖增加、凋亡减少及ＲＯＳ累积,但不造成细胞衰老状况加剧,说明ＡＲＰＥ-１９细胞衰老不受ＳＴＡＴ３调节。结论ＳＴＡＴ３在细胞氧化损伤中能独立地发挥抗氧化应激作用,提示了ＳＴＡＴ３在ＡＭＤ治疗过程中的应用前景。     

Protective effects of upregulated HO-1 gene against the apoptosis of human retinal pigment epithelial cells in vitro

AIM: To assess the correlation between disorganization of the retinal inner layers (DRIL) and best-corrected visual acuity (BCVA) in patients with uveitis and macular edema (UME) who underwent systemic treatment using optical coherence tomography (OCT). METHODS: A retrospective clinical study of 23 patients (30 eyes) with DRIL and 23 patients (31 eyes) without DRIL secondary to UME were included. All patients underwent comprehensive ophthalmic examinations at baseline, 3, 6, and 12mo after local and systemic treatment. The OCT-based parameters included: foveal center point thickness (FCPT), mean thickness (MT), and diameters of DRIL in horizontal and vertical directions. BCVA and OCT-based parameters were compared between the two groups. The relationship between each OCT parameter and BCVA was evaluated using linear correlation and regression analysis. RESULTS: At the initial visit, the mean baseline FCPT was 441.03±128.68 μm in the eyes with DRIL and 337.26±99.31 μm in the eyes without DRIL (P=0.001). No significant differences were observed in MT (P=0.357). The mean size of transverse and vertical diameters of DRIL was 684.07±267.51 μm, and 267.07±104.61 μm at baseline, respectively. There was significant improvement in BCVA and OCT-based parameters at baseline, 3, 6, and 12mo in all cases (P<0.001 for each timepoint). In addition, significant differences were detected in BCVA and OCT parameters between eyes with and without DRIL at each time point (P<0.01 for each timepoint). A greater DRIL range at baseline was associated with a worse baseline BCVA (transverse diameter of DRIL: r=0.875, P<0.001; vertical diameter of DRIL: r=0.622, P<0.001). The transverse diameter of baseline DRIL was found to be significantly correlated with the final BCVA (P=0.003). CONCLUSION: The improvement in BCVA is associated with DRIL in patients with UME. DRIL is an easy-to-determine and robust imaging biomarker that could help predict BCVA prognosis in eyes with UME.     

CERKL通过激活SIRT1/E2F1轴减轻蓝光导致的人视网膜色素上皮细胞氧化应激损伤

目的：探讨神经酰胺类似蛋白(CERKL)是否通过激活沉默信息调节因子1(SIRT1)/E2F转录因子1(E2F1)轴减轻蓝光导致的视网膜色素上皮(RPE)细胞氧化应激损伤。

方法：培养人视网膜色素上皮-19(ARPE-19)细胞，蓝光照射后观察细胞形态变化，PCR与蛋白免疫印迹法测定细胞CERKL的表达情况； 分别采用siRNA-CERKL与pcDNA3.1-CERKL转染ARPE-19细胞，蓝光暴露处理后，采用MTT法测定细胞活力，TUNEL法检测细胞凋亡情况，分析氧化应激标志物的含量与SIRT1/E2F1轴的表达情况； 随后转染siRNA-SIRT1至ARPE-19细胞，再次测定蓝光照射下细胞氧化应激损伤状况。

结果：蓝光照射后，ARPE-19细胞逐渐收缩成圆球状，且出现空泡； 蓝光照射导致CERKL表达水平升高(P<0.05)，同时观察到细胞活力降低(P<0.05)，凋亡率升高(P<0.05)，活性氧、丙二醛与8-羟基脱氧鸟苷含量升高(P<0.05)； 沉默CERKL会加剧此现象，而上调CERKL则能缓解此变化(P<0.05)； 上调CERKL同时激活了SIRT1表达，促进了E2F1的脱乙酰化(P<0.05)，而沉默SIRT1能逆转上调CERKL对蓝光导致ARPE-19细胞氧化应激损伤的缓解作用(P<0.05)。

结论：CERKL通过激活SIRT1表达，促进E2F1脱乙酰化，从而减轻蓝光诱发的ARPE-19细胞氧化应激损伤。