缺氧对人视网膜色素上皮细胞蛋白质表达的影响

目的 为揭示缺氧对视网膜色素上皮(RPE)细胞的影响及与脉络膜新生血管(CNV)生成的关系提供实验依据.方法 实验研究.分别从对数生长期的正常及缺氧状态人RPE细胞中提取蛋白样本后行等电聚焦电泳(IEF),随后采用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)进行第二次分离.采用考马斯亮蓝染色法对凝胶进行显色固定,Image Master 2D Elite软件行分析配比,筛选出正常和缺氧RPE细胞表达明显差异的蛋白质斑点,随机选取差异斑点进行质谱分析,数据经蛋白质组数据库检索得出蒡异表达蛋白.结果 正常组分离蛋白斑点578个,组内匹配率92.90%;缺氧组559个,匹配率91.41%;两组间蛋白斑点匹配率为85.47%.发现volume值改变超过2倍的点共92个,其中32个点表达卜调,60个点表达下调.选择7个蛋白斑点行质谱分析,成功鉴定5种差异表达蛋白质(3个斑点为相同的蛋白):表达上调为热休克蛋白70、热休克蛋白60;表达下调的为β肌动蛋白、β微管蛋白以及过氧化还原蛋白3.结论 缺氧状态下RPE细胞表达差异蛋白可引起细胞应激能力明显增强,而主要细胞骨架蛋白下调,细胞维持形态、保持内部结构有序性的能力下降;本研究首次发现人RPE细胞在缺氧状态下热休克蛋白60表达上调,此蛋白的进一步研究可能能够发现新的CNV致病途径;蛋白质组学方法为CNV相关疾病的研究提供了-个很好的参考平台.     

应用二维电泳法对视网膜色素上皮细胞光损伤后蛋白质组学的初步研究

目的 应用二维电泳法观察光损伤后视网膜色素上皮细胞蛋白质组的表达。 方法 运用冷白光以（2200±300）Lx光照人视网膜色素上皮细胞(ARPE 19)6 h，建立光损伤模型；提取细胞可溶性蛋白并通过二维电泳分离和凝胶图像分析，寻找光损伤细胞的蛋白质谱变化。 结果 软件分析显示全细胞可溶性蛋白在图谱上出现（390±10）个清晰斑点,11个蛋白斑点有明显表达差异。光损伤细胞内8种蛋白表达上调,1种下调，2种蛋白表达缺失。 讨论 二维电泳实验能够找出光损伤RPE细胞与正常细胞的蛋白表达差异，为进一步研究在此改变中发挥重要作用的蛋白质奠定基础。     

siRNA作用后培养的人视网膜色素上皮细胞蛋白质谱分析

目的 通过抑制人视网膜色素上皮(retinal pigment epithelium,RPE)细胞缝隙连接蛋白43(connexin 43,cx43)基因的表达,寻找差异蛋白,初步探讨ex43基因对RPE细胞生命活动的意义.方法 提取细胞总蛋白并定量分析,第一向电泳是用DH 3～10线性IPG预制胶条进行等电聚焦,第二向电泳是用SDS-PAGE凝胶再分离蛋白.银染PAGE凝胶、干燥,Powerlook 2100XL扫描凝胶.利用Image Master 2D Elite4.01图像分析软件进行分析,蛋白斑点相对分子质量和等电点采用二维校正法确定.随机选取图像分析中siRNA转染组与RPE细胞对照组凝胶图中volume值相差≥2倍的7个蛋白质斑点进行胶内酶切,经胶上原位酶切和质谱分析得到肽质量指纹图谱,查寻蛋白质数据库并鉴定差异蛋白质,分析其生物学意义.结果 RPE细胞对照组3块凝胶共分离出蛋白质斑点861个,组间匹配率达到92%;siRNA转染组3块凝胶共分离出蛋白质斑点887个,组间匹配率达到93%.经过软件图像分析,发现volume值相差≥2倍的点共78个.其中上调的16个,下调的18个.有19个点在RPE细胞对照组存在而siRNA转染组不存在,25个在siRNA转染组存在而RPE细胞对照组不存在.随机选取7个点进行蛋白质谱分析:SP-H抗原、P27BBP、有丝分裂活性蛋白激酶、次黄嘌呤核苷酸脱氢酶和热休克蛋白70表达上调,β-aetin表达下降,电压依赖阴离子通道蛋白在转染siRNA后出现表达.结论 通过蛋白质谱发现ex43基因沉默后引起多种蛋白的表达失调,ex43可能参与RPE细胞的多种生命活动.     

抑制光损伤人视网膜色素上皮细胞血管内皮生长因子A的表达对趋化因子受体3配体表达的影响

目的 观察抑制光损伤人视网膜色素上皮(RPE)细胞血管内皮生长因子A(VEGF-A)的表达对趋化因子受体3(CCR3)配体嗜酸细胞活化趋化因子(eotaxin)表达的影响.方法 体外培养人RPE细胞,取第8～12代生长良好的传代细胞用于实验.将同代细胞随机分为正常对照组、光照损伤组和光照干预组.采用波长400 nm的蓝色节能灯以(600±100) Lux持续光照RPE细胞12h建立光损伤模型,光照干预组采用0.125 mg/ml的VEGF-A受体拮抗剂雷珠单抗处理光损伤的RPE细胞;正常对照组以锡箔纸包裹细胞培养皿避光培养.结束光照24 h时,应用实时聚合酶链反应、酶联免疫吸附测定、蛋白免疫印迹法及免疫组织化学染色法检测3组细胞中VEGF-A、eotaxin-1、eotaxin-2、eotaxin-3及核因子(NF)-κB的mRNA和蛋白表达情况.结果 光照损伤组VEGF-A、eotaxin-1、eotaxin-2、eotaxin-3、NF-κB的mRNA和蛋白表达较正常对照组明显增加,差异均有统计学意义(P＜0.05).光照干预组VEGF-A、eotaxin-1、eotaxin-2、eotaxin-3、NF-κB的mRNA和蛋白表达较光照损伤组明显降低,差异也有统计学意义(P＜0.05).结论 抑制光损伤后人RPE细胞VEGF-A的表达可以下调CCR3配体eotaxin的表达,其机制可能与阻断转录因子NF-κB的激活有关.     

内质网应激对光照诱导的人视网膜色素上皮细胞损伤的促进作用

背景 视网膜色素上皮(RPE)细胞的光损伤模型是多种视网膜变性类疾病的研究工具,光诱导RPE细胞损伤的主要病理基础是凋亡及炎症反应,但是内质网应激(ERS)反应是否参与其病理机制的研究国内外少有报道. 目的 探讨ERS对光损伤诱导的人RPE细胞凋亡的作用及其机制.方法 体外培养人RPE细胞株(ARPE-19),将培养的细胞分为正常对照组及光照3、6、12和24 h组,各光照组在培养箱内以(2 000± 500) lx的白色荧光灯光照细胞建立光损伤模型,正常对照组细胞在暗环境中培养且不给予光照射,筛选实验最适光照时间.将细胞分为正常对照组、光照组(光照12h)和苯基丁酸(4-PBA)预处理+光照组,4-PBA预处理+光照组先用ERS抑制剂4-PBA培养细胞30 min,然后光照细胞12h.采用流式细胞仪检测各组人RPE细胞的凋亡率和细胞内活性氧(ROS)的荧光强度;采用ELISA法检测各组细胞上清液中炎性因子白细胞介素-1β(IL-1β)和肿瘤坏死因子-α(TNF-α)质量浓度;分别采用实时荧光定量PCR和Western blot法检测人RPE细胞中ERS标志物活化转录因子-6(ATF-6)、C/增强结合蛋白同源蛋白(CHOP)和细胞凋亡标志物半胱氨酸天冬氨酸蛋白酶-12(caspase-12) mRNA及其蛋白的表达. 结果 光照后细胞形态呈长梭形改变,边界不清,细胞质脱颗粒,细胞碎片增多,且随光照时间的延长而加重.流式细胞仪检测发现,随光照时间的延长,人RPE细胞内ROS含量逐渐增加,细胞凋亡率逐渐升高,差异均有统计学意义(F=763.00、119.30,均P＜0.01).ELISA法检测发现,与正常对照组比较,光照后6h细胞上清液中IL-1β和TNF-α质量浓度均明显升高,12h达峰.实时荧光定量PCR和Western blot检测显示,与正常对照组比较,光照后人RPE细胞中ATF-6、CHOP和caspase-12 mRNA及其蛋白的相对表达量均明显升高,均于光照后12h达峰或升高,故选择光照12 h为最适光照时间作为后续研究.4-PBA预处理+光照组细胞中ATF-6、CHOP和caspase-12 mRNA的相对表达量均明显低于光照组,差异均有统计学意义(F=281.69、473.88、308.45,均P＜0.01);ATF-6、CHOP和caspase-12蛋白的相对表达量均明显低于光照组,差异均有统计学意义(F=47.86、57.93、106.59,均P＜0.01);细胞凋亡率、细胞上清液中IL-1β和TNF-α质量浓度均明显低于光照组,差异均有统计学意义(F=88.64、245.47、101.01,均P＜0.01). 结论 (2 000±500)lx的光照可诱导人RPE细胞内ROS增加,并激活细胞的ERS反应,导致RPE细胞凋亡及炎症反应.ERS抑制剂4-PBA可抑制光损伤导致的ERS反应,进而降低RPE细胞凋亡率并抑制炎症反应过程.     

杞黄颗粒对H2O2诱导的人视网膜色素上皮细胞炎症损伤的保护作用

目的　探讨杞黄颗粒（QHG）对H₂O₂诱导的人视网膜色素上皮（RPE）细胞系ARPE-19炎症损伤的保护作用。方法　常规培养ARPE-19细胞,随机分为对照组、H₂O₂损伤组(200 μmol?L^-1 H₂O₂处理细胞）、QHG低剂量组（1 g?L^-1 QHG作用24 h后再加入H₂O₂处理细胞）和QHG高剂量组（2 g?L^-1QHG作用24 h后再加入H₂O₂处理细胞）。使用相差显微镜观察各组ARPE-19细胞形态;流式细胞仪检测各组ARPE-19细胞凋亡率,Real-time PCR检测各组ARPE-19细胞β淀粉样蛋白（Aβ)mRNA表达,ELISA检测各组ARPE-19细胞Aβ以及攻膜复合物（MAC）的蛋白表达水平。结果　相差显微镜下可见,QHG低剂量组、QHG高剂量组ARPE-19细胞形态改变均较H₂O₂损伤组轻。对照组、H₂O₂损伤组、QHG低剂量组、QHG高剂量组ARPE-19细胞凋亡率分别为（6.85±0.14）%、（43.60±1.80）%、（23.23±1.43）%、（17.90±0.78）%,H₂O₂损伤组细胞凋亡率较对照组明显增加,QHG低剂量组及QHG高剂量组细胞凋亡率均较H₂O₂损伤组明显降低,且QHG高剂量组较QHG低剂量组降低更明显,差异均有统计学意义（均为P<0.05）。与对照组相比,H₂O₂损伤组ARPE-19细胞Aβ mRNA及Aβ、MAC蛋白表达水平均升高,差异均有统计学意义（均为P<0.01）。与H₂O₂损伤组相比,QHG低剂量组及QHG高剂量组均可明显降低ARPE-19细胞Aβ mRNA 相对表达量及Aβ、MAC蛋白的表达水平,差异均有统计学意义（均为P<0.01）。结论　QHG能有效抑制H₂O₂诱导的人RPE细胞凋亡,减少细胞Aβ及MAC表达,从而起到保护人RPE细胞的作用。     

地塞米松及Lat—A对人眼小梁细胞蛋白质表达的影响

目的探讨地塞米松（Dex）及latrunculin—A（Lat-A）处理后人眼小梁细胞蛋白质表达的变化。方法正常供体人眼小梁细胞培养后用Dex及Lat—A处理,经二维凝胶电泳分离小梁细胞蛋白质,分析凝胶电泳图谱,选取部分差异凝胶斑点并采用基质辅助激光解析电离飞行时间质谱（MALDI—TOFMS）鉴定蛋白质。结果采用24cm IPG胶条获得正常人眼小梁细胞对照组、Dex组、Dex＋Lat—A组以及Lat—A组的二维电泳凝胶图谱,得出不同培养条件下人眼小梁细胞蛋白质表达的差异。应用GDPi MALDI—TOFMS得出不同培养条件下差异表达的蛋白质斑点并成功鉴定出其中的24种蛋白质,主要包括细胞骨架相关的蛋白、热休克蛋白、氧化还原相关的蛋白和糖代谢相关的蛋白4类蛋白质。ADLH和Rab蛋白在Lat—A组的人小梁细胞中表达增加,但在Dex组表达减少,而热休克蛋白27（HSP27）和人CRMP2呈现相反的结果。结论成功建立Dex及Lat—A诱导人眼小梁细胞蛋白质表达图谱。Dex及Lat—A能诱导人眼小梁细胞的蛋白质表达变化,可能与青光眼致病的分子机制相关。     

年龄相关性白内障和正常晶状体蛋白质组的差异分析

目的 运用双向电泳和基质辅助的激光解吸电离飞行时间质谱（matrix-assisted laser desorption/ionization time-of-flight mass spectrometry,MALDI-TOF-MS）的方法,鉴定年龄相关性白内障和正常晶状体之间差异表达的蛋白质.方法 用固相pH梯度双向凝胶电泳技术分离16例年龄相关性白内障和正常晶状体之间的总蛋白质,建立二维电泳图谱,再利用MALDI-TOF-MS分析及数据库搜索相结合对差异表达的蛋白质点进行鉴定.结果 双向电泳胶图显示年龄相关性白内障和正常晶状体共有84个差异点,其中52个上调表达,32个下调表达.有和无的差异表达点10个,相差3倍以上差异表达点10个.利用MALDI-TOF-MS对差异表达的15个蛋白质点进行肽指纹图谱分析,经数据库检索后鉴定出5个蛋白质.结论 年龄相关性白内障和正常晶状体之间蛋白表达存在差异,蛋白质组的分析结果为年龄相关性白内障的基础研究提供依据和新实验方法.     

曲安奈德对色素上皮细胞衍生因子表达的影响

目的 观察曲安奈德(TA)对人视网膜色素上皮细胞衍生因子(PEDF)表达的影响.方法 体外培养人视网膜色素上皮(RPE)细胞,取第4～6代细胞用于实验.分别以浓度为40、400、4×103、4×104μg/L的TA干预12、24、48 h后,采用蛋白质免疫印迹法(Western blot)检测培养液及细胞浆中PEDF蛋白表达水平.采用20 ng/ml的肿瘤坏死因子-α(TNF-α)预干预RPE细胞24 h后,再加入400μg/L的TA进行干预(TNF-α预处理组);分别选用20 ng/ml的TNF-α和400μg/L的TA单独干预RPE细胞作为单独干预组.分别作用1、6、24 h后,测定3组细胞培养液及细胞浆中PEDF及细胞浆中磷酸化p38丝裂原活化蛋白激酶(p-p38MAPK)的蛋白表达水平.结果 各TA浓度组RPE细胞培养液及细胞浆中PEDF蛋白表达均升高,其中400μg/L组PEDF蛋白表达最高(P＜0.05).干预1、6、24 h时,TNF-α预处理组和TA单独干预组PEDF蛋白表达均增高、p-p38MAPK蛋白表达均降低(P＜0.01);TNF-α单独干预组PEDF蛋白表达降低、p-p38MAPK蛋白表达增高(P＜0.01).结论 TA能够上调体外培养的人RPE细胞PEDF蛋白表达,下调p-p38MAPK蛋白表达.     

光损伤后人视网膜色素上皮细胞血管内皮生长因子A及其受体的表达

目的 观察光损伤后人视网膜色素上皮(RPE)细胞血管内皮生长因子A(VEGF-A)及其受体可溶性fm样酪氨酸激酶受体(sFlt-1)和包含激酶植入区域受体(KDR)的表达.方法 体外培养人RPE细胞,取第8～12代生长良好的传代细胞用于实验.将同代细胞随机分为对照组和光损伤组.以18 W冷白色荧光灯作为光源,自制光照器.采用双层高压消毒锡纸包裹对照组细胞,与光损伤组细胞一同置于自制光照器下,控制光照强度在(2200±300) Lux,持续光照12 h建立光损伤模型.于光损伤后0、6、12、24 h终止培养,采用逆转录聚合酶链反应(RT-PCR)和蛋白免疫印迹法(Western blot)检测VEGF-A、sFlt-1及KDR的mRNA及蛋白表达.结果 光损伤组VEGF-A mRNA及蛋白表达在光损伤后6h时明显增加,12h时达到高峰,明显高于对照组(t=2.74,2.93;P＜0.05),随后降低.sFlt-1的mRNA及蛋白表达在光损伤后12h达到高峰,明显高于对照组(t=4.32,P＜0.01);24 h时明显低于对照组(t=2.41,P＜0.05).KDR的mRNA及蛋白表达在光损伤后6、12 h时较对照组无明显变化(t=1.84,P＞0.05),24 h时高于对照组(t=2.89,P＜0.05).结论 光损伤后6、12h时,RPE细胞VEGF-A及sFlt-1的表达明显增高,KDR的表达相对稳定.光损伤后24 h时,RPE细胞VEGF-A及sFlt-1的表达降低,KDR的表达增高.     

Effect of EGb761 on light-damaged retinal pigment epithelial cells

AIM:To investigate the protective mechanism of Gingko Biloba extract (EGb761) on the ability of retinal pigment epithelial (RPE) cells to resist light-induced damage in a comparative proteomics study.METHODS:Human RPE cells (ARPE-19) were randomly distributed to one of three groups:normal control (NC group) and light-damaged model without or with EGb761 group (M and ME groups, respectively). The light-damaged model was formed by exposing to white light (2 200±300)lx for 6h. The RPE cells in ME group were conducted with EGb 761 (100μg/mL) before light exposure. The soluble cellular proteins extracting from each groups were separated by two-dimensional electrophoresis and stained by silver staining. Different proteins in the profiles of the gels were analyzed by Image Master Software. Two-fold expressing protein spots were identified by Matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) mass spectrometry.RESULTS: NC, M and ME groups displayed 1 892±71, 2 145±23 and 2 216±85 protein spots, respectively. We identified 33 proteins with different expression levels between the NC and M groups, 25 proteins between the M and ME groups, and 11 proteins between the NC and ME groups. MALDI-TOF/TOF mass spectrometry successfully identified 16 proteins, including metabolic enzymes, cytoskeletal proteins, anti-oxidation proteins, and others.CONCLUSION:Differences in some important proteins, such as cathepsin B, heat shock protein, and cytochrome creductase, indicated that multiple pathways may be induced in light-damaged RPE cells and the protective effect of EGb761.     

银杏叶提取物对视网膜神经节细胞保护作用的实验研究

目的探讨银杏叶提取物(EGb761)对大鼠视神经夹伤模型视网膜神经节细胞(RGC)的保护作用。设计实验研究。研究对象SPF级SD大鼠83只。方法右眼行视神经夹伤,于球后2mm处用40克压力微型视神经夹夹持视神经60秒,左眼作为正常对照。夹伤后2小时及此后每日一次灌胃给药,各组分别给予生理盐水5ml/kg(n=18)、1%灯盏细辛5ml/kg(n=16)、0.25% EGb761 5ml/kg(n=15)、1% EGb761 5ml/kg(n=18)和4% EGb761 5ml/kg(n=16)。视神经夹伤后第23天用荧光金作上丘逆行标记,第28天取眼球标本做视网膜铺片并拍摄照片,计数RGC并计算RGC的存活率。主要指标RGC存活率。结果生理盐水组、灯盏细辛组、0.25% EGb761组、1% EGb761组和4% EGb761组RGC存活率分别为60.59%、72.21%、69.10%、71.60%和74.20%。各剂量EGb761组与生理盐水组之间均有显著性差异(F=11.33,P<0.01),灯盏细辛组与生理盐水组之间也有显著性差异(P<0.01)。不同浓度EGb761各组之间无显著性差异(F=2.25,P>0.05),灯盏细辛组与不同浓度EGb761各组之间均无显著性差异(F=1.16.P>0.05)。结论EGb761能有效保护大鼠视神经夹伤模型的RGC。(眼科,2007,16:418-421)     

Functional protection of photoreceptors from light-induced damage by dimethylthiourea and Ginkgo biloba extract

PURPOSE: To investigate the functional protective effect of a synthetic (dimethylthiourea, DMTU) and a natural antioxidant (Ginkgo biloba extract, EGb 761) against light-induced retinal degeneration. METHODS: Wistar rats were exposed for 24 hours to 1700-lux light after treatment with DMTU or EGb 761. Electroretinograms were recorded before and on day (D)1, D3, D8, D15, D22, and D29 after light exposure. The b-wave amplitude was plotted against log L (ganzfeld luminance), providing the b-wave sensitivity curve. The Naka-Rushton function fitted to the sensitivity curve enabled derivation of the parameters Bmax (saturated amplitude) and K (luminance-inducing Bmax/2). In addition, rats from each group were killed for retinal morphometric analyses. RESULTS: In the untreated group, light exposure caused collapse of the b-wave sensitivity curves. Bmax was reduced by 51% at D1 without subsequent recovery. K increased temporarily, reverting to normal values 8 days later. The outer nuclear layer thicknesses decreased markedly in the superior retina. In the treated groups, light exposure had a weaker effect on sensitivity curves. The values of Bmax were not significantly different from those in the unexposed-untreated group, although K increased temporarily. Retinal morphometry was preserved. CONCLUSIONS: Dimethylthiourea and EGb 761 afford functional protection against light-induced retinal damage.     

Intraperitoneal injection of Ginkgo biloba extract enhances antioxidation ability of retina and protects photoreceptors after light-induced retinal damage in rats

PURPOSE: To investigate the effect of Ginkgo biloba extract EGb 761, a free-radical scavenger, on the antioxidation capability of retina after light-induced retinal damage in rats in an attempt to understand the mechanism by which EGb 761 protects the photoreceptors after light-induced retinal damage. METHODS: Seventy-two female Sprague-Dawley (SD) rats were evenly randomized into normal control group (NC group), light-induced retinal damage model group (M group), model + normal saline group (MN group), and model + EGb 761 group (ME group). Light-induced retinal damage model was induced via exposure to white light at 2740 +/- 120 lux for 6 hr. Rats in MN group and ME group were intraperitoneally injected daily with normal saline and 0.35% EGb 761 (100 mg/kg), respectively, 1 week before and 2 weeks after light exposure. The levels of malondialdehyde (MDA), total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) in the retinal tissues were determined 24 hrs after light exposure; photoreceptor apoptosis was detected 4 days after light exposure. One and 2 weeks after light exposure, histopathologic examination was carried out, and the outer nuclear layer (ONL) thickness (number of nuclei) in the superior and inferior retina was counted. RESULTS: Twenty-four hours after exposure, the MDA levels in the other three groups were significantly higher than that in the NC group (p < 0.05); those in the M and MN groups were similar to each other (p > 0.05); and that of the ME group was significantly lower than those in the M and MN group (p < 0.05). The activities of T-SOD, GSH-Px, and CAT were similar in the M and MN groups (p > 0.05); the activities in the M and MN groups were significantly lower than those in the NC and ME groups (p < 0.05); and the activities in the ME group were significantly higher than those in the M and MN groups (p < 0.05). Four days after exposure, the apoptotic photoreceptors within the ONL in the ME group were obviously fewer than those in the M and MN groups. One week and 2 weeks after exposure, the ONL thickness (number of nuclei) in the ME group was more than that in the M and MN groups but less than that in the NC group. CONCLUSIONS: Intraperitoneal injection of EGb 761 can enhance the antioxidation ability of retina and partially inhibit the apoptosis of photoreceptors, thus exert a protective effect on photoreceptors.     

The Ginkgo biloba extract (EGb 761) provides a neuroprotective effect on retinal ganglion cells in a rat model of chronic glaucoma

PURPOSE: To investigate the effect of Ginkgo biloba extract (EGb 761) against neurotoxicity of retinal ganglion cells of rats with chronic moderately elevated intraocular pressure (IOP). METHODS: Unilateral chronic moderately elevated IOP was produced in rats by cautery of three episcleral vessels. Secondary degeneration was measured with and without EGb 761 for 5 months. At 5 months, retinal ganglion cells were labeled with a fast blue tracer applied to both superior colliculi. Densities of surviving retinal ganglion cells were estimated by counting fast blue labeled cells in whole mounted retinas. RESULTS: When compared with their contralateral control eyes with normal IOP, in the peripheral retina, retinal ganglion cell loss in eyes with chronic, moderately elevated IOP was 29.8 +/- 1.5% (n=5) at 5 months in untreated animals and 4.6 +/- 4.5% (n=5) at 5 months in treated animals with EGb 761. CONCLUSIONS: Pretreatment and early posttreatment with EGb 761 is an effective neuroprotectant in a rat model of chronic glaucoma.     

Molecular and cellular assessment of ginkgo biloba extract as a possible ophthalmic drug

We have investigated the biochemical and cell biological basis of the reported beneficiary effects of the leaf extracts of the plant Ginkgo biloba, which has been used as a possible ophthalmic drug. The antioxidant, antimicrobial, anti-apoptotic and cytoprotective properties of the standardized extract called EGb761 were assayed. Chemical stresses were induced in cells using alloxan or dexamethasone, and the effect of EGb761 on them was studied using the MTT and TUNEL assays. Its ability to modulate the activities of some antioxidant enzymes was tested in vitro. In addition, cataract was induced in rats through selenite injection, and the effect of EGb761 administration on the progression of cataract was studied using slit lamp examination. Ginkgo biloba was found to be an excellent antioxidant. It readily scavenges reactive oxygen and nitrogen radicals and inhibits oxidative modifications that occur to proteins in vitro. It enters intact cells and protects them from alloxan-mediated and light-mediated stress, and the nuclear DNA from single strand breaks. It also effectively inhibits chemically induced apoptosis. It does not modulate the activities of endogenous antioxidant enzymes, nor does it have any significant antimicrobial activity. Unlike some other plant extracts, it is not phototoxic. In experiments wherein selenite cataract was induced in laboratory rats, treatment with the extract significantly retards the progression of lens opacification in vivo. Ginkgo biloba's inherent antioxidant, antiapoptotic and cytoprotective action and potential anticataract ability appear to be some of the factors responsible for its beneficial effects.     

银杏叶提取物对大鼠视网膜光损伤后感光细胞的保护作用

目的:探讨银杏叶提取物（EGb 761）对视网膜光损伤后感光细胞 的保护作用。 方法:72只Sprague-Dawley(SD)大鼠随机分为正常对照组 、模型组、模型+生理盐水组和模型+ EGb 761组，每组 各18只大鼠。模型组、模型+生理盐水组和模型+EGb 761组暗适应24 h后持续光照6 h，光照强度为（2740±120）lx ，建立光损伤模型。模型+EGb 761组和模型+生理盐水组分别于光照前7 d每日腹腔注射0.35 % EGb 761（100 mg/kg）和相应体积的生理盐水，光照后继续给药14 d；于光照后4 d对 各组进行视网膜原位凋亡细胞检测，并于光照后7、14 d作组织病理学检查并计数视盘 上、下方视网膜外核层（ONL）感光细胞层数。 结果:光照后4 d，除 正常对照组外，其余 各组ONL均出现凋亡感光细胞，但模型+ EGb 761组ONL凋亡感光细胞数明显少于模型组和模 型+生理盐水组。光照后7 d，模型组和模型+生理盐水组感光细胞核层数均为3~4层，模型+ EGb 761组为7~8层；模型+ EGb 761组平均感光细胞核层数（6.92 ± 0.82）少于正常对照 组（8.40±0.95）（t=-1.416，P＜0.05），但显著多于模型组（5.96±1.36）和 模型+生理盐水组（5.90±1.40）（t=1.024，1.084；P＜0 .05）。光照后14 d，模型 组和模型+生理盐水组感光细胞核为0~1层，而模型+ EGb 761组仍存有3~4层；模型+ EGb 76 1组平均感光细胞核层数（5.52±1.06）仍显著多于模型组（3.44±2.15）和模型+ 生理 盐水组（3.37±1.91）（t=2.082，2.146；P＜0.05）。 结论:EGb 761能部分抑制视网膜光损伤后感光细胞凋亡，对感光细胞具 有一定的保护作用。     

Influence of Ginkgo biloba on ocular blood flow

PURPOSE: To investigate the effect of Ginkgo biloba extract (EGb761) on ocular blood flow. METHODS: This randomized, double-masked, placebo-controlled, two-way crossover study included 15 healthy male volunteers. Measurements were taken with laser Doppler flowmetry, laser Doppler velocimetry, a retinal vessel analyser, laser interferometry and applanation tonometry, before and up to 3 hours after oral intake of 240 mg EGb761. RESULTS: At baseline, no significant differences in ocular and systemic haemodynamic parameters were observed between the two study days. Ginkgo biloba significantly decreased retinal venous diameters (p < 0.05 versus baseline), but there was no significant difference between the two groups. Blood pressure, retinal arterial and venous diameters, choroidal blood flow, fundus pulsation amplitude, intraocular pressure and retinal blood flow remained unchanged in both groups and did not differ between groups. Optic nerve head blood flow significantly increased in response to Ginkgo biloba (p < 0.002 versus baseline), but this effect was not significant compared with that of placebo. CONCLUSIONS: The results of this study indicate that a single administration of Ginkgo biloba does not influence ocular blood flow to a relevant degree. Whether the drug may influence ocular blood flow in patients with ocular vascular disease after longterm treatment remains to be investigated in a randomized, placebo-controlled clinical trial.     

银杏叶提取物增强大鼠视网膜光损伤模型抗氧化应激能力

目的研究银杏叶提取物(extract of Ginkgo biloba,EGb761)对大鼠视网膜光损伤后抗氧化应激状态的影响,探讨EGb761对视网膜光损伤后感光细胞的保护机制。方法 24只雌性SD大鼠随机分为正常对照(NC)组、模型(M)组、模型+生理盐水(MN)组和模型+EGb761(ME)组,每组6只。MN组和ME组分别于造模前1周腹腔注射相应体积的生理盐水和3.5g·L-1EGb761(100mg·kg-1);于光照造模后24h进行神经视网膜组织丙二醛(malondialdehyde,MDA)、总超氧化物歧化酶(total superoxide dismutase,T-SOD)、谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)、过氧化氢酶(catalase,CAT)检测。结果光照24h后M组、MN组和ME组视网膜MDA水平分别为(33.87±1.19)nM·g-1、(31.13±1.50)nM·g-1、(24.33±3.23)nM·g-1,均高于NC组的(20.00±1.99)nM·g-1,差异均有统计学意义(均为P<0.05)。M组和MN组之间MDA水平相近,差异无统计学意义(P>0.05);ME组MDA水平显著低于M组和MN组,差异均有统计学意义(均为P<0.05);M组和MN组T-SOD、GSH-Px、CAT活性均相近,差异均无统计学意义(均为P>0.05),均显著低于NC组和ME组,差异均有统计学意义(均为P<0.05),而ME组T-SOD、GSH-Px、CAT活性均低于NC组,差异均有统计学意义(均为P<0.05)。结论腹腔注射银杏叶提取物EGb761能增强光损伤后视网膜抗氧化应激能力,从而保护视网膜光损伤后光感受器细胞。     

Rolle des Energiestoffwechsels im retinalen Pigmentepithel

The universal energy source adenosine triphosphate (ATP)is reduced by approximately 30 % in the retinal pigment epithelium (RPE) of elderly persons. Increased oxidative stress and decreased antioxidative capacity, such as glutathione in aging eyes cause impairment of energy-dependent RPE processes and lead to loss of visual function. We developed a cell culture model of aging RPE using atractyloside to inhibit mitochondrial ATP synthesis and tert-butyl hydroperoxide as oxidant. The ATP levels were reduced by 30 % and oxidative damaged proteins and DNA increased whereas antioxidative glutathione decreased. Autophagy as an internal cellular repair mechanism and phagocytosis of photoreceptors were impaired. Antioxidative and mitochondria-activating Ginkgo biloba extract EGb 761® increased the intracellular ATP level and antioxidative glutathione. This cell culture model seems to be suitable to investigate in vitro the effect of protective substances and their compounds on aging processes in RPE.