Superoxide dismutase,catalase and glutathione peroxidase in the human cataractous lens

The activities of the protective enzymes, superoxide dismutase, catalase and glutathione peroxidase have been measured in the cortical and nuclear sections of 76 human cataractous lenses as well as in calf, rabbit and rat lenses.No changes was observed in the activity of catalase with the progressive development of cataract. However, a precipitous decrease (70%) in both superoxide dismutase and glutathione peroxidase in the nuclear region of the lens was found at the onset of nuclear cataract. Further decreases accompanied the progression of the cataract and similar, but less marked, decreases were observed in the cortical region of the lens.It is suggested that the inactivation of these enzymes may result in an elevation of the H₂O₂ and O₂^.? levels in the lens and that this may be responsible for the oxidative modification of lens proteins observed in nuclear cataracts.     

阿司匹林对半乳糖性白内障抑制作用的实验研究

目的观察阿司匹林对大鼠半乳糖性白内障的抑制作用。方法将60只Wistar大鼠分为3组：半乳糖组每日腹腔注射80%的D-半乳糖（20mL/kg）,连续10d,制成白内障动物模型;阿司匹林组同半乳糖组处理的同时每日给予阿司匹林混悬液150mg/kg灌胃至实验结束;对照组无特殊处理。实验前及造模起第3、6、10、14、20天行裂隙灯显微镜观察晶状体情况并拍照;造模起第5天各组随机处死8只大鼠,右眼晶状体匀浆检测超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-PX）、过氧化氢酶（CAT）的活性,左侧晶状体行扫描电镜观察并定量分析。结果对照组晶状体始终透明,实验第3、6、10、14、20天阿司匹林组白内障的发生率分别为0、25%、41.67%、58.33%、83.33%,大多数为囊泡初期,而半乳糖组第3天白内障发生率达65%,第6天后晶状体均发生混浊,最终发展为成熟期白内障;实验第5天扫描电镜下见对照组组织结构正常,半乳糖组损伤严重,阿司匹林组损伤较轻微;与对照组比较,半乳糖组SOD、GSH-PX、CAT活性明显降低（P〈0.05）,阿司匹林组各酶活性强于半乳糖组（P〈0.05）。结论阿司匹林能增强晶状体中SOD、GSH-PX、CAT的活性,对大鼠半乳糖性白内障有抗氧化作用,从而延缓早期白内障的发生发展。     

糖尿病性白内障大鼠晶状体谷胱甘肽含量的变化

目的:分析谷胱甘肽和糖尿病性白内障的相关性。方法:复制链脲佐菌素(streptozocin,STZ)糖尿病大鼠模型,成模后采用裂隙显微镜观察并记录晶状体混浊的程度,使用二硫硝基苯法测定糖尿病性白内障大鼠晶状体中谷胱甘肽的含量。结果:与正常大鼠相比糖尿病性白内障大鼠晶状体中谷胱甘肽的含量降低70%(P<0.05),它们之间差异具有统计学意义。并且糖尿病性白内障大鼠晶状体中谷胱甘肽的含量与它的晶状体混浊程度呈负相关(rs=-0.783)。结论:随着糖尿病性白内障的发生和进展,晶状体中的谷胱甘肽含量明显减少。     

槲皮素对H2O2诱导人RPE细胞氧化应激损伤的保护作用

目的：探讨槲皮素对H2 O2诱导的人视网膜色素上皮细胞( retina pigment epithelium,RPE)氧化应激损伤的保护作用及可能机制。
　　方法：RPE细胞传代培养,分为阴性对照组：以正常培养液培养;氧化损伤组：100μmol/L的H2 O2作用12h;槲皮素低浓度组：100μmol/L 槲皮素孵育24h 后,加入 H2 O2作用12 h;槲皮素高浓度组：500μmol/L槲皮素孵育24 h后,加入H2 O2作用12h。 MTT比色法检测细胞活力,流式细胞仪检测细胞凋亡率, Hochest33258染色观察凋亡细胞形态,比色法检测细胞中过氧化氢酶( catalase,CAT)、超氧化物歧化酶( superoxide dismutase,SOD)和谷胱甘肽过氧化物酶( glutathione peroxidase,GSH-Px)活性。
　　结果：槲皮素能明显抑制H2 O2诱导的RPE细胞活力的下降,用不同浓度槲皮素处理后,RPE细胞活性分别提高到79.67%±4.98%和83.00%±3.60%,与氧化损伤组(48.93%±3.39%)比较,差异具有统计学意义(P<0.05);经不同浓度槲皮素处理后, RPE细胞凋亡率分别下降至23.23%±3.29%和16.23%±1.94%,与氧化损伤组(38.03%±4.76%)比较,差异具有统计学意义(P<0.05);此外,槲皮素还能增加细胞中CAT、SOD、GSH-Px活性,与氧化损伤组比较,差异均具有统计学意义(P<0.05)。
　　结论：槲皮素通过改善细胞中抗氧化酶活性有效抑制了H2 O2对RPE细胞的损伤,从而为其用于治疗RPE细胞损伤提供可靠的实验±据。     

中药对大鼠晶状体老化过程中脂质过氧化和抗氧化能力的干预作用

目的:分析抗氧化中药参乌胶囊(Shenwu capsule,SW)、二苯乙烯苷(tetrahydroxystilbene glucoside,TSG)对晶状体老化过程中脂质过氧化及抗氧化能力的影响。方法:雄性SD大鼠96只,分为增龄组及中药干预组,增龄组按月龄不同分为1,3,6,12,18,24共6组,每组10只;中药干预组分为参乌胶囊低剂量[SW(L)]、高剂量[SW(H)]、二苯乙烯苷低剂量[TSG(L)]、高剂量[TSG(H)]4组,分别为10,8,10,8只;药物干预组大鼠均为24mo龄,从21mo龄开始,SW(L),SW(H)组分别给予参乌胶囊0.8g/kg和1.6g/kg灌胃;TSG(L),TSG(H)组分别给予TSG0.03g/kg和0.06g/kg灌胃;老年对照组给予等体积蒸馏水灌胃,每日1次,至24mo,共3mo。处死动物取出眼球,自赤道部切开眼球,完整取出晶状体,采用硫代巴比妥酸比色法与化学发光法检测各组大鼠晶状体组织中谷胱甘肽(glutathione,GSH)、超氧化物岐化酶(superoxide dismutase,SOD)、丙二醛(malondialdehyde,MDA)含量。结果:大鼠晶状体GSH含量1,3,6,12,18,24mo龄、SW(L),SW(H),TSG(L),TSG(H)分别为30.5±7.4,33.3±6.1,29.0±3.1,28.6±4.8,22.0±4.2,20.5±3.2,21.9±3.2,23.1±5.1,20.2±4.4,18.8±2.5mg/gpr;增龄组GSH含量F检验P<0.01,GSH含量与月龄负相关(P<0.01),各用药组与24mo龄组GSH含量F检验P>0.05。大鼠晶状体SOD含量1,3,6,12,18,24mo龄、SW(L),SW(H),TSG(L),TSG(H)分别为41.1±2.1,42.3±2.7,38.6±3.0,38.7±5.2,34.1±1.8,31.9±4.9,30.2±3.2,32.7±3.3,29.8±3.3,32.5±6.0μkat/gpr;增龄组SOD含量F检验P<0.01,SOD含量与月龄负相关(P<0.01),各用药组与24mo组SOD含量F检验P>0.05。大鼠晶状体MDA含量1,3,6,12,18,24mo龄、SW(L),SW(H),TSG(L),TSG(H)分别为49.6±4.4,52.2±3.8,53.1±6.4,53.6±4.7,59.8±4.2,62.2±3.9,61.6±2.9,63.6±5.8,58.1±13.6,57.9±7.6nmol/gpr;增龄组MDA含量F检验P<0.01,MDA含量与月龄正相关(P<0.01),各用药组与24mo组MDA含量F检验P>0.05。结论:大鼠晶状体GSH,SOD及MDA随年龄增长分别呈降低、降低、升高趋势,抗氧化能力下降之后抗氧化药物对此无预防作用。     

决明退障丸对亚硒酸钠性白内障大鼠晶状体中谷胱甘肽过氧化物酶和6-磷酸葡萄糖脱氢酶活性的影响

目的 探讨中药决明退障丸对硒性白内障大鼠晶状体脂质过氧化反应的抑制作用及其机制，寻求决明退障丸治疗老年性白内障的疗效机制。方法 参照张家萍等学者亚硒酸钠性白内障大鼠模型制作方法和不同时段（造模10天、20天、30天、40天）晶状体混浊程度裂隙灯显微镜分级记分，以及化学比色法测定晶状体谷胱甘肽过氧化物酶（GSH～Px）、6-磷酸葡萄糖脱氢酶（G-6-PD）含量的方法，对80只Wistar大鼠随机分为正常对照组、模型对照组、中药预防组和中药治疗组同时进行中药预防和治疗作用的比较研究。结果 注射亚硒酸钠后，大鼠晶状体混浊程度分级记分不断增高，GSH-Px、G-6-PD活性逐渐降低，与正常晶状体比较差异有显著性（P〈0．001或P〈0．05）。中药预防组和中药治疗组晶状体混浊程度分级记分明显低于模型对照组，而GSH-Px、G-6-PD活性则明显高于模型对照组（P〈0．05或P〈0．001），其中以中药预防组作用优于治疗组（P〈0．05），其造模40天的GSH-Px、G-6-PD活性接近正常对照组水平（P〉0．05）。结论 决明退障丸具有明显的阻抑大鼠晶状体混浊发生发展的作用，能显著提高和恢复GSH-Px、G-6-PD活性，增强晶状体的抗氧化能力，阻止氧自由基连锁反应，抑制硒性白内障的形成，以早期预防用药作用更为突出，决明退障丸防治白内障的作用机制可能与其提高晶状体抗氧化损伤能力，清除氧自由基，抑制脂质过氧化反应有关。     

谷胱甘肽乙酯滴眼液防治大鼠糖尿病性白内障的研究

目的:探讨谷胱甘肽乙酯滴眼液抑制糖尿病性白内障的效果及作用机制。方法:复制链脲佐菌素(streptozocin,STZ)糖尿病大鼠模型,成模前药物治疗组每日给予2次谷胱甘肽乙酯滴眼液治疗,而对照组给予磷酸钠缓冲液。采用英国牛津大学晶状体分级方法记录晶状体混浊的程度。并于实验过程中观察大鼠的一般状况,了解还原型谷胱甘肽乙酯对糖尿病大鼠一般状况的影响。结果:糖尿病诱导的晶状体混浊进展呈双向趋势:前6wk缓慢增长,后7wk快速进展。治疗组大鼠的混浊进展相对于未治疗组较慢,并且在第4wk时两者差别具有统计学意义(治疗组vs未治疗组,P<0.05)。同时药物治疗组大鼠与未治疗组相比较,精神、毛色等一般状况较好。结论:谷胱甘肽乙酯滴眼液抑制了早期晶状体混浊的进展,它作为合成谷胱甘肽的前体,进而发挥其抗白内障的作用。     

Effect of alpha-ketoglutarate against selenite cataract formation

We have previously shown that pyruvate protects against reactive oxygen species (ROS) induced damage to lens in vitro. It has also a significant effect against cataract development. Its effectiveness has been ascribed to the presence of alpha-keto-carboxylate group in the molecule, acting as a scavenger of ROS. Hence, it was felt desirable to determine if other alpha-keto-acids could have similar effects. These studies have hence been conducted with alpha-ketoglutarate (alpha-KG), a compound with greater stability and without any known significant effect on the glycolysis. Its effectiveness has been assessed by monitoring cataract development in rat pups given sodium selenite. A large percentage of such animals (about 80%) developed nuclear opacity 7-8 days after its administration. In animals treated with alpha-ketoglutarate, the incidence of cataracts was only 23%. The agent therefore has a very substantial anticataractogenic effect, as apparent by direct slit lamp examination followed by photography, as well as by examination of the isolated lenses through transillumination. The significance of the ophthalmologic findings was apparent also by better physiological maintenance of the tissue, reflected by higher levels of ATP and GSH. In view of these in vivo beneficial effects, studies are in progress to identify the biochemical and metabolic sites of its action. Whether the effectiveness is related only to its action as a ROS scavenger or it could be contributed also by some metabolic effects independent of ROS remains to be determined.     

DNA氧化损伤修复基因ERCC6对过氧化氢诱导的氧化损伤的晶状体上皮细胞增殖和凋亡的影响

目的　探讨DNA氧化损伤修复基因ERCC6对过氧化氢（H₂O₂）诱导的氧化损伤的晶状体上皮细胞增殖和凋亡的影响。方法　采用不同浓度H₂O₂处理SRA01/04细胞,构建氧化损伤模型。取氧化损伤的SRA01/04细胞分为H₂O₂组、H₂O₂+空白质粒转染组和H₂O₂+pcDNA3.1-ERCC6质粒转染组进行转染处理。采用实时荧光定量PCR和免疫印迹实验检测转染后氧化损伤的SRA01/04细胞中ERCC6 mRNA和科凯恩综合征互补B蛋白（CSB）的表达变化。采用免疫印迹实验和EdU染色实验分别检测转染后各组SRA01/04细胞凋亡相关蛋白表达变化和细胞增殖能力。结果　当H₂O₂处理浓度为200 μmol·L^-1时, SRA01/04细胞ERCC6 mRNA和CSB蛋白的相对表达量最低,后续转染实验H₂O₂处理浓度均采用200 μmol·L^-1。与H₂O₂组和H₂O₂+空白质粒转染组相比,H₂O₂+pcDNA3.1-ERCC6质粒转染组SRA01/04细胞ERCC6 mRNA和蛋白表达水平均显著增高（均为P<0.05）。进一步实验发现,与另外两组相比,H₂O₂+pcDNA3.1-ERCC6质粒转染组SRA01/04细胞中的促凋亡蛋白Bax表达均明显降低,而抑制凋亡的蛋白Bcl-2表达则均明显增加（均为P<0.05）。EdU染色实验检测结果显示,与H₂O₂+空白质粒转染组相比,H₂O₂+pcDNA3.1-ERCC6质粒转染组SRA01/04细胞内绿色荧光亮度增高,阳性细胞数量增加,表明SRA01/04细胞的增殖能力增强。结论　ERCC6基因可能通过减弱DNA的核苷酸切除修复作用抑制氧化损伤的晶状体上皮细胞增殖、促进其凋亡从而导致老年性白内障的发生。     

老年性白内障患者房水中氧化应激物质含量的测定

目的测定老年性白内障患者房水中的氧化应激物质的含量。方法收集59例老年性白内障患者并测定患者房水中蛋白质的浓度,超氧化物歧化酶(superoxide dis-mutase,SOD)、过氧化氢酶(catalase,CAT)、谷胱甘肽过氧化物酶(glutathione-peroxide,GSH-PX)的活性。结果 59例患者中术前蛋白质的浓度、SOD、CAT、GSH-PX的活性平均分别为(2.572±0.176)g·L-1、(0.156±0.180)U·mL-1、(1.118±0.015)U·mL-1、(0.062±0.022)U·mL-1,术前对数视力是0.402±0.070。随着白内障的核硬度的分级增加可以观察到蛋白质的浓度、SOD、GSH-PX的活性显著性增加(P<0.05),没有发现明显年龄相关性氧化应激物质的差异。结论随着白内障核硬度的分级增加,蛋白质浓度、SOD、GSH-PX的活性显著性增加,而不是随着患者的年龄增加而增加。表明白内障进展中大分子物质蛋白质、SOD是从晶状体囊分泌的。     

白内障病人晶状体上皮细胞DNA损伤初探

目的欲研究白内障病人晶状体上皮细胞是否存在DNA损伤及损伤的程度.方法用单细胞电泳测定白内障手术中取下的白内障病人晶状体上皮细胞DNA损伤的情况.结果11例白内障病人晶状体上皮细胞中有6例确实存在DNA损伤,占54.54%.白内障病人晶状体上皮细胞中DNA损伤程度与年龄无关.每例白内障病人晶状体上皮细胞中DNA损伤程度的分布是轻＞中＞重.结论白内障的发生与晶状体上皮细胞DNA损伤有关,但只是众多诱因之一.     

糖尿病性白内障患者血清和房水中MDA与SOD的变化

目的:观察糖尿病性白内障(diabetic cataract,DC)患者血清和房水中丙二醛(MDA)与超氧化物歧化酶(superoxide dismutase,SOD)的变化,探讨氧化应激与DC的关系。方法:分别测定68例DC患者(DC组)、62例单纯糖尿病患者(D组)、60例单纯白内障患者(C组)和同期50例非糖尿病非白内障眼科手术者(NS组)血清和房水中MDA与SOD的水平。结果:与NS组比较,D组和C组患者血清和房水中MDA水平升高,SOD水平下降(均P<0.05),但D组和C组之间差异无统计学意义;与D组或C组比较,DC组MDA升高,SOD下降更明显(均P<0.05)。Pearson相关分析显示,MDA与SOD呈负相关(r=-0.835,P<0.05);Logistic回归分析显示,MDA和SOD是DC的影响因素(均P<0.05)。结论:糖尿病性白内障患者血清和房水中MDA水平升高,SOD水平下降,氧化应激可能参与了DC的发生发展。     

The effects of sub-solar levels of UV-A and UV-B on rabbit corneal and lens epithelial cells

The purpose of this work was to establish whether exposing cultured rabbit corneal and lens epithelial cells to ultraviolet radiation equivalent to several hours under the sun would damage the cells. Confluent rabbit corneal epithelial cells were irradiated with broadband UV-A or UV-B, and confluent lens epithelial cells were irradiated with broadband UV-A. The maximum dose of UV-A was 6.3 J cm(-2) and that of UV-B was 0.60 J cm(-2). Damage to corneal epithelial cell was studied using the terminal deoxynucleotidyl transferase mediated dUTP-X nick end labeling (TUNEL) assay and damage to lens epithelial cell was studied using the single cell gel electrophoresis (comet) assay and trypan blue exclusion assay. Lipid peroxidation was assayed using the thiobarbituric acid reaction. Both UV-B and UV-A induced cell death in corneal epithelial cells with different latent periods. UV-A damage included cell death, decreased viability and increased lipid peroxidation of lens epithelial cell. In addition, UV irradiation of the corneal and lens epithelial cells decreased the activity of catalase to thirty to fifty percent of its original value, while the activities of glutathione peroxidase and superoxide dismutase did not decrease within experimental error. Thus, even sub-solar UV radiation can cause irreversible damage to corneal and lens epithelial cells.     

正常眼和老年性白内障晶状体中抗氧化酶活性的测定

采集了较大量的正常眼和老年性白内障眼的晶状体，根据氧自由基生物学和脂质过氧化学说进行研究，发现同正常晶状体眼相比，老年性白内障眼的晶状体中超氧化物歧化酶、谷胱甘肽过氧化物酶和过氧化氢酶活性均非常显著下降，而丙二醛含量则升高非常显著，结果提示氧自由基和脂质过氧化可能是导致老年性白内障形成的直接原因之一。     

茶多酚对体外培养鼠晶状体抗氧化损伤作用的研究

目的:观察茶多酚(tea polyphenols,TP)对氧化损伤鼠晶状体的形态学及抗氧化系统的变化,研究其对晶状体氧化损伤的保护作用.方法:采用体外培养鼠晶状体的氧化损伤模型,设置正常对照组、氧化损伤(H2O2)组和TP(H2O2+TP)组,分别于12,24,48h后观察各组晶状体的混浊情况,并检测各组晶状体的抗氧化酶系统中的超氧化物歧化酶(SOD)及谷胱甘肽过氧化物酶(GSH-Px)活性以及脂质过氧化反应终产物丙二醛(MDA)的含量.结果:正常对照组晶状体均保持透明,未见白内障形成;H2O2组大鼠离体晶状体随作用时间的延长,晶状体的混浊程度逐渐明显增加;TP组的晶状体混浊较H2O2组明显减轻,白内障形成不明显.晶状体混浊相对灰度值组间比较,差异有统计学意义(P<0.05).H2O2组大鼠晶状体组织中MDA 含量较对照组明显增高(P<0.05),而GSH-Px和SOD明显下降(P<0.05),TP组与H2O2组相比,其晶状体中MDA降低(P<0.05),但仍高于对照组,而GSH-Px和ATP含量明显升高,差异均有统计学意义(P<0.05).结论:TP可提高氧化损伤晶状体的抗氧化能力,降低脂质过氧化物水平,从而延缓白内障的发生和发展,为临床白内障的诊疗提供了新的理论基础.     

白藜芦醇对过氧化氢诱导人晶状体上皮细胞氧化损伤的保护作用

目的 探讨白藜芦醇(resveratrol,RES)对过氧化氢(H2O2)诱导人晶状体上皮细胞(human lens epithelial cells,HLEC)氧化损伤的保护作用.方法 HLEC传代培养24h后,分别加入不同浓度(5μmol·L-1、10 μmol·L-1、20 μmol·L-1、40 μmol·L-1) RES预处理12 h后,加入100 μmol·L-1 H2O2继续孵育24 h,倒置相差显微镜观察细胞形态改变,MTT比色法检测RES对H2O2诱导的HLEC活力的影响,流式细胞仪检测HLEC细胞凋亡率,比色法检测凋亡相关因子caspses-3及caspase-9的表达.结果 氧化损伤可以诱导HLEC形态改变,RES处理后,细胞形态逐渐得到改善.MTT结果显示RES对HLEC活性无抑制作用,RES(5μmol·L-1、10 μmol· L-1、20 μmol· L-1、40 μmol·L-1)孵育24 h后细胞存活率分别为(101.30±4.49)％、(100.31±3.53)％、(101.71±3.33)％、(99.30±3.00)％,与对照组(99.67±2.67)％比较,差异均无统计学意义(均为P＞0.05);模型组HLEC经氧化损伤处理后,细胞存活率(34.33±3.71)％明显下降,用20 μmol·L-1及40 μmol·L-1 RES处理后,HLEC存活率分别提高到(57.33±5.61)％和(72.67±6.98)％,与模型组比较差异均有统计学意义(均为P＜0.05).流式细胞计数结果显示:对照组HLEC凋亡率为(1.99±0.17)％,经H2O2处理后,模型组HLEC凋亡率为(51.73±4.97)％,20μmol·L-1、40 μmol·L-1 RES处理后,HLEC凋亡率分别为(34.43±3.67)％、(26.55±2.07)％,与模型组比较,差异均有统计学意义(均为P＜0.05).此外,RES还可以减少H2O2所致HLEC内caspses-3及caspase-9的表达.结论 RES可以明显抑制HLEC凋亡,其抑制凋亡的作用可能是其防止和延缓白内障发生发展的细胞学基础,从而为寻求有效的防治白内障药物提供可靠的实验依据.     

Effect of H(2)O(2)on human lens epithelial cells and the possible mechanism for oxidative damage repair by thioltransferase

Human lens epithelial (HLE) B3 cells were used to study the oxidative damage and cellular repair with respect to the redox homeostasis, the oxidative defense enzymes and the glucose metabolic pathway. The effect of oxidative stress on cell growth was initially analyzed by culturing the cells with a bolus amount (0.02--0.1m M) of hydrogen peroxide (H(2)O(2)) in minimal essential medium (MEM) containing 20% fetal bovine serum (FBS) for 1 week. Concentration of H(2)O(2)greater than 0.03m M showed progressive inhibition of cell growth. However, the cells were also shown to tolerate H(2)O(2)concentrations up to 0.5m M by detoxifying the exogenous oxidant within 3hr without any detectable DNA damage. Therefore, this short-term H(2)O(2)exposure model was chosen to study the effect of oxidative stress on the cellular redox homeostasis. HLE B3 cells were first grown to confluence in MEM with 20% FBS. Approximately 1.6 million cells were gradually weaned off serum by subculturing in 2% FBS overnight, followed by serum-free medium for 30 min before subjecting to a bolus of 0.1m M H(2)O(2)for up to 180 min. These cells were used for biochemical analysis, which included H(2)O(2)detoxification (H(2)O(2)in the medium), glutathione (GSH) level and lactate production. Activity measurements were conducted on the oxidation defense enzymes: glutathione-S-transferase (GST), glutathione reductase (GR) and glutathione peroxidase (GPx); the dethiolating enzyme, thioltransferase (TTase); and a key glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (G-3PD). While the B3 cells were shown to tolerate and detoxify 0.1m M H(2)O(2)within 60 min, the GSH pool was transiently depleted in the first 60 min before fully recovered. GPx suffered more than 80% loss in activity and was unable to recover fully. GST showed slight inactivation but neither GR nor TTase was affected. G-3PD was inactivated to < 50% within 15 min of oxidative stress and was reactivated gradually to 80% of normal at the end of 180 min, concurrent with the transient loss of lactate production in the same cells. The reactivation of G-3PD was both temperature- and GSH-dependent, occurring only at physiological temperature and failing to reactivate when the intracellular GSH pool was depleted by BCNU (GR inhibitor) pretreatment. The inactivated cellular G-3PD in the cell extract could be partially reactivated by DTT (6m M) or by recombinant human lens thioltransferase (RHLT) but not by GSH (1m M), GR or GST. HLE cells cultured in the presence of L-(35)S-cystine and cycloheximide displayed an extra radiolabelled protein band on the autoradiograph in the H(2)O(2)treated cells. The labelled band was positively reacted with G-3PD antibody and could be removed by RHLT, indicating that S-thiolation of G-3PD occurred. The H(2)O(2)pre-exposed cells also transiently accumulated proteins modified by thiolation, including protein-S-S-glutathione (PSSG) and protein-S-S-cysteine (PSSC). It can be concluded that HLE could endure up to 0.1m M of H(2)O(2)oxidative stress since the cell could be protected by its effective repair systems, including dethiolating the inactivated key SH-sensitive enzymes. TTase may play a role in this. One of the mechanisms may be through preserving glucose metabolism and supplying ATP needed for maintaining cell viability.     

Biochemical changes induced by intravitreally-injected doxorubicin in the iris-ciliary body and lens of the rabbit eye

The aim of this study was to examine the chronic effects and mode of action of doxorubicin in ocular tissues. A dose of 10 μg (17.24 nanomoles) of doxorubicin hydrochloride in 20 μl sterile saline were intravitreally injected, under local anaesthesia, in one eye of 13 rabbits and 50 μg (86.20 nanomoles) were similarly injected in one eye of 3 rabbits. The contralateral eye received 20 μl of saline only. The dose of 50 μg induced initially mild uveal inflammation which became chronic and turned into circular iritis. Both doses of the drug induced cataract of the lens and clouding of the cornea within 2-3 months. The activity of superoxide dismutase, in iris-ciliary bodies and lenses treated with either 10 or 50 μg of the compound, was significantly lower relative to that in respective control tissues. In contrast to superoxide dismutase, catalase showed an increased activity in experimental tissues relative to control. The lysosomal hydrolases acid phosphatase, N-acetyl-B-D-glucosaminidase, aryl sulphatase and acid cathepsin, all showed significantly elevated activities in iris-ciliary body tissues one year after injection with the 50 μg doxorubicin. The reduction in superoxide dismutase activity may render ocular tissues susceptible to peroxidative attack and the increased activities of lysosomal hydrolases may contribute to chronic cell injury and inflammation. This revised version was published online in July 2006 with corrections to the Cover Date.     

DNA damage in lens epithelium of cataract patients in vivo and ex vivo

Purpose: DNA damage has been described in the human cataractous lens epithelium, and oxidative stress generated by UV radiation and endogenous metabolic processes has been suggested to play a significant role in the pathogenesis of cataract. In this study, the aim was to explore the quality and relative quantity of DNA damage in lens epithelium of cataract patients in vivo and after incubation in a cell culture system. Methods: Capsulotomy specimens were analysed, before and after 1 week of ex vivo cultivation, using the comet assay to measure DNA strand breaks, oxidized purine and pyrimidine bases and UV‐induced cyclobutane pyrimidine dimers. Results: DNA strand breaks were barely detectable, oxidized pyrimidines and pyrimidine dimers were present at low levels, whereas there was a relatively high level of oxidized purines, which further increased after cultivation. Conclusion: The observed levels of oxidized purines in cataractous lens epithelium may support a theory consistent with light damage and oxidative stress as mediators of molecular damage to the human lens epithelium. Damage commonly associated with UV‐B irradiation was relatively low. The levels of oxidized purines increased further in a commonly used culture system. This is of interest considering the importance and versatility of ex vivo systems in studies exploring the pathogenesis of cataract.     

雷公藤甲素对人视网膜色素上皮细胞氧化损伤的抑制作用

目的　探讨雷公藤甲素对人视网膜色素上皮（retinal pigment epithelium,RPE）细胞氧化损伤的抑制作用及其机制。方法　RPE细胞传代培养,分为对照组、氧化损伤组（100 μmol·L^-1 H₂O₂）和雷公藤甲素（100 μmol·L^-1、200 μmol·L^-1）干预组。应用MTT比色法检测细胞增殖率,流式细胞技术测定细胞内活性氧（reactive oxygen species,ROS）含量,Hoechst33258染色观察细胞凋亡,Western-blot检测RPE细胞内超氧化物歧化酶（superoxide dismutase,SOD）及丙二醛（malondialdehyde,MDA）蛋白的表达水平。结果　雷公藤甲素能明显抑制H₂O₂诱导的RPE细胞活性的下降,用不同浓度雷公藤甲素处理后,RPE细胞存活率分别提高到（56.00±2.76）%和（70.33±3.85）%,与氧化损伤组［（32.67±3.08）%］相比差异均有统计学意义（均为P<0.05）。雷公藤甲素能减少细胞内ROS生成,抑制细胞凋亡,不同浓度雷公藤甲素处理后,RPE细胞凋亡率分别下降到（50.33±4.61）%和（46.67±4.73）%,与氧化损伤组（67.00%±3.42%）相比差异均有统计学意义（均为P<0.05）。雷公藤甲素组SOD蛋白表达显著高于氧化损伤组,MDA蛋白表达显著低于氧化损伤组（P<0.05）。结论　雷公藤甲素对RPE细胞氧化损伤具有抑制作用,其作用机制与上调SOD的表达及下调MDA的表达有关;雷公藤甲素有望成为治疗视网膜病变的有效药物。