Increased stress-induced generation of reactive oxygen species and apoptosis in human keratoconus fibroblasts

PURPOSE: To determine whether keratoconus (KC) corneal fibroblast cultures have increased reactive oxygen species (ROS) production and are more susceptible to stress-related challenges. METHODS: Normal (n = 9) and KC (n = 10) stromal fibroblast cultures were incubated in either neutral- or low-pH conditions, with or without hydrogen peroxide. Catalase activities were measured with a fluorescent substrate assay. Superoxide and ROS/reactive nitrogen species (RNS) productions were determined with an amine-reactive green-dye assay and 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) dye assay, respectively. Cell viability was analyzed by a dye-exclusion assay. Caspase 3 activity was measured by a fluorochrome inhibitor of caspase (FLICA) assay. A cationic (green) dye was used to measure the mitochondrial membrane potential (delta psi m). RESULTS: KC fibroblasts had increased superoxide and ROS/RNS production (6.2-fold, P < 0.001 and 1.8-fold, P < 0.001, respectively) and catalase activity (P < 0.01) with higher concentrations of H2O2 compared with normal cultures (P = 0.16). After a low-pH stress challenge, KC fibroblasts maintained higher ROS/RNS levels (3.3-fold, P < 0.02), showed higher caspase-3 activity (7.5-fold, P < 0.02) and decreased delta psi m (2.6-fold, P < 0.04), and had decreased cell viability (37%, P < 0.005 vs. 20%, P < 0.27) compared with normal fibroblasts. CONCLUSIONS: Under identical conditions, KC fibroblasts had increased basal generation of ROS/RNS and were more susceptible to stressful challenges (low-pH and/or H2O2 conditions) than were normal fibroblasts. In addition, the stressed KC fibroblasts possessed characteristics similar to those found in the intact KC corneas (increased catalase activity, ROS production, and apoptosis). These properties may play a role in the pathogenesis of KC.     

Hyperoxia and thyroxine treatment and the relationships between reactive oxygen species generation, mitochondrial membrane potential, and cardiolipin in human lens epithelial cell cultures

Thyroxine-treated human lens epithelial cells, HLE B-3, were grown in either a normoxic or hyperoxic atmosphere as a first step in identifying factors related to their increased viability. Reactive oxygen species, ROS, and the apparent mitochondrial membrane potential, Delta Psi(m), were measured using fluorescent probes. ROS were significantly higher in HLE B-3 cells grown in a hyperoxic atmosphere for both thyroxine-treated and untreated samples. However, treatment with thyroxine produced 40% lower ROS levels than untreated cells. A mitochondrial uncoupler concomitantly reduced ROS generation. In cells that were grown in a hyperoxic atmosphere, the Delta Psi(m) was significantly higher for samples treated with thyroxine compared to those that were untreated. ROS generation correlated inversely with the apparent Delta Psi(m) and the amount of cardiolipin, and correlated with the amount of lipid oxidation products. These correlations were valid whether cardiolipin and the Delta Psi(m) were decreased as a result of oxygen or increased as a result of thyroxine treatment. Therapies that protect mitochondria from damage and reduce damaging ROS generation may potentially ameliorate the effects of oxidation that occur in aging tissues and in diseases such as cataract.     

Nanoparticle-delivered biosensor for reactive oxygen species in diabetes

The cell's own antioxidant response element (ARE) can be used to evaluate the complications of diabetes mellitus. The hypothesis that a synthetic ARE could be used as a genetic switch, or biosensor, to turn on and off therapeutic genes is tested herein. Mitochondrial oxidative stress (MOS) has been hypothesized as one of the earliest insults in diabetes. Fluorescent probes used to monitor MOS revealed that the addition of glucose at physiological levels to cultures of endothelial cells was able to induce MOS above normal levels and in a dose-dependant manner. Additional data showed that increased glucose levels activated the ARE-GFP in a dose-dependant manner. These data support the hypothesis that the induction of MOS is more sensitive to hyperglycemia than the induction of the ARE. Delivery of an ARE-GFP construct with nanoparticles to the eye was successful using sub-retinal injection. This ARE-GFP/nanoparticle construct was functional and reported the activation of the ARE in diabetic rat retinal pigment epithelium (RPE). These data support the use of nanoparticle-delivered biosensors for monitoring the oxidative status of tissues in vivo.     

Heat shock induces apoptosis through reactive oxygen species involving mitochondrial and death receptor pathways in corneal cells

Although many studies have been performed to elucidate the molecular consequences of ultraviolet irradiation, little is known about the effect of infrared radiation on ocular disease. In addition to photons, heat is generated as a consequence of infrared irradiation, and heat shock is widely considered to be an environmental stressor. Here, we are the first to investigate the biological effect of heat shock on Statens Seruminstitut Rabbit Cornea (SIRC) cells. Our results indicate that heat shock exhibits effective cell proliferation inhibition by inducing apoptosis. Heat shock triggers the mitochondrial apoptotic pathway indicated by a change in Bax/Bcl-2 ratios, resulting in caspase-9 activity. In addition, heat shock triggered the death receptor apoptotic pathway indicated by a change in Fas ligand expression, resulting in caspase-8 activity. Furthermore, we also found that generation of reactive oxygen species (ROS) is a critical mediator in heat shock-induced apoptosis. In addition, the antioxidant vitamin C significantly decreased heat shock-mediated apoptosis. Taken together, these findings suggest a critical role for ROS involving mitochondrial and death receptor pathways in heat shock-mediated apoptosis of cornea cells.     

莱菔硫烷对高糖条件下大鼠晶状体上皮细胞活性氧产生与线粒体膜电位的影响及机制研究

目的 观察莱菔硫烷(sulforaphane,SFN)对高糖条件下大鼠晶状体上皮细胞(lens epithelial cell,LEC)活性氧(reactive oxygen species,ROS)和线粒体膜电位(mitochondrial membrane potential,MMP)的影响,并探讨其可能的分子机制.方法 人LEC系SRA01/04细胞分别用含5.5 mmol·L-1葡萄糖(正常对照组)和30.5 mmol·L-1葡萄糖(高糖处理组)的培养基培养.SFN干预组在高糖处理组培养基基础上加入不同浓度SFN.处理24h后,荧光探针法检测ROS产生量,罗丹明123染色检测MMP的改变,Western blot观察血红素氧合酶-1(heme oxygenase-1,HO-1)的表达.为了评价SFN对LEC的保护作用与HO-1的关系,SFN干预组(30 μmol·L-1)中分别加入不同浓度的HO-1激动剂钴原卟啉和抑制剂锡原卟啉,24h后观察ROS产生量及MMP变化.结果 与正常对照组相比,高糖处理组LEC中ROS的产生量增加了29.0％,差异有统计学意义(P＜0.05);SFN干预组ROS的产生量随着SFN浓度的增高而逐渐降低,当SFN浓度为30 μmol·L-1时,ROS产生量接近正常对照组,差异无统计学意义(P＞0.05).高糖处理组MMP水平与正常对照组相比下降了61.0％,差异有统计学意义(P＜0.05),30μmol·L-1 SFN使MMP水平恢复到95.4％,与高糖处理组相比,差异有显著统计学意义(P＜0.01).高糖处理组HO-1表达轻微增加;SFN干预组与高糖处理组相比HO-1表达进一步增高.HO-1特异性抑制剂锡原卟啉能减弱SFN对ROS的抑制作用并降低MMP水平,而HO-1激动剂钴原卟啉则能进一步协同SFN降低高糖诱导的ROS产生,并提高MMP水平.结论 SFN能在一定程度上抑制高糖诱导的人LEC内ROS产生及MMP改变,机制可能与其诱导HO-1的表达有关.     

蓝光诱导的光感受器细胞凋亡与c-Fos蛋白的表达

目的:观察宽谱蓝光诱导Lewis大鼠视网膜光损伤后光感受器细胞的凋亡及c-Fos蛋白的表达。方法:8～10wk龄雌性Lewis大鼠24只,在循环光环境下饲养并随机分为6组。暗适应24h后,5组接受3012×115Lux的宽谱蓝光(400～500nm)照射1h,光照后予暗适应并于0,6,12,24及48h颈椎脱位法处死大鼠,摘除眼球;另1组为正常对照组,不予光照。采用透射电镜及TUNEL试剂盒检测视网膜细胞凋亡,免疫组化法检测视网膜内c-Fos蛋白的表达。结果:蓝光可特异性引起大鼠光感受器细胞凋亡和光感受器细胞内c-Fos蛋白的表达上调。光照后外核层细胞开始出现凋亡,24h达峰值,大量光感受器细胞出现核固缩并可见凋亡小体形成。c-Fos蛋白的表达在时间与空间分布上与TUNEL阳性细胞基本一致,在同一时间点,二者呈正相关(r =0.905,P <0.05)。结论:蓝光可诱导大鼠光感受器细胞凋亡,视网膜外核层中c-Fos蛋白的表达上调对视网膜蓝光损伤后光感受器细胞凋亡可能具有重要作用。     

Early disappearance of alpha-transducin in light-induced photoreceptor degeneration in albino rats.

Progressive degeneration of retinal photoreceptor cells occurs in albino rats when exposed to continuous lighting. Three proteins involved in the phototransduction cascade were immunodetected in these cells after various durations of continuous illumination. We found that S-antigen (arrestin) and rhodopsin immunoreactivities persisted for 1-2 months during the degenerative process, whilst immunoreactivity of the alpha subunit of transducin totally disappeared between day 2 and day 4 of continuous light exposure. This suggests that continuous illumination could impair alpha-transducin synthesis, a possible causal factor of photoreceptor damage.     

低氧预适应对小鼠视网膜光感受器细胞光损伤的防护作用

目的研究低氧预适应对光损伤后小鼠视网膜光感受器细胞的保护作用,并探讨其可能的基因调控机制。方法将54只BALB／c小鼠随机分成单纯光照组、低氧预适应组和正常对照组,并将前两组动物在自制光照箱中连续光照3h,制成光损伤动物模型。应用光镜、核苷酸末端转移酶介导的dUTP缺口标记法观察各组小鼠视网膜组织结构的改变;应用免疫组织化学方法检测各组小鼠视网膜c-fos和caspase-1的表达。结果单纯光照组小鼠视网膜组织学改变出现的早且明显,光照后视网膜出现不同程度的病理学改变,且损伤主要发生在光感受器细胞层。随着光照后时间的延长,光感受器细胞凋亡数增加,视网膜光损伤逐渐加重,c-fos和caspase-1在该层出现了不同程度的阳性表达：低氧预适应组同单纯光照组相比,各时间段损伤均较轻,视网膜光感受器细胞层明显得到了保护,同单纯光照组比较,caspase-1的阳性表达明显减少,而c-fos无明显变化;正常对照组小鼠视网膜组织结构层次清楚,外核层排列规则,无c-fos和caspase-1的阳性表达。结论低氧预适应通过抑制凋亡相关基因的表达而对光感受器细胞有神经保护作用,且caspase-1参与了低氧预适应的保护机制。(中华眼科杂志,2005,41：631-635)     

Elevated retinal zeaxanthin and prevention of light-induced photoreceptor cell death in quail

PURPOSE: Inferential evidence indicates that macular pigments (lutein and zeaxanthin) protect photoreceptors and/or retard age-related macular degeneration. These experiments tested the hypothesis that retinal zeaxanthin prevents light-induced photoreceptor cell death. METHODS: Retinal damage was assessed in quail fed a carotenoid-deficient (C-) diet for 6 months. Groups of 16 birds (8 male, 8 female) were fed a C- diet supplemented with 35 mg 3R,3'R-zeaxanthin for 1, 3, or 7 days; one group was continued on C- diets. Half of each group was exposed to intermittent 3200-lux white light (10 1-hour intervals separated by 2 hours in dark). After 14 additional hours in the dark, one retina of each quail was collected for HPLC analysis, and the contralateral retina was embedded in paraffin for counts of apoptotic nuclei. RESULTS: After 7 days' supplementation, concentrations of zeaxanthin in serum, liver, and fat had increased by factors of 50.8, 43.2, and 6.5, respectively (all P < 0.001). In contrast, retinal zeaxanthin fluctuated significantly upward on day 3, but there was no net change on day 7. The number of apoptotic rods and cones in light-damaged eyes correlated significantly and inversely with zeaxanthin concentration in the contralateral retina (r = -0.61; P < 0.0001 and r = -0.54; P < 0.002), but not with serum zeaxanthin. Similar correlations were observed with retinal lutein, which correlated strongly with retinal zeaxanthin (r = 0.95; P < 0.0001). CONCLUSIONS: Retinal zeaxanthin dose dependently reduced light-induced photoreceptor apoptosis; elevated serum levels did not. These data provide the first experimental evidence that xanthophyll carotenoids protect photoreceptors in vivo.     

Blue light-induced apoptosis in cultured retinal pigment epithelium cells of the rat

Background: We previously demonstrated that phagosome-free retinal pigment epithelium (RPE) cells in culture can be damaged directly by blue light (wavelength 440±10 nm) as observed by electron microscope. A low intensity (1.0 mW/cm²) of light induced only swelling of mitochondria, while a high intensity (4.0 mW/cm²) induced necrosis in the RPE. The aim of the present study was to investigate what intensity of blue light could induce apoptosis in cultured phagosome-free RPE. Methods: Primary cultured RPE cells, harvested from Long-Evans rats, that contained no phagosomes were exposed to a cool blue light (wavelength 440±10 nm). After exposure, transmission electron microscopy (TEM) and TdT-mediated dUTP nick-end labeling (TUNEL) staining were used to detect apoptosis in the RPE cells. To assess the relationship of oxidation to apoptosis by blue light, we added N-acetyl-cysteine (NAC) as a free radical scavenger and investigated its inhibitory effect on apoptosis. Results: In RPE cells exposed to blue light of 2.7 mW/cm² for 24 h, apoptotic bodies were found by TEM. In RPE cells exposed to blue light of 2.0 mW/cm² for 60 h, apoptotic bodies, nuclear condensation and nuclear segmentation were observed by TEM and some RPE cells showed positive TUNEL staining. When 30 mM of NAC was added, TUNEL staining was negative. Conclusion: Our findings demonstrate that apoptotic cell death is induced by blue light exposure in cultured RPE cells in vitro. The findings of our previous experiments and those of the present study suggest that a higher intensity of blue light could induce necrosis, and moderately intense blue light could induce non-necrotic cell death or apoptosis, in RPE cells. Furthermore, it is suggested that blue light caused cell death by a free-radical-associated mechanism. Received: 10 April 2000 Revised: 30 June 2000 Accepted: 29 August 2000     

An immunohistochemical study of opsin in photoreceptor cells following light-induced retinal degeneration in the rat

Light-induced retinal degeneration has been hypothesized to be rhodopsin-mediated. However, the alterations induced in the opsin moiety of the rhodopsin molecule and its distribution in the rod cell after a photic insult have not been definitively established. We used light and electron immunohistochemistry to study the alterations in retinal opsin immunoreactivity in a rat model of retinal photic injury. In normal unexposed rat retinas, opsin immunoreactivity was restricted to the rod outer segments. At 6 h after a 24-h light exposure, opsin immunoreactivity was present in the rod outer segments in both the superior and inferior retina, but in addition marked immunoreactivity was present in the inner segments in the superior quadrant of the light-damaged retina. At 6 days after exposure, intense immunoreactivity was noted around the severely degenerating rod nuclei and inner segments. However, at 21 days following light exposure, opsin immunoreactivity in areas of recovery was again restricted to the short regenerated rod outer segments. It appears that, despite severe light-mediated retinal degeneration, anti-opsin immunoreactivity persisted in the photoreceptor cells but with an altered pattern in damaged rod outer segments and photoreceptor perikarya. However, opsin immunoreactivity relocated to the regenerated rod outer segments in the recovery phase.Supported in part by grant R01 EY01903 (Pathology of Retinal Dysfunction) and Core Grant EY01792 from the National Eye Institute, Bethesda, Md.; a grant from the Lions of Illinois Foundation, Maywood Ill., and gifts from the Clifford Sawyer Estate and the McGraw Foundation, Arlington Heights, Ill.     

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。     

Calcium-induced lens protein aggregation accelerated by reactive oxygen species photosensitized in the presence of hydroxykynurenines

Photo-oxidation with 3-OH L-kynurenine O-beta-glucoside or 3-OH L-kynurenine accelerates the aggregation of water-soluble protein in the rat lens due to the presence of calcium ion. The present report demonstrates that hydrogen peroxide and superoxide anion mediate a large part of the photodynamic acceleration with hydroxykynurenine compounds. In addition, 3-OH L-kynurenine O-beta-glucoside seems to photosensitize more of the two reactive oxygen species than 3-OH L-kynurenine. The findings are discussed together with other photodynamic effects of hydroxykynurenine compounds.     

Protection of photoreceptor cells in adult rats from light-induced degeneration by adaptation to bright cyclic light

Light history has been shown to affect the susceptibility of the albino rat retina to the damaging effects of constant light exposure. Retinas of animals raised in relatively bright cyclic light are protected against light-induced degeneration compared with dim-reared animals. These effects were observed in animals raised from birth in bright cyclic light and are part of an adaptive response that protects photoreceptors from stress-induced degeneration. To determine if retinas of adult animals are capable of such adaptive changes or flexibility by switching between different light environments which do not pathologically damage photoreceptor cells, albino rats were maintained in less than 250 lux cyclic light for more than 3 weeks. At 12-13 weeks of age, they were placed into 800 lux cyclic light for 1 week, after which they were exposed to constant illumination of 1500-lux for 1, 3 or 7 days. Retinal function was evaluated by electroretinography and photoreceptor cell death was quantified by measuring outer nuclear layer thickness. After 1 week in bright cyclic light, the retinas were completely protected against 1 day constant light exposure that significantly damaged retinas of animals without 800 lux cyclic light adaptation. Significant protection was also observed in 3 day constant light exposed animals; limited protection occurred after 7 days exposure. These results indicate that the retinas of adult rats adapted to bright cyclic light within certain ranges that did not significantly damage photoreceptor cells are protected from constant light challenge. This phenomenon is a post-developmental response that demonstrates a remarkable plasticity of the retina. The mechanism(s) underlying the ability of this adaptation/flexibility in protecting photoreceptors could involve endogenous molecules that encompass many aspects of retinal cell and molecular biology and physiology. Identification of these molecules may provide insight into the development of therapeutic approaches to treat retinal degeneration.