Oxidative damage in the retinal mitochondria of diabetic mice: possible protection by superoxide dismutase

PURPOSE: Superoxide levels are elevated in the retina in patients with diabetes, and cytochrome c is released from the mitochondria. The purpose of this study was to elucidate the mechanism involved in the oxidative damage of retinal mitochondria in diabetes and to determine whether mitochondrial superoxide dismutase (MnSOD) provides protection. METHODS: Effects of diabetes were investigated on superoxide and GSH levels, electron transport complexes I and III, and membrane permeability in the isolated mitochondria prepared from the retinas of streptozotocin diabetic mice. To investigate the effect of MnSOD, retinal mitochondrial oxidative stress and electron transport complexes were determined in mice overexpressing MnSOD (MnSOD-Tg). Histopathology was evaluated in trypsin-digested retina. RESULTS: Retinal mitochondria had twofold increase in superoxide levels in nontransgenic (wild-type [WT]) diabetic mice compared with WT nondiabetic mice. In the same retina, diabetes decreased mitochondrial GSH levels by 40% and complex III activity by approximately 20%, and it increased mitochondrial membrane permeability (swelling) by more than twofold; however, complex I activity was not affected. Overexpression of MnSOD inhibited diabetes-induced increases in mitochondrial superoxide levels and membrane permeability and the decrease in complex III activity. GSH values, however, were not statistically different in WT and MnSOD-Tg diabetic mice. In contrast to the diabetes-induced increase in the number of degenerate (acellular) capillaries in WT diabetic mice, the numbers of acellular capillaries in MnSOD-Tg nondiabetic and diabetic mice were similar to those in WT nondiabetic mice. CONCLUSIONS: Retinal mitochondria experience increased oxidative damage in diabetes, and complex III is one of the sources of increased superoxide. MnSOD protects the retina from diabetes-induced abnormalities in the mitochondria and prevents vascular histopathology, strongly implicating the role for MnSOD in the pathogenesis of retinopathy in diabetes.     

Diabetes-induced mitochondrial dysfunction in the retina

PURPOSE: Oxidative stress is increased in the retina in diabetes, and antioxidants inhibit activation of caspase-3 and the development of retinopathy. The purpose of this study was to investigate the effect of diabetes on the release of cytochrome c from mitochondria and translocation of Bax into mitochondria in the rat retina and in the isolated retinal capillary cells. METHODS: Mitochondria and cytosol fractions were prepared from retina of rats with streptozotocin-induced diabetes and from the isolated retinal endothelial cells and pericytes incubated in 5 or 20 mM glucose medium for up to 10 days in the presence of superoxide dismutase (SOD) or a synthetic mimetic of SOD (MnTBAP). The release of cytochrome c into the cytosol and translocation of the proapoptotic protein Bax into the mitochondria were determined by the Western blot technique and cell death by caspase-3 activity and ELISA assay. RESULTS: Diabetes of 8 months' duration in rats increased the release of cytochrome c into the cytosol and Bax into the mitochondria prepared from the retina, and this phenomenon was not observed at 2 months of diabetes. Incubation of isolated retinal capillary cells with 20 mM glucose increased cytochrome c content in the cytosol and Bax in the mitochondria, and these abnormalities were accompanied by increased cell apoptosis. Inclusion of SOD or its mimetic inhibited glucose-induced release of cytochrome c, translocation of Bax, and apoptosis. CONCLUSIONS: Retinal mitochondria become leaky when the duration of diabetes is such that capillary cell apoptosis can be observed; cytochrome c starts to accumulate in the cytosol and Bax into the mitochondria. Inhibition of superoxides inhibits glucose-induced release of cytochrome c and Bax and inhibits apoptosis in both endothelial cells and pericytes. Identifying the mechanism by which retinal capillary cells undergo apoptosis may reveal novel therapies to inhibit the development of retinopathy in diabetes.     

氧化应激与糖尿病视网膜病变

糖尿病视网膜病变（DR）是严重的糖尿病眼部并发症之一。研究表明，氧化应激和应激激活的信号通路与DR有着密切关系。高糖通过多种机制刺激活性氧类物质的产生，从而生长因子和细胞因子的基因表达。正常情况下，过多的活性氧物质可被抗氧化防御系统清除，但持续性高血糖情况下过多的活性氧物质可导致细胞损害，包括视网膜内皮细胞、周细胞和视网膜节细胞。现就氧化应激在DR发生发展中所起的作用做一综述。     

Role of interleukin-1beta in the development of retinopathy in rats: effect of antioxidants

PURPOSE: Diabetic retinopathy is shown to share many similarities with chronic inflammatory disease, and in diabetes, accelerated apoptosis of retinal capillary cells is evident before histopathology can be seen. The purpose of this study was to examine the effect of interleukin (IL)-1beta on capillary cell apoptosis in rat retina and to determine the effect of antioxidants on diabetes-induced changes in retinal IL-1beta. METHODS: The effect of injection of IL-1beta into the vitreous (5 ng/5 microL) of normal rats on capillary cell apoptosis (detected by terminal transferase dUTP nick-end labeling [TUNEL]) and formation of acellular capillaries was investigated in the trypsin digested retinal microvessels. The levels of IL-1beta were quantified (by ELISA and Western blot) in the retina of rats diabetic for 2 months, and the effect of administration of antioxidants on diabetes-induced changes in retinal IL-1beta was determined. RESULTS: The number of TUNEL-positive capillary cells in the retinal microvessels obtained from normal rats that received intravitreal injection of IL-1beta was increased by more than threefold and that of acellular capillaries by more than twofold, compared with the microvessels obtained from rats that received an intravitreal injection of PBS (5 microL) or BSA (5 ng/5 microL). IL-1beta also increased the levels of 8-hydroxy-2'-deoxyguanosine (an indicator of oxidative stress) and nitric oxide by more than 40% and activated NF-kappaB by 35% to 55%. Two months of diabetes in rats increased retinal IL-1beta levels by more than twofold, and antioxidants inhibited such increases. CONCLUSIONS: IL-1beta, by activation of NF-kappaB and an increase in oxidative stress, plays an important role in the retinal microvascular disease that is characteristic of diabetic retinopathy. Antioxidants inhibit diabetes-induced increases in retinal IL-1beta. These studies offer a possible rationale to test IL-1beta-targeted therapies to inhibit the development of retinopathy in diabetes.     

Hydrogen Sulfide: A Potential Therapeutic Target in the Development of Diabetic Retinopathy

PurposeHyperglycemia damages the retinal mitochondria, and the mitochondrial damage plays a central role in the development of diabetic retinopathy. Patients with diabetes also have higher homocysteine levels, and abnormalities in homocysteine metabolism result in decreased levels of hydrogen sulfide (H₂S), an endogenous gasotransmitter signaling molecule with antioxidant properties. This study aimed to investigate the role of H₂S in the development of diabetic retinopathy.MethodsStreptozotocin-induced diabetic mice were administered a slow releasing H₂S donor GYY4137 for 6 months. The retina was used to measure H₂S levels, and their retinal vasculature was analyzed for the histopathology characteristic of diabetic retinopathy and oxidative stress, mitochondrial damaging matrix metalloproteinase-9 (MMP-9), and mitochondrial integrity. These parameters were also measured in the isolated retinal endothelial cells incubated in high glucose medium containing GYY4137.ResultsAdministration of GYY4137 to diabetic mice ameliorated decrease in H₂S and prevented the development of histopathology, characteristic of diabetic retinopathy. Diabetes-induced increase in oxidative stress, MMP-9 activation, and mitochondrial damage were also attenuated in mice receiving GYY4137. Results from isolated retinal endothelial cells confirmed the results obtained from diabetic mice.ConclusionsThus, supplementation of H₂S donor prevents the development of diabetic retinopathy by ameliorating increase in oxidative stress and preserving the mitochondrial integrity. H₂S donors may provide a novel therapeutic strategy to inhibit the development of diabetic retinopathy.     

Diabetic retinopathy. Pathophysiology and therapy of hypoxia-induced inflammation

Diabetic retinopathy is the most common chronic microvascular complication associated with diabetes mellitus. The development of diabetic retinopathy is a consequence of metabolic dysregulation. Hyperglycemia is a critical factor which is involved in basement membrane thickening, loss of pericytes and endothelial cells, and retinal capillary nonperfusion. We review the molecular basis of diabetic retinopathy and maculopathy and elaborate the role of growth factors and cytokines in the development of diabetic vascular alterations, their specific influence on the cellular interaction between retinal endothelial cells and pericytes, and the role of intravascular blood components.     

褪黑素对早期糖尿病大鼠视网膜锰超氧化物歧化酶和铜锌超氧化物歧化酶表达的影响

目的观察褪黑素对早期糖尿病大鼠视网膜锰超氧化物歧化酶（MnSOD）、铜锌超氧化物歧化酶（Cu/ZnSOD）活性及基因表达变化的影响,以揭示褪黑素对早期糖尿病大鼠视网膜的抗氧化作用机制。方法 54只SD大鼠按随机数字表法分为正常对照组、糖尿病组及褪黑素组,每组18只。糖尿病组及褪黑素组大鼠一次性腹腔注射链脲佐菌素（STZ）60mg/kg,升高血糖至〉16.7mmol/L,建立糖尿病大鼠模型,褪黑素组大鼠于STZ注射后第2天给予褪黑素灌胃,每日10mg/kg。于造模后4、8、12周3个时间点处死各组6只大鼠制备视网膜匀浆。应用黄嘌呤氧化酶法检测视网膜组织中总SOD、Cu/ZnSOD及MnSOD的活性;应用实时荧光定量PCR法检测视网膜组织中MnSODmRNA及Cu/ZnSODmRNA的表达量。结果模型建立后4、8、12周时糖尿病组和褪黑素组大鼠血糖水平明显高于正常对照组大鼠,差异均有统计学意义（P〈0.01）。造模后4、8、12周时各时间点糖尿病组大鼠视网膜中总SOD、MnSOD活性明显低于正常对照组,8周及12周时褪黑素组大鼠视网膜中总SOD、MnSOD活性明显高于糖尿病组,差异均有统计学意义（P〈0.01）。糖尿病组大鼠视网膜中Cu/ZnSOD活性在8周及12周时明显降低,与正常对照组比较差异均有统计学意义（P〈0.01）,12周时褪黑素组较糖尿病组有所升高,差异有统计学意义（P〈0.01）。各时间点糖尿病组大鼠MnSODmRNA在视网膜中的表达量与正常对照组相比明显下降,褪黑素组MnSODmRNA的表达明显高于糖尿病组,差异均有统计学意义（P〈0.01）;8周及12周时糖尿病组Cu/ZnSODmRNA的表达量较正常对照组明显下降,12周时褪黑素组Cu/ZnSODmRNA的表达明显高于糖尿病组,差异有统计学意义（P〈0.01）。结论糖尿病早期,褪黑素主要通过提高视网膜MnSOD的活性及增强其基因表达减轻视网膜的氧化损伤。     

Pathogenetic potential of leukocytes in diabetic retinopathy

Recently, increasing interest has been directed toward the role of leukocytes in microvascular disorders including diabetic retinopathy because of their large cell volume, high cytoplasmic rigidity, natural tendency to stick to the vascular endothelium, and capacity to generate toxic superoxide radicals and proteolytic enzymes. Leukocytes in diabetes are reported to be less deformable and more activated, and may be involved in capillary non-perfusion, endothelial cell damage, and vascular leakage in the retinal microcirculation. In fact, histological studies show many capillary occlusions by leukocytes and capillary dropout or degeneration associated with leukocytes in the diabetic retina. Serial acridine orange leukocyte fluorography and fluorescein angiography studies also identify trapped leukocytes directly associated with areas of downstream non-perfusion in the diabetic retinal microcirculation. More recent studies suggest that adhesion molecules may mediate retinal leukocyte stasis (leukostasis) in diabetes and a reduction in the leukostasis by anti-adhesion antibodies can suppress retinal vascular leakage. In addition, some agents inhibiting leukostasis are reported to improve retinal abnormalities induced by diabetes. Thus, leukostasis in the retinal microcirculation can be a new promising target in the treatment of diabetic retinopathy.     

LIN28A attenuates high glucose-induced retinal pigmented epithelium injury through activating SIRT1-dependent autophagy

AIM: To evaluate the effects of LIN28A (human) on high glucose-induced retinal pigmented epithelium (RPE) cell injury and its possible mechanism. METHODS: Diabetic retinopathy model was generated following 48h of exposure to 30 mmol/L high glucose (HG) in ARPE-19 cells. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot tested the expression of the corresponding genes and proteins. Cell viability as well as apoptosis was determined through cell counting kit-8 (CCK-8) and flow cytometry assays. Immunofluorescence assay was adopted to evaluate autophagy activity. Caspase 3 activity, oxidative stress markers, and cytokines were appraised adopting their commercial kits, respectively. Finally, ARPE-19 cells were preincubated with EX527, a Sirtuin 1 (SIRT1) inhibitor, prior to HG stimulation to validate the regulatory mechanism. RESULTS: LIN28A was downregulated in HG-challenged ARPE-19 cells. LIN28A overexpression greatly inhibited HG-induced ARPE-19 cell viability loss, apoptosis, oxidative damage as well as inflammatory response. Meanwhile, the repressed autophagy and SIRT1 in ARPE-19 cells challenged with HG were elevated after LIN28A overexpression. In addition, treatment of EX527 greatly inhibited the activated autophagy following LIN28A overexpression and partly abolished the protective role of LIN28A against HG-elicited apoptosis, oxidative damage as well as inflammation in ARPE-19 cells. CONCLUSION: LIN28A exerts a protective role against HG-elicited RPE oxidative damage, inflammation, as well as apoptosis via regulating SIRT1/autophagy.     

Expression modification of uncoupling proteins and MnSOD in retinal endothelial cells and pericytes induced by high glucose: the role of reactive oxygen species in diabetic retinopathy

Uncoupling proteins (UCPs) are mitochondrial transporters present in the inner membrane of mitochondria. They belong to the family of anion mitochondrial carriers. UCPs could act as proton carriers activated by metabolites and create a shunt between complexes of the respiratory chain and ATP synthase. The increased leakiness of the mitochondrial inner membrane to protons may be to minimize superoxide production by limiting the maximum Deltamu(H+). The purpose of this study was to detect UCP expression in retinal capillary cells and their modification in high levels of glucose. The role of reactive oxygen species (ROS) of mitochondria and UCPs in pathogenesis of diabetic retinopathy was investigated. Bovine retinal capillary endothelial cells and pericytes were cultured with selective culture media, respectively. Passage cells were cultured in three different glucose concentrations (5, 23, 30 mM) until passage four. ROS changes in mitochondria of these cells in different glucose concentrations were detected with scanning laser confocal microscopy (SLCM). The mitochondria membrane potential (Deltapsi), cell death rate and apoptosis rate were measured with flowing cytometry. UCP expression in retinal capillary cells was detected by immunocytochemistry. Expression and modification of MnSOD and uncoupling proteins (UCPs) in different concentrations of glucose were detected by means of semi-quantitative RT-PCR. ROS in mitochondria of both endothelial cells and pericytes increased as the glucose concentration of media increased. Deltapsi and cell death rate of endothelial cells increased also. ROS was correlated to Deltapsi and cell death rate positively in endothelial cells. No difference in Deltapsi and cell death rate among different glucose levels was found in pericytes. Apoptosis rate of endothelial cells and pericytes in high glucose levels was higher than that in lower glucose levels. UCP1 and UCP2 were expressed in cultured retinal capillary cells whereas UCP3 was not. At high levels of glucose, expression of UCP1, UCP2 and MnSOD increased to accommodate ROS production compensatively. The compensative mechanism disappeared when glucose concentration was too high (30 mM). The results of this study showed that increasing mitochondrial ROS could be induced by high glucose concentration. Those proteins related to antioxidation mechanism, such as MnSOD and UCPs, could exert compensative action to a certain extent. This compensative action was insufficient when the glucose concentration was too high.     

Response of capillary cell death to aminoguanidine predicts the development of retinopathy: comparison of diabetes and galactosemia

PURPOSE: To examine the relationship between early retinal capillary cell apoptosis and late histologic lesions of diabetic retinopathy and to compare the effects of aminoguanidine (AMG) on the retinopathies caused by diabetes and galactose feeding. METHODS: Rats with alloxan-induced diabetes and rats fed a 30% galactose diet (known to induce diabetic-like retinopathy) were assigned randomly to receive diet with (2.5 g/kg diet) or without AMG. After 6 to 8 months of diabetes or galactosemia, retinal trypsin digests were prepared, and capillary cell apoptosis was quantitated using the Tdt-mediated dUTP nick-end labeling (TUNEL) reaction in association with morphologic evidence of nuclear fragmentation. At 18 months duration, pericyte ghosts and acellular capillaries were quantitated in the isolated vasculature. Several advanced glycation end products (AGEs) were measured at 4 months of study and at 18 months of study by established methods to assess biochemical effects of AMG. RESULTS: As expected, both diabetic and galactosemic rats showed increased frequency of TUNEL-positive capillary cells at 6 to 8 months and vascular lesions characteristic of retinopathy at 18 months. AMG inhibited both the early apoptosis and late histopathology in the diabetic rats, but neither of these abnormalities in the galactosemic rats. In contrast to its preventative effect on retinopathy in the diabetic rats, AMG showed no inhibitory effect on levels of hemoglobin AGE, or tail collagen pentosidine, fluorescence, and thermal breaking time. Diabetes of 4 months' duration did not cause a detectable increase in retinal levels of several AGEs. CONCLUSIONS: The frequency of early apoptosis in retinal microvascular cells predicted the development of the histologic lesions of retinopathy in diabetes as well as in galactosemia. The beneficial effect of AMG on retinal lesions in diabetes is exerted on pathways that are either not operative or are less important in galactosemia and that may not relate to the accumulation of AGEs.     

Effects of pancreatic islet implantation on the morphology of retinal capillaries in alloxan diabetic mice

Intrasplenic implantation of syngeneic islets normalized the hyperglycaemia of alloxan-diabetic mice irrespective of whether the implantation was performed 2 weeks or 4 months after the induction of diabetes. Islet implantation soon after the onset of diabetes prevented the development of retinal capillary disease as evidenced by a normal ratio between endothelial cells and pericytes. The increase of the cell count ratio (endothelial cells/pericytes) induced by long-term diabetes could not, however, be restored by islet implantation. The present results further underline the importance of optimal treatment for prevention of diabetic retinopathy.     

早期糖尿病大鼠视网膜毛细血管细胞凋亡及其特征

目的：研究早期糖尿病大鼠视网膜毛细血管细胞凋亡及其特征。方法：腹腔内注射链脲佐菌素诱导40只糖尿病大鼠模型,随机分为正常对照组（CON）、糖尿病1月组（DM1）、3月组（DM3）、6月组（DM6）。应用PAS染色、末端脱氧核苷酸转移酶介导的dUTP缺口标记（TUNEL）法标记和染射电镜观察进行研究。结果：于糖尿病3月和6月组,见周细胞和内皮细胞鬼影。周细胞和内皮细胞核异染色质聚集靠边,并随病程进展而加重。被TUNEL法标记的细胞核染色质分布不均,表现为环形核、新月形核等。结论：早期糖尿病大鼠视网膜毛细血管周细胞丧失的性质为细胞凋亡,内皮细胞亦存在调亡。     

糖尿病早期大鼠视网膜神经细胞凋亡与锰超氧化物歧化酶活性及mRNA表达变化的关系

目的 探讨糖尿病早期大鼠视网膜神经细胞凋亡与锰超氧化物歧化酶(MnSOD)活性及mRNA表达变化的关系.方法 对照实验研究.健康雄性8周龄SD大鼠72只,分为正常对照组及糖尿病组.糖尿病组大鼠给予一次性腹腔注射链脲佐菌素(STZ)60 mg/kg体重,以诱导糖尿病大鼠模型;建模成功后再于建模后4、8、12周3个时间点分为3组,每组各12只大鼠.正常对照组不进行干预,与糖尿病组大鼠同时饲养,也按相同时间点分为4、8、12周3组,每组各12只大鼠.采用TdT介导DNA缺口末端的dUTP标记(TUNEL)法检测视网膜神经细胞凋亡指数,免疫组织化学方法检测半胱氨酸天冬氨酸蛋白酶3(caspase-3)蛋白表达水平,黄嘌呤氧化酶法检测MnSOD及铜一锌超氧化物歧化酶(Cu-ZnSOD)活性,实时荧光定量聚合酶链反应(PCR)法检测MnSOD mRNA、Cu-ZnSODmRNA及easpase-3 mRNA表达情况.正常对照组与糖尿病组大鼠各时间点视网膜神经细胞凋亡指数、视网膜caspase-3 mRNA表达水平、Cu-ZnSOD和MnSOD活性及mRNA表达水平比较,均采用两因素方差分析,进一步两两比较采用LSD检验法.结果 (1)正常对照组4、8、12周大鼠视网膜均未见神经细胞凋亡.糖尿病组大鼠于建模后4周即出现神经细胞凋亡,凋亡指数为(0.5±1.5)%;8和12周大鼠神经细胞凋亡指数为(5.7±3.9)%和(11.8±5.1)%.糖尿病组与正常对照组大鼠各时间点视网膜神经细胞凋亡指数差异有统计学意义(F=19.5412,P＜0.05).进一步两两比较,8和12周糖尿病大鼠视网膜神经细胞凋亡指数较同期正常对照组大鼠均明显升高(均P=0.000).(2)正常对照组4、8、12周大鼠视网膜均未见caspase-3蛋白阳性表达,糖尿病组大鼠4周即可见caspase-3蛋白阳性表达,8和12周时表达增强.正常对照组4、8、12周大鼠caspase-3 mRNA表达RQ值分别为1.649±0.586、1.526±0.486及1.614±0.296;糖尿病组4、8、12周大鼠caspase-3 mRNA表达RQ值分别为5.672±1.193、12.566±2.272及14.297±2.11;正常对照组与糖尿病组大鼠各时间点视网膜caspase-3 mRNA表达RQ值差异有统计学意义(F=105.175,P＜0.05).进一步两两比较,糖尿病组大鼠各时间点视网膜caspase-3 mRNA表达RQ值均较同期正常对照组明显升高(均P=0.000).(3)正常对照组4、8、12周大鼠MnSOD活性分别为(47.118±5.018)、(46.033±6.835)及(45.813±6.859)U/mg,MnSOD mRNA表达RQ值分别为0.973±0.123、0.974±0.085及0.994±0.074,CuZnSOD活性分别为(113.884±9.07)、(112.301±5.24)及(117.52±7.982)U/mg,Cu-ZnSOD mRNA表达RQ值分别为1.067±0.109、1.055±0.119及1.092±0.180.糖尿病组4、8、12周大鼠MnSOD活性分别为(33.863±6.909)、(22.877±7.875)及(20.034±6.796)U/mg,MnSOD mRNA表达RQ值分别为0.627±0.083、0.333±0.080及0.256±0.057;8和12周大鼠Cu-ZnSOD活性分别为(98.588±9.212)和(78.168±12.180)U/mg,Cu-ZnSOD mRNA表达RQ值为0.829±0.048和0.621±0.033.正常对照组与糖尿病组大鼠各时间点视网膜MnSOD、Cu-ZnSOD活性及mRNA表达RQ值差异有统计学意义(MnSOD:活性F=19.709,P＜0.05;mRNA F=93.352,P＜0.05;Cu-ZnSOD:活性F=16.708,P＜0.05;mRNA F=16.332,P＜0.05).进一步两两比较,糖尿病组大鼠各时间点视网膜MnSOD、Cu-ZnSOD活性及mRNA表达RQ值均较同期对照组明显下降(MnSOD活性:4周P=0.002,8和12周均P=0.000;MnSOD mRNA:4、8、12周均P=0.000;Cu-ZnSOD活性:8周P=0.011,12周P=0.000;Cu-ZnSOD mRNA:8周P=0.001,12周P=0.000).结论 糖尿病早期视网膜神经细胞凋亡可能与MnSOD活性及MnSOD mRNA表达水平下降有关.     

糖尿病早期大鼠视网膜毛细血管细胞凋亡研究

目的：研究早期糖尿病视网膜病变大鼠视网膜毛细血管细胞凋亡及其特征。方法：腹腔内注射链脲佐菌素诱导糖尿病大鼠模型。应用PAS染色、末端脱氧核苷酸转移酶介导的dUTP缺口标记（TUNEL）法标记和透镜观察进行研究。结果：在糖尿病3月和6月,可见周细胞和内皮细胞鬼影;周细胞和内皮细胞核异染色质聚集靠边并随病程进展而加重;被TUNEL法标记的细胞核染色质分布不均,表现为环形核、新月形核等。结论：早期糖尿病视网膜病变大鼠视网膜毛细血管周细胞丧失的性质为细胞凋亡,内皮细胞亦存在凋亡。     

高糖对牛视网膜微血管内皮细胞和周细胞线粒体活性氧的影响

目的探讨高糖条件下培养的牛视网膜微血管内皮细胞、周细胞线粒体活性氧产生量的变化及导致此种变化的原因和所致影响。方法采用选择性培养方法培养牛视网膜微血管内皮细胞、用细胞,通过共聚焦显微镜检测不同葡萄糖浓度下内皮细胞、周细胞线粒体活性氧产生量的变化,同时采用流式细胞仪检测线粒体膜电化、细胞死亡率的变化,RT—PCR检测培养细胞锰超氧化物歧化酶及解耦联蛋白（UCP）1、2、3表达情况和不同葡萄糖浓度下的变化。结果（1）随着培养基中葡萄糖浓度的增加,内皮细胞、周细胞线粒体活忆氧的产生呈增多趋势。（2）随着培养基中葡萄糖浓度的增加,内皮细胞线粒体膜电位逐渐升高,细胞死亡率逐渐增多,而周细胞则无此变化趋势。（3）培养的视网膜微血管内皮细胞、周细胞中,通过RT—PCR检测到UCPI、2mRNA,未检测到UCP3。UCP1、2及锰超氧化物歧化酶表达量随培养基中葡萄糖浓度的变化而变化。结论高糖可以诱导培养的视网膜微血管内皮细胞、周细胞线粒体活性氧产生增多;内皮细胞活性氧产生的增多与线粒体膜电位增高间存在反馈调节;高糖条件下UCP1、2及锰超氧化物歧化酶表达量代偿性增多调节活性氧的产生,但是当糖浓度达到一定程度时,代偿机制消失;内皮细胞、周细胞在高糖条件下表现出的不同特征,说明其在糖尿病性视网膜病变的发生中起着不同的作用。     

Captopril inhibits capillary degeneration in the early stages of diabetic retinopathy

PURPOSE: This study was conducted to examine the effect of angiotensin-converting enzyme (ACE) inhibitors on the development of early stages of diabetic retinopathy. METHODS: Rats were made diabetic by injection of streptozotocin and treated with the ACE inhibitor captopril and the AT1 antagonist losartan. The extent of capillary degeneration and leukostasis in the retina were determined. RESULTS: Acellular capillaries and pericyte ghosts in the retina of diabetic animals were increased by approximately two-fold after 8 months of diabetes compared with the nondiabetic control, and captopril completely inhibited this capillary degeneration. Captopril and losartan also inhibited hyperglycemia-induced leukostasis at 6 weeks and 1 week in the retinal vasculature, respectively. In cultured retinal endothelial cells, angiotensin II-induced VCAM-1 expression was inhibited by losartan. CONCLUSIONS: Inhibition of the renin-angiotensin system can block retinal capillary degeneration and inflammation in the early stages of diabetic retinopathy.     

Inducible nitric oxide synthase mediates retinal DNA damage in Goto-Kakizaki rat retina

PURPOSE: To examine the nitrosative and oxidative DNA damage induced by 8-nitroguanine and 8-hydroxy-2-deoxy guanosine (8-OHdG), and to determine the role played by inducible nitric oxide synthase (iNOS) in damage to DNA in the retina of the Goto-Kakizaki (GK) rat. METHODS: Experiments were performed on GK rats, an animal model of spontaneous type 2 diabetes without obesity or visible diabetic vascular lesions. Immunohistochemistry was used to determine the retinal distribution of 8-nitroguanine, 8-OHdG, and iNOS in GK rats and control rats. The change in the expression of 8-nitroguanine and 8-OHdG in GK rats was also determined following an intravitreal injection of 1400W, an inhibitor of iNOS activity. RESULTS: Immunohistochemical analysis showed that 8-nitroguanine and 8-OHdG were expressed strongly in the inner nuclear layer of GK retinas but only weakly in control retinas. This expression was correlated with an increase in the expression of iNOS in GK retinas, which was confirmed by the inhibition of iNOS activity by 1400W. CONCLUSION: These findings demonstrate that iNOS plays a crucial role in nitrosative and oxidative DNA damage in GK rats, suggesting a retinal neurotoxic role of nitric oxide and superoxide in diabetic retinas.     

Abnormalities of retinal metabolism in diabetes or experimental galactosemia VIII. Prevention by aminoguanidine

PURPOSE: Aminoguanidine has been found to inhibit the development of some retinal lesions in diabetic rats and diabetic dogs, thereby raising a possibility that the formation of glycation end products (AGEs) may be an essential step in the pathogenesis of the retinopathy. The purpose of this study is to investigate the effect of aminoguanidine administration on other metabolic abnormalities which might be involved in the development of retinopathy in two models of the retinopathy, alloxan diabetes and experimental galactosemia. METHODS: Oxidative stress, nitric oxide (NO) and the activity of protein kinase C (PKC, total activity) were measured in the retina of the rats experimentally diabetic or galactosemic for 2 months. Effect of aminoguanidine administration on the inhibition of hyperglycemia-induced retinal dysmetabolism was investigated. RESULTS: Two months of diabetes or experimental galactosemia in rats resulted in elevation of retinal oxidative stress (increase in thiobarbituric acid reactive substances, TBARS, and decrease in glutathione, GSH), NO, and PKC activity. Aminoguanidine supplementation (2.5 g aminoguanidine/kg rat diet) significantly inhibited each of these abnormalities in retinas of diabetic rats and galactosemic rats, and did so without lowering the blood hexose levels of these animals. CONCLUSIONS: The ability of aminoguanidine to normalize the hyperglycemia-induced increases in retinal oxidative stress, NO and PKC in diabetic rats and galactose-fed rats suggests that these abnormalities may be inter-related in the retina, and that the biochemical mechanism by which aminoguanidine inhibits retinal microvascular disease in diabetes may be complex.