Prevention of Selenite-Induced Cataractogenesis by an Ethanolic Extract of Cineraria maritima: An Experimental Evaluation of the Traditional Eye Medication

In the present study, the antioxidant potential of an ethanolic extract of Cineraria maritima and its efficacy in preventing selenite-induced cataractogenesis were assessed in vitro and in vivo. In the in vitro phase of the study, lenses dissected out from the eyes of Wistar rats were incubated for 24 h at 37°C in Dulbecco’s modified Eagle medium (DMEM) alone (group I), in DMEM containing 100 μM of selenite only (group II), or in DMEM containing 100 μM of selenite and 300 μg/ml C. maritima extract added at the same time (group III). Gross morphological examination of the lenses revealed dense opacification in group II, minimal opacification in group III, and no opacification in group I lenses. The mean activities of the antioxidant enzymes catalase, glutathione peroxidase, and superoxide dismutase were significantly lower in group II than in group I or group III lenses, while malondialdehyde concentration was significantly higher in group II lenses than in group I and group III lenses. In the in vivo phase of the study, dense opacification of lenses was noted in all rat pups (100%) that had received a single subcutaneous injection of sodium selenite alone (19 μM/kg body weight) on postpartum day 10, whereas cataract formation occurred in only 33.3% of rat pups that had received selenite as well as an intraperitoneal injection of the extract of C. maritima (350 mg/kg body weight) for five consecutive days. These observations suggest that the ethanolic extract of C. maritima may prevent experimental selenite-induced cataractogenesis.     

TAT-mediated PRDX6 protein transduction protects against eye lens epithelial cell death and delays lens opacity

A diminished level of endogenous antioxidant in cells/tissues is associated with reduced resistance to oxidative stress. Peroxiredoxin 6 (PRDX6), a protective molecule, regulates gene expression/function by controlling reactive oxygen species (ROS) levels. Using PRDX6 protein linked to TAT, the transduction domain from human immunodeficiency virus type 1 TAT protein, we demonstrated that PRDX6 was transduced into lens epithelial cells derived from rat or mouse lenses. The protein was biologically active, negatively regulating apoptosis and delaying progression of cataractogenesis by attenuating deleterious signaling. Lens epithelial cells from cataractous lenses bore elevated levels of ROS and were susceptible to oxidative stress. These cells harbored increased levels of active transforming growth factor (TGF)-beta 1 and of alpha-smooth muscle actin and beta ig-h3, markers for cataractogenesis. Importantly, cataractous lenses showed a 10-fold reduction in PRDX6 expression, whereas TGF-beta1 mRNA and protein levels were elevated. The changes were reversed, and cataractogenesis was delayed when PRDX6 was supplied. Results suggest that delivery of PRDX6 can postpone cataractogenesis, and this should be an effective approach to delaying cataracts and other degenerative diseases that are associated with increased ROS.     

Lipid peroxidation in cataract of the human

Lipid peroxidation was investigated as one of the possible mechanisms of cataractogenesis in the human. Malondialdehyde (MDA), a major breakdown product of lipid peroxides, was significantly higher in cataractous lenses as compared to that in normal lenses. 2-Thiobarbituric acid-reactive material, isolated from cortical cataracts and purified by Sephadex G-10 column chromatography, was identified as MDA. In cataractous lenses the enzymic defenses against reactive species of O2 were impaired as evidenced by the significant decrease in activities of superoxide dismutase, catalase and glutathione peroxidase. Hydrogen peroxide in aqueous humor and vitreous humor of human eyes associated with cataract was increased 2-3 fold. It is possible that carbonyl groups of MDA could interact with primary amino groups of proteins and phospholipids of lenticular plasmalemmae by a cross-linking reaction forming Schiff-base conjugates and these mechanisms might be involved in the pathogenesis of cataract.     

Expression changes in mRNAs and mitochondrial damage in lens epithelial cells with selenite

An overdose of sodium selenite induces cataracts in young rats. The mid-stage events producing the cataract include calpain-induced hydrolysis and precipitation of lens proteins. Apoptosis in lens epithelial cells has been suggested as an initial event in selenite cataracts. Expression levels of two genes associated with apoptosis were altered in lens epithelial cells from selenite-injected rats. The purpose of the present experiment was to perform a more comprehensive search for changes in expression of mRNAs in lens epithelial cells in order to more fully delineate the early events in selenite-induced cataracts. Lens epithelial cells were harvested at 1 and 2 days after a single subcutaneous injection of sodium selenite (30 mumol/kg body weight) into 12-day-old rats. Gene expression was analyzed using a commercial DNA array (Rat Genome U34A GeneChip array, Affymetrix). Of approximately 8000 genes assayed by hybridization, 13 genes were decreased and 27 genes were increased in the rat lens epithelial cells after injection of selenite. Some of the up-regulated genes included apoptosis-related genes, and a majority of the down-regulated genes were mitochondrial genes. Previously observed changes in expression of EGR-1 mRNA were also confirmed. Changes in the expression patterns of mRNAs were also confirmed by RT-PCR. To determine the mechanism for damage of lens epithelial cells (alpha TN4 cell) by culture in selenite, leakage of cytochrome c from mitochondria was measured. Selenite caused significant leakage of cytochrome c into the cytosol of alpha TN4 cells. Our data suggested that the loss of integrity of lens epithelial cells by selenite might be caused by preferential down-regulation of mitochondrial RNAs, release of cytochrome c, and impaired mitochondrial function. Up-regulation of mRNAs involved in maintenance of DNA, regulation of metabolism, and induction of apoptosis may also play roles.     

The effect of melatonin on eye lens of rats exposed to ultraviolet radiation

We investigated the influence of exogenously administered melatonin on adult rats eye lenses exposed to ultraviolet radiation (UV) A and B ranging from 356-254 nm irradiation at 8 microW/cm(2). Rats exposed to this range of UV for 15 min for one week showed a significant (P<0.05) reduction in antioxidant enzymes activities; superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and elevated (P<0.001) lipid peroxidation served as an index of cellular damage by free radicals. UV-radiation significantly (P<0.001) elevated calcium ions (Ca(2+)) and lactate dehydrogenase (LDH) activity in lenses. Depleting animals of their stores of important intracellular antioxidant and elevating lenticular Ca(2+) by UV irradiation, may be the main cause of lens opacification. Melatonin injection with radiation significantly reduced (P<0.05) lipid peroxidation, Ca(2+) and (P<0.001) for LDH. When melatonin was injected after radiation, SOD and GSH-Px enzyme activities increased significantly (P<0.01), and lipid peroxidation, Ca(2+) levels and LDH activities were reduced significantly. Melatonin injection after UV radiation was as effective as melatonin treatment concurrent with UV irradiation. We conclude that melatonin may protect the eye lens from the damaging effects of UV exposure, and its actions protect lens from oxidative stress, elevating Ca(2+) levels, which are considered as an important causes of cataractogenesis.     

Acetyl-<Emphasis Type="SmallCaps">l</Emphasis>-carnitine prevents carbon tetrachloride-induced oxidative stress in various tissues of Wistar rats

Acetyl-l-carnitine (ALCAR) has been shown to prevent experimental selenite cataractogenesis, a manifestation of oxidative stress, but little is known about its potential in other settings of oxidative stress. The present study was based on the hypothesis that ALCAR prevents carbon tetrachloride (CCl₄)-induced oxidative stress in vital tissues. Male albino Wistar rats were divided into three groups, each of six rats. Group I (control) rats received only vehicle (1 ml/kg b.w.) for 4 days; Group II (CCl₄-exposed, untreated) rats received CCl₄ (2 ml/kg b.w.) on the second and third days and vehicle on the first and fourth days; Group III (CCl₄-exposed, ALCAR-treated) rats received ALCAR (200 mg/kg b.w.) for 4 days and CCl₄ on the second and third days. All administrations were made intraperitoneally. After the experimental period, significantly (P < 0.05) elevated mean serum levels of aspartate transaminase, alanine transaminase, alkaline phosphatase, and lactate dehydrogenase were observed in Group II rats when compared to Group I and Group III rats. The mean levels of vitamin C, vitamin E, and reduced glutathione and the mean activities of superoxide dismutase, catalase, and glutathione peroxidase were significantly (P < 0.05) lower in samples of hemolysate and of liver, kidney, and brain tissues of Group II rats than those in Group I and Group III rats. The mean level of lipid peroxidation was significantly (P < 0.05) higher in Group II rats than that in Group I and Group III rats. Moreover, the CCl₄-induced upregulation of inducible nitric oxide synthase expression was prevented by ALCAR in the liver and brain tissues. These results suggest that ALCAR is able to prevent the CCl₄-induced oxidative stress.     

Prevention of selenite induced oxidative stress and cataractogenesis by luteolin isolated from Vitex negundo

Free radical mediated oxidative stress plays a crucial role in the pathogenesis of cataract and the present study was to determine the efficacy of luteolin in preventing selenite induced oxidative stress and cataractogenesis in vitro. Luteolin is a bioactive flavonoid, isolated and characterized from the leaves of Vitex negundo. Lenses were extracted from Sprague-Dawley strain rats and were organ cultured in DMEM medium. They were divided into three groups with eight lenses in each group as follows: lenses cultured in normal medium (G I), supplemented with 0.1mM sodium selenite (G II) and sodium selenite and 2 μg/ml luteolin (G III). Treatment was from the second to fifth day, while selenite administration was done on the third day. After the experimental period, lenses were taken out and various parameters were studied. The antioxidant potential of luteolin was assessed by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity. In the selenite induced group, morphological examination of the lenses showed dense cortical opacification and vacuolization. Biochemical examinations revealed a significant decrease in activities of antioxidant enzymes and enzymes of the glutathione system. Additionally decreased glutathione level and increased reactive oxygen species (ROS) and thiobarbituric acid reactive substances (TBARS) were observed. Luteolin treatment abated selenite induced oxidative stress and cataractogenesis by maintaining antioxidant status, reducing ROS generation and lipid peroxidation in the lens. These finding demonstrated the anticataractogenic effect of luteolin by virtue of its antioxidant property, which has been reported in this paper for the first time.     

Age-associated oxidative damage leads to absence of gamma-cystathionase in over 50% of rat lenses: relevance in cataractogenesis

Oxidative damage to lens proteins and glutathione depletion play a major role in the development of senile cataract. We previously found that a deficiency in gamma-cystathionase activity may be responsible for glutathione depletion in old lenses. The aims of this study were: (1) to investigate the mechanism that causes the age-related deficiency in gamma-cystathionase activity in the eye lens, and (2) to determine the role of gamma-cystathionase deficiency in cataractogenesis. Two populations of old rats were found, one (56%) whose lenses lacked gamma-cystathionase activity and the rest that exhibited detectable enzyme activity. gamma-Cystathionase protein was absent in lenses from old rats without gamma-cystathionase activity. Oxidative stress targeted gamma-cystathionase in the eye lens upon aging, since the enzyme contained more carbonyl groups in old lenses than in young ones. gamma-Cystathionase mRNA was also markedly reduced in old lenses, thus contributing to the age-associated deficiency in gamma-cystathionase. Inhibition of gamma-cystathionase activity caused glutathione depletion in lenses and led to cataractogenesis in vitro. In conclusion, the lack of gamma-cystathionase activity in over 50% of old lenses is due to decreased gene expression and proteolytic degradation of the oxidized enzyme. This results in a high risk for the development of senile cataract.     

C-Phycocyanin Modulates Selenite-Induced Cataractogenesis in Rats

The present investigation is aimed to evaluate the anticataractogenic potential of C-phycocyanin (C-PC), extracted and purified from Spirulina platensis. Enucleated rat lenses were maintained in vitro in Dulbecco’s modified Eagle medium (DMEM). Group I contained DMEM, Group II and Group III contained 100 μM of sodium selenite, Group III was subdivided into three viz IIIa, IIIb, IIIc supplemented with 100, 150, 200 μg of C-PC respectively. In the in vivo study, on tenth day post partum: Group I rat pups received an intraperitoneal injection of saline, Group II, IIIa, IIIb, and IIIc rat pups received a subcutaneous injection of sodium selenite (19 μmol/kg bodyweight) Group IIIa, IIIb, IIIc also received an intraperitoneal injection of 100, 150, 200 mg/kg body weight of C-PC, respectively, from postpartum days?9–14. On termination of the experiment, the lenses from both in vitro and in vivo studies were subjected to morphological examination and subsequently processed to estimate the activities of antioxidant enzymes namely superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, levels of reduced glutathione and lipid peroxidation products. Sodium selenite-exposed, C-PC-treated rat lenses (Group IIIc), showed significant restoration of antioxidant enzyme activity (p?<?0.05) when compared to their counterpart Group II. Group IIIc conserved the levels of GSH and lipid peroxidation products at near to normal levels as compared with Group II. Results conclude the possible role of C-PC in modulating the antioxidant enzyme status, thereby retarding sodium selenite-induced cataract incidence both in vitro and in vivo.     

Oral consumption of α-glucosyl-hesperidin could prevent lens hardening,which causes presbyopia

Presbyopia is one of the most well-known diseases of the eye, predominantly affecting the adult population after 50 years’. Due to hardening of the lens and failure of accommodative change, patients lose the ability to focus on near objects. This eye symptom is reported to be an early symptom of age-related nuclear cataract, and we have previously reported that hesperetin treatment could delay the onset of nuclear cataractogenesis induced by sodium selenite. In this study, we examined whether oral intake of α-glucosyl-hesperidin (G-Hsd), which has greater water solubility than hesperetin, could delay the onset of presbyopia. G-Hsd treatment protected lens elasticity, upregulated the mRNA expression of anti-oxidative enzymes like glutathione reductase and thioredoxin reductase 1 in the plasma and lens, and prevented premature cataract symptoms in selenite-induced cataract rat lens. Thus, the anti-presbyopic effects of G-Hsd were attributed, at least in part, to its antioxidant effects. G-Hsd represents the first oral treatment agent with anti-presbyopia and/or anti-cataract properties.     

紫外线对大鼠晶状体抗氧化酶mRNA表达水平的影响

为探讨紫外线对晶状体的损伤机制,用ＲＴ－ＰＣＲ方法（ｒｅｖｅｒｓｅｔｒａｎｓｃｒｉｐｔｉｏｎ－ｐｏｌｙｍｅｒａｓｅｃｈａｉｎｒｅａｃｔｉｏｎ,反转录聚合酶链反应）,研究经紫外线照射后大鼠晶状体抗氧化相关酶,包括铜锌－超氧化物歧化酶（ｃｏｐｐｅｒ－ｚｉｎｃ－ｓｕｐｅｒｏｘｉｄｅｄｉｓｍｕｔａｓｅ,Ｃｕ－Ｚｎ－ＳＯＤ）,谷胱甘肽过氧化物酶（ｇｌｕ－ｔａｔｈｉｏｎｅｐｅｒｏｘｉｄａｓｅ,ＧＳＨ－Ｐｘ）和过氧化氢酶（ｃａｔａｌａｓｅ,ＣＡＴ）等ｍＲＮＡ的表达．结果显示,短时间的照射（２～５ｍｉｎ）,抗氧化相关酶的ｍＲＮＡ表达水平有增高表现,随后其ｍＲＮＡ表达水平开始下降,１５ｍｉｎ时抗氧化相关酶ｍＲＮＡ的表达下降更为明显,与对照组相比有非常显著性差异（Ｐ＜０．００１）．照射后２４ｈ,抗氧化相关酶的ｍＲＮＡ表达有不同程度的恢复;照射后４８ｈ,其ｍＲＮＡ表达水平基本恢复,与对照组相比没有显著性差异．从而从基因水平上初步探讨了紫外线的氧化损伤机制