Rutin ameliorates free radical mediated cataract by enhancing the chaperone activity of α-crystallin.

BACKGROUND: Cataract, the leading cause of blindness, is associated with oxidative damage and protein modification in the lens. The present study was carried out to assess the efficacy of rutin on rat-lens crystallins in selenite-induced in-vivo cataract models. METHODS: Eight-day-old Sprague-Dawley rat pups were grouped as control (G I), experimental (G II) and rutin-treated (G III). The rat pups in G II, and G III received a single subcutaneous injection of sodium selenite (4 µg/g body weight) and G I received a single subcutaneous injection of sterile water on the 10th day. The treatment groups (G III) were administered with rutin (1 µg/g body weight) respectively from the 8th to 15th day. Cataract was visualized from the 16th day. Lens crystallins (α, ß, and γ) were isolated by size exclusion chromatography. Chaperone activity of isolated crystallins was measured by heat, DTT, and oxidation-induced aggregation and refolding assays. Concentration of total protein (soluble and insoluble) and SDS-PAGE analysis of soluble proteins were also done. RESULTS: Treatment with rutin prevented the loss of α crystallin chaperone property, and protein insolubilization prevailed during selenite-induced cataract. CONCLUSIONS: These results suggest the therapeutic potential of rutin, a bioflavonoid, against selenite-induced cataract, which has been reported in this paper for the first time. The work assumes significance, as this is a novel approach in modulating the chaperone activity of lens crystallins in selenite-induced cataract by a natural product.

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.

Analysis on the alterations of lens proteins by Vitex negundo in selenite cataract models.

PURPOSE: Cataract is the leading cause of blindness and is associated with oxidative damage and protein modification in the lens. In the present study, we have employed proteomic and microscopic approaches to investigate the attenuation of selenite cataract by the flavonoids from Vitex negundo (FVN). METHODS: To demonstrate this attenuation, Sprague-Dawley rat pups were divided into control (G I), selenite induced (G II), and selenite + FVN treated (G III). Cataract was induced by single subcutaneous injection of sodium selenite (4 mg/Kg bodyweight) on the 10th day and FVN (1 mg/Kg bodyweight) administered intraperitoneally from the 8th to the 15th day. RESULTS: Our study indicated that chaperone property of α-crystallin and soluble protein levels were reduced in the selenite induced group. Post translational modifications identified by two dimensional-polyacrylamide gel electrophoresis (2D-PAGE) and immunoblot analysis revealed the loss of cytoskeletal proteins in selenite induced group. Damage of lenticular membrane and abnormal fiber structure were observed by electron microscopy. CONCLUSIONS: The results of this study suggest that FVN modulated selenite induced cataractogensis in rat pups by preventing loss of chaperone property, various changes in lens proteins, and lens structure, further strengthening its protective role.

Amelioration of selenite toxicity and cataractogenesis in cultured rat lenses by Vitex negundo.

PURPOSE: Recent evidence suggests that oxidative stress plays a significant role in the development of cataract. The present study sought to evaluate the efficacy of flavonoid fraction of Vitex negundo (FVN) in preventing the toxicity induced by sodium selenite in vitro culture condition. METHODS: Enucleated rat lenses were maintained in organ culture containing DMEM medium alone (G I), supplemented with 0.1mM sodium selenite (G II), sodium selenite + 20 µg/ml quercetin (G III) and sodium selenite + 50 µg/ml FVN (G IV). 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 the activities of superoxide dismutase (SOD), catalase, Ca(2+) ATPase, levels of reduced glutathione (GSH), calcium, reactive oxygen species (ROS), thiobarbituric acid reactive substances (TBARS), and sulfhydryl content were studied. RESULTS: Morphological examination revealed dense vacuolization and loss of cortical transparency in G II compared to control and treated group. The mean activities of the enzymes SOD, catalase and Ca(2+) ATPase, levels of GSH and sulfhydryl content were significantly reduced in lenses of G II compared to control. In addition, the mean levels of ROS, calcium and TBARS were elevated in G II compared to control. However, these changes were modulated by FVN treatment to further strengthen its protective role over selenite cataract. CONCLUSION: These results suggest that FVN treatment prevented selenite toxicity and cataractogenesis by maintaining antioxidant status, calcium homeostasis, protecting sulfhydryl group, and decreasing oxidative stress in lens, which may be due to its protective effects.

Moringa oleifera prevents selenite-induced cataractogenesis in rat pups.

PURPOSE: The aim of this study was to investigate the protective effects of the flavonoid fraction of Moringa oleifera leaves (FMO) on selenite cataract in vivo. METHODS: Rat pups of Sprague-Dawley strain initially weighing 10­12 g on day 8 were used for the study and grouped as control (G I), selenite induced (G II), and FMO treated (G III). The rat pups in G II and G III received a single subcutaneous injection of sodium selenite (4 µg/g body weight) on day 10 and G III was administered with FMO (2.5 µg/g body weight) from day 8 to 15. Cataract was visualized from day 16. The development of cataract was assessed and rat lenses were analyzed for the activities of antioxidant enzymes (superoxide dismutase and catalase), generation of reactive oxygen species, reduced glutathione, protein oxidation, and lipid peroxidation. FMO was subjected to in vitro antioxidant assays (2,2-diphenyl-picrylhydrazyl and superoxide scavenging assays). RESULTS: The total phenolic content of FMO was 4.4 mg of catechin equivalent/g dried plant material. The extract showed remarkable activity on 2,2-diphenyl-picrylhydrazyl (IC50 36 µg/mL) and in superoxide radical (IC50 33.81µg/mL) scavenging assays. FMO effectively prevented the morphological changes and oxidative damage in lens. FMO maintained the activities of antioxidant enzymes and sulfhydryl content and prevented reactive oxygen species generation and lipid peroxidation. CONCLUSIONS: FMO was effective in preventing cataractogenesis in selenite model by enhancing the activities of antioxidant enzyme, reducing the intensity of lipid peroxidation, and inhibiting free radical generation.

Isorhamnetin-3-glucoside alleviates oxidative stress and opacification in selenite cataract in vitro.

Oxidative stress has long been recognized as an important mediator in the pathogenesis of cataract and the goal of this study was to determine the efficacy of isorhamnetin-3-glucoside (IR3G) in alleviating the toxicity induced by sodium selenite in in vitro culture condition. IR3G is the bioactive flavonoid isolated and characterized from the leaves of Cochlospermum religiosum. Enucleated rat lenses were maintained in organ culture containing M-199 medium alone (G-I), supplemented with 0.1 mM selenite (G-II) and selenite + 25 microg/ml IR3G (G-III). Treatment to G-III was from the second to fifth day while selenite administration to G-II & III was done on the third day. The antioxidant potential of the compound was assessed by Cu(2+) induced lipoprotein diene formation and superoxide scavenging assays. Morphological examination of the lenses also gave a supporting data. Antioxidant enzymes-superoxide dismutase (SOD), catalase and concentration of reduced glutathione (GSH) were significantly lower, while TBARS showed an increase in G-II than that in G-III and G-I lenses. Activity of Ca(2+)-ATPase was decreased and level of calcium was increased in G-II than G-III and G-I lenses. These data suggest that IR3G is able to significantly retard selenite cataract in vitro by virtue of its antioxidant property.

Vitex negundo modulates selenite-induced opacification and cataractogensis in rat pups.

Recently, much interest has been generated in the search for phytochemical therapeutics, as they are largely free from adverse side effects and economical. The goal of this study was to determine the efficacy of Vitex negundo in modulating the selenite-induced oxidative stress in vivo model. Sprague-Dawley rat pups of 8 days old were used for the study and divided into control (G I), selenite induced (G II), and selenite + V. negundo treated (G III). Cataract was induced by the single subcutaneous injection of sodium selenite (4 mg/kg body weight) on the tenth day and V. negundo (2.5 mg/Kg body weight) administered intraperitoneally from eighth to 15th day. Morphological examination of the rat lenses revealed no opacification in G I and mild opacification in G III whereas dense opacification in G II (stages 4-6). Levels of selenium in G II and G III showed no significant changes. The activities of superoxide dismutase, catalase, and Ca(2+)ATPase were significantly increased in G III compared to G II (p < 0.05), while lower level of reactive oxygen species, Ca(2+), and thiobarbituric acid reactive substances were observed in G III compared G II (p < 0.05). These results indicate the therapeutic potential of methanolic extract of V. negundo on modulating biochemical parameters against selenite-induced cataract, which have been reported in this paper for the first time.

Vitex negundo attenuates calpain activation and cataractogenesis in selenite models.

Recent investigations have shown that phytochemical antioxidants can scavenge free radicals and prevent various diseases. Cataract is the leading cause of blindness and is associated with oxidative damage of the lens. Selenite-induced cataract in rat pups is an excellent mimic of oxidative stress-induced cataract. Selenite cataract is associated with oxidative stress, loss of calcium homeostasis, calpain activation and protein insolubilization in the lens. Our present study focuses on the isolation of flavonoids from Vitex negundo and to assess its efficacy in preventing these changes in the lens of selenite-induced cataract models. Eight-day-old Sprague-Dawley rat pups were used for the study and divided into four groups: Control (G I), Sodium selenite-induced (G II), Sodium selenite+quercetin treated (G III), Sodium selenite+flavonoids from Vitex negundo (FVN) (G IV). Cataract was induced by a single subcutaneous injection of Sodium selenite (4 mg/Kg body weight) on the 10th day. Treatment groups received quercetin (1.0mg/Kg body weight) and FVN (1.0mg/Kg body weight) intraperitoneally from 8th to 15th day. Cataract was visualized from the 16th day. Morphological examination of the rat lenses revealed no opacification in G I and mild opacification in G III and G IV (stage 1) whereas dense opacification in G II (stage 4-6). The activities of superoxide dismutase (SOD), catalase, Ca(2+)ATPase, concentration of reduced glutathione (GSH) and protein sulfhydryl content were significantly increased in G III and G IV compared to G II, while decreased activities of calpains, lower concentration of calcium and thiobarbituric acid reactive substances (TBARS) were observed in G III and IV as compared to G II. Lens protein profile of water soluble proteins showed normal levels of expression in treated groups compared to that of selenite-induced rats. These results indicate good antioxidant and therapeutic potential of FVN in modulating biochemical parameters against selenite-induced cataract, which have been reported in this paper for the first time.

Drevogenin D prevents selenite-induced oxidative stress and calpain activation in cultured rat lens.

PURPOSE: Selenite-induced cataractogenesis is mediated by oxidative stress, accumulation of calcium and activation of lenticular calpains. Calpains are a super family of Ca2+ dependent proteases, which are involved in lens protein proteolysis and insolubilization. Many inhibitors could prevent calpain-induced proteolysis of alpha- and beta-crystallins in rodent cataracts. Evaluating natural sources with antioxidant property and subsequent prevention of calpain activation may lead to the development of safer and more effective agents against cataractogenesis. There are no reports on the protective role of bioactive components against calpain-mediated proteolysis and subsequent cataractogenesis. The purpose of the study was to evaluate the role of Drevogenin D, a triterpenoid aglycone, isolated from Dregea volubilis in preventing selenite-induced, calcium-activated, calpain-mediated proteolysis in cultured rat lenses. METHODS: Lenses were extracted from Sprague-Dawley strain rats at the age of one month and were organ cultured in M-199 medium with HEPES buffer. The lenses were divided into three groups with eight lenses in each group as follows: lenses cultured in a normal medium (GI), lenses cultured in a sodium selenite supplemented medium (GII), and lenses cultured in a medium supplemented with sodium selenite and Drevogenin D-treated (GIII). Changes to transparency and opacity formation of lenses were monitored under microscopic observation. At the end of the experiment, biochemical parameters such as activity of lens superoxide dismutase (SOD), lens Ca2+ ATPase, concentration of Ca2+, levels of sulfhydryl content, and thiobarbituric acid reacting substances (TBARS) were determined. Changes to casein zymography for calpains, immunoblot for Lp82, and SDS-PAGE of lens water soluble protein fraction (WSF) were also done. RESULTS: Microscopic evaluation of lens morphology showed that Drevogenin D prevented the opacification in G-III. Drevogenin D inhibited the accumulation of calcium, the activation of calpain system, and lipid peroxidation. Activity of Ca2+ ATPase, SOD, and SDS-PAGE profile of water soluble proteins was normalized following treatment with Drevogenin D. CONCLUSIONS: Selenite-induced cataractogenesis is mediated by oxidative stress leading to a decrease in the activity of Ca2+ ATPase, resulting in the accumulation of calcium and the subsequent activation of lenticular calpains. The results obtained indicated that Drevogenin D treatment was effective in protecting the lens proteins by controlling stress-induced protein oxidation, maintenance of Ca2+ ATPase activity, calcium accumulation, lipid peroxidation, and prevention of calpain activation. Hence, Drevogenin D can be used as a potential therapeutic agent against oxidative stress-induced cataract.