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1.
Curr Eye Res ; 45(6): 696-704, 2020 06.
Article in English | MEDLINE | ID: mdl-31770036

ABSTRACT

Purpose: To evaluate whether a methanolic extract of Ocimum basilicum (OB) leaves prevented lenticular protein alterations in an in-vitro model of selenite-induced cataractogenesis.Materials and Methods: Transparent lenses extirpated from Wistar rats were divided into three groups: control; selenite only; treated. Control lenses were cultured in Dulbecco's modified Eagle's medium (DMEM) alone, selenite only lenses were cultured in DMEM containing sodium selenite only (100 µM selenite/ml DMEM) and treated lenses were cultured in DMEM containing sodium selenite and the methanolic extract of OB leaves (200 µg of extract/ml DMEM); all lenses were cultured for 24 h and then processed. The parameters assessed in lenticular homogenates were lenticular protein sulfhydryl and carbonyl content, calcium level, insoluble to soluble protein ratio, sodium dodecyl sulphate-polyacrylamide gel electrophoretic (SDS-PAGE) patterns of lenticular proteins, and mRNA transcript and protein levels of αA-crystallin and ßB1-crystallins.Results: Selenite only lenses exhibited alterations in all parameters assessed. Treated lenses exhibited values for these parameters that were comparable to those noted in normal control lenses.Conclusions: The methanolic extract of OB leaves prevented alterations in lenticular protein sulfhydryl and carbonyl content, calcium level, insoluble to soluble protein ratio, SDS-PAGE patterns of lenticular proteins, and expression of αA-crystallin and ßB1-crystallin gene and proteins in cultured selenite-challenged lenses. OB may be further evaluated as a promising agent for the prevention of cataract.


Subject(s)
Cataract/prevention & control , Lens, Crystalline/drug effects , Ocimum basilicum/chemistry , Plant Extracts/pharmacology , Sodium Selenite/toxicity , alpha-Crystallin A Chain/metabolism , beta-Crystallin B Chain/metabolism , Animals , Calcium/metabolism , Cataract/chemically induced , Cataract/metabolism , Electrophoresis, Polyacrylamide Gel , Immunoblotting , Lens, Crystalline/metabolism , Methanol , Plant Leaves/chemistry , Protein Carbonylation , Rats , Rats, Wistar , Real-Time Polymerase Chain Reaction , Sulfhydryl Compounds/metabolism
2.
Mol Vis ; 22: 401-23, 2016.
Article in English | MEDLINE | ID: mdl-27168717

ABSTRACT

PURPOSE: Selenite-induced cataract is associated with oxidative stress, loss of calcium homeostasis, activation of calpain enzymes, and apoptotic cell death in the lens. An evaluation of naturally occurring antioxidants that also restrict calcium influx into the lens and calpain activation and thus prevent lenticular cell death may lead to the development of safe and effective anticataractogenic drugs. This study focuses on a naturally occurring flavone, chrysin, and its efficacy in preventing cataractogenic changes in in vitro cultured Wistar rat lenses. METHODS: Lenses from Wistar rats incubated for 24 h at 37 °C in Dulbecco's modified Eagle's medium (DMEM) were categorized into four main groups: Group I (control, incubated in DMEM alone); Group II (selenite-challenged and untreated, incubated in DMEM that contained 100 µM/ml of sodium selenite only); Group III (selenite-challenged and chrysin-treated, incubated in DMEM that contained sodium selenite [100 µM/ml of DMEM] and chrysin [200 µM/ml of DMEM]); and Group IV (chrysin-treated, incubated in DMEM that contained chrysin [200 µM/ml of DMEM] only). The Group III (selenite-challenged and chrysin-treated) lenses were further categorized into five sub-groups: Group IIIa (incubated for 24 h in DMEM that contained sodium selenite and chrysin added simultaneously), Group IIIb (first incubated for 2 h in DMEM that contained chrysin only and then for up to 24 h in fresh DMEM that contained sodium selenite only), Group IIIc (first incubated for 30 min in DMEM that contained sodium selenite only and subsequently for up to 24 h in DMEM that contained chrysin only), and Groups IIId and IIIe (first incubated for 1 h and 2 h, respectively, in DMEM that contained sodium selenite only and subsequently for up to 24 h in DMEM that contained chrysin only). RESULTS: Gross morphological assessment revealed dense opacification (Grade +++) in the selenite-challenged, untreated lenses (Group II); however, seven of the eight selenite-challenged and simultaneously chrysin-treated (Group IIIa) lenses showed no opacification (Grade 0) after 24 h incubation, while the remaining single lens exhibited only a slight degree of opacification (Grade +). In the Group IIIa lenses, the reduced glutathione, protein sulfhydryl, and malondialdehyde concentrations appeared to have been maintained at near-normal levels. The mean lenticular concentration of calcium was significantly lower in the Group IIIa lenses than that in the Group II lenses and approximated the values observed in the normal control (Group I) lenses. The Group IIIa lenses also exhibited significantly (p<0.05) higher mean lenticular activity of calpain, significantly higher mean mRNA transcript levels of genes that encode m-calpain and lenticular preferred calpain (Lp82), and significantly higher mean levels of the m-calpain and Lp82 proteins than the corresponding values in the Group II lenses. Casein zymography results suggested that chrysin prevented calpain activation and autolysis. Significantly (p<0.05) lower mean levels of mRNA transcripts of the genes that encode calcium transporter proteins (plasma membrane Ca(2+)-ATPase-1 and sarco/endoplasmic reticulum Ca(2+)-ATPase-2) and lenticular apoptotic-cascade proteins (early growth response protein-1, caspase-3, caspase-8, and caspase-9) and significantly (p<0.05) lower mean concentrations of the proteins themselves were seen in the Group IIIa rat lenses in comparison to the values noted in the Group II rat lenses. CONCLUSIONS: Chrysin appears to prevent selenite-induced cataractogenesis in vitro by maintaining the redox system components at near-normal levels and by preventing the abnormal expression of several lenticular calcium transporters and apoptotic-cascade proteins, thus preventing accumulation of calcium and subsequent calpain activation and lenticular cell death in cultured Wistar rat lenses.


Subject(s)
Apoptosis Regulatory Proteins/metabolism , Calpain/metabolism , Cataract/prevention & control , Flavonoids/pharmacology , Lens, Crystalline/drug effects , TRPV Cation Channels/metabolism , Animals , Antioxidants/pharmacology , Apoptosis Regulatory Proteins/genetics , Calcium/metabolism , Calpain/genetics , Cataract/chemically induced , Cataract/metabolism , Cataract/pathology , Glutathione/metabolism , Lens, Crystalline/metabolism , Lens, Crystalline/pathology , Lipid Peroxidation , Malondialdehyde/metabolism , Organ Culture Techniques , RNA, Messenger/genetics , Rats , Rats, Wistar , Real-Time Polymerase Chain Reaction , Sodium Selenite/toxicity , TRPV Cation Channels/genetics
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