Effects And Mechanisms Of Milk On Naphthalene-induced Cataract

PARTⅠEffect of calpain II on oxidative damage naphthalene-induced cataractPurpose Naphthalene-induced cataract has been extensively used to test potential anticataract drugs. We sought to find out whether naphthalene cataractogenesis was a valuable animal model to study etiology of age-related cataract, and tried to clarify the possible mechanism for these changes.Methods Thirty female Sprague-Dawley rats were divided into two groups:control, and naphthalene group. Cataracts were induced by oral administration of 1 g naphthalene/kg body weight/day. The development of naphthalene-initiated cataract was monitored every week by slit lamp microscopy for 9 weeks, then Concentrations of reduced glutathione (GSH), and malondialdehyde (MDA) in rat lenses were measured to determine the role of oxidative stress in cataract induction. The total concentrations of Ca2+ were measured by atomic absorption spectrometry and the intro-cell Ca2+ were measured by co-focal microscope. Activity of calpain II was assayed fluorometrically with Fluorescence-labeled casein. The lens proteins were separated by HPLC, and peaks corresponding to alpha-crystallins were resolved on 2-DE. The spots of 2-DE were subjected to mass spectrometry to identify PTMs. Chaperone activity of alpha-crystallins was measured by heat-induced aggregation of PL-crystallin.Results The lenses of rats fed with naphthalene exhibited higher light scattering than that fed with only naphthalene at 3 weeks after treatment (P< 0.01). GSH concentrations in lenses of rats fed with naphthalene were lower than in controls (p<0.05)。MDA concentrations in naphthalene group were significantly higher than that in control group (p<0.05), indicating increased lipid peroxidation with naphthalene treatment. C-terminal truncatedαA-crystallin was detected in naphthalene-induced cataract and Several other post-translational modifications were identified including methylation, phosphorylation, acetylation, carbamylation, and oxidation.Conclusions The morphology as well as toxic manifestation of naphthalene were similar as that of age-related cataract. Activity of calpainⅡwas raised by naphthalene in our study. Our data showed for the first time that chaperone activity ofα-crystallins decreased in naphthalene-induced cataract.By mass spectrometry, we detected C-terminal truncation of 16 amino acids and other posttranslational modifications (PTMs) such as acetylation, phosphorylation, oxidation and carbamylation ofα-crystallins. Furthermore, our study imply from proteomics level that naphthalene-induced is a valuable animal mode for study age-related cataract. PARTⅡMolecular mechanism of calpainⅡon naphthoquinone-induced oxidative damage of lens epithelial cellsPurpose To investigate the effects of naphthoquinone on expression and activity of calpainⅡin lens epithelial cells, and tried to clarify the possible mechanism for these changes.Methods The effect of naphthoquinone on apoptosis of lens epithelial cells was measured by flow cytometry. The level of reactive oxygen species (ROS) was measured by Fluorescence probe DCFH-DA, and the level of intro-cell Ca2+ was measured by con-focal microscope. The expression of calpainⅡon protein level was measured by western blot and immunofluorescence, and the expression of calpainⅡon mRNA level was measured by Real Time-PCR. The activity of calpainⅡwas evaluated by quantity ofaB measured with western blot test.Results The apoptosis rates of lens epithelial cells were raised in naphthoquinone group by 12,24 and 36 hours after treatment, which were inhibited byE64D. The level of ROS were also elevated in naphthoquinone by12,24 and 36 hours after treatment, which were inhibited byE64D,and there was no significant difference between naphthoquinone and E64D group and control group at 12 hours after treatment. The level of intro-cell Ca2+ were elevated in naphthoquinone by 12,24 and 36 hours after treatment, which were inhibited byE64D,and there was no significant difference between naphthoquinone and E64D group and control group at 12 hours after treatment. The expression of calpainⅡon protein level and the expression of calpainⅡon mRNA level were that naphthoquinone group> naphthoquinone and E64D group>control group. The quantity ofaB was that control group> naphthoquinone and E64D group> naphthoquinone group.Conclusions Oxidative damage of naphthoquinone could lead to apoptosis of lens epithelial cells by elevation of calpainⅡwhich was inhibited by E64D. PARTⅢExcessive intake of milk as a risk factor for naphthalene-initiated cataract is probably associated with oxidative stressPurpose To determine whether excessive milk consumption induces naphthalene-initiated cataract, a common animal model of age-related human cataract.Methods Ninety rats were fed a natural diet supplemented with either:water (control), normal milk, excessive mil,naphthalene, and milk plus naphthalene. Cataract development was monitored weekly using slit lamp and lens gray value analysis. Concentrations of reactive oxygen species (ROS), reduced glutathione (GSH), and malondialdehyde (MDA) in rat lenses were measured to determine the role of oxidative stress in cataract induction.Results By week 3, the cortical gray value was significantly higher in rats fed milk plus naphthalene than in rats fed naphthalene, while after 8 weeks, no significant difference was observed between rats fed milk or water without naphthalene. ROS concentrations in lenses of rats fed milk were slightly higher than in controls, but ROS concentrations in rats fed milk plus naphthalene were significantly higher than in those fed water plus naphthalene. GSH concentrations in lenses of rats fed milk were lower than in controls, while GSH concentrations in rats fed milk plus naphthalene were significantly lower than in rats fed water plus naphthalene. MDA concentrations in rats given milk plus naphthalene were significantly higher than in those given water plus naphthalene, indicating increased lipid peroxidation with milk plus naphthalene intake. The concentration of galactose in plasma, aqueous humor, and lens were all increased in the milk group compared with the control group. There was a significantly higher concentration of galactose in the naphthalene plus milk group than in the milk group.There was a slight difference in Ca2+ concentration in plasma between the milk group and the control group (p=0.08), however, there was a significant difference in Ca2+ concentration in plasma, aqueous humor, and lens between the naphthalene group and the control group.Conclusions Our results provide quantitative evidence that excessive milk intake induces naphthalene-initiated cataract due, in part, to oxidative damage caused by ROS that may result from metabolic processing of milk components.