Effects of ultraviolet-B laser irradiation on lens α-crystallin and protection of indole-3-carbinol to chaperone activity of α-crystallin / 中华实验眼科杂志

ABSTRACT

Background Ultraviolet radiation is one of factors of the formation of age-related cataract.Indole-3-carbinol(I3C) is a plant chemical material with inhibitory effect on oxidative-induced cell damage and formation of amyloid fibrils,and the oxidative damage and amyloid fibrils are associated with cataract.However,the relationship between I3C and α-crystallin is in study. Objective This study was to evaluate the effects of ultraviolet-B laser irradiation on the secondary structure of α-crystallin and to explore the protection of I3C to chaperone activity of α-crystallin. Methods The fresh eyeballs were obtained from 1-year-old cattle to prepare the purified lens α-crystallin by gel chromatography.α-Crystallin was isolated from cattle lenses using gel chromatography.The purified α-crystallin was collected using fast protein liquid chromatography ( FPLC ) and exposed to 1308 nmultraviolet-B at different irradiation intensities ( 23.75,118.75,475.00,1187.50,2375.00,4750.00,11 875.00,23 750.00 mJ/cm2 ) and then to ultraviolet-B 2308 nm with irradiation intensities of 28 535.00,6730.00,3435.00,1910.00,1040.00 mJ/cm2.Ultraviolet-absorbance spectra,tryptophan fluorescence and N-formylkynurenine (N-FK)fluorescence spectra of both irradiated and non-irradiated α-crystallin were measured.I3C at the concentrations of 50 μmol/L and 100 μmoL/L were added to the α-crystallin solution to perform a catalase (CAT) thermal aggregation to confirm the chaperone activity of the α-crystallin,and the α-crystallin solution without any I3C was used as control.The ratios of A360 between various intervene groups with control group were calculated using spectrophotometry.Results The A280 values of the α-crystallin declined to 10％ at the ultraviolet-B irradiation intensity of 1187.5 mJ/cm2 and that at the intensity of 23.75 J/cm2 lowed to 2％.A negative correlation was seen between the ultraviolet-B irradiation intensity and the A280 value of the α-crystallin (R2=0.925 ) and a positive correlation was found between ultraviolet-B with N-FK ( R2 =0.949 ).Ultraviolet-B irradiation intensity showed a negative correlation with Trp fluorescence intensity (R2 =0.996 ).CAT hot condensed experiment revealed that after addition of different concentrations of indole-3-carbinol,the relative A360 values at various ultraviolet-B irradiation group were significantly higher than those of the control group (P =0.000),and the decreasing degree of chaperone activity of α-crystallin was lower than that of the control group ( P =0.000 ). Conclusions The study suggests that I3C can protect the chaperone activity of α-crystallin from photooxidation,and the ultraviolet-B laser may be a good exposure source compared with ultraviolet lamp.The ultraviolet-B laser irradiation causes the alteration of structure and chaperone activity of α-crystallin.