ABSTRACT
Objeetive To observe the damage of human lens epithelial cells (LECs) induced by ultraviolet (UV) B radiation and the expression changes of glutaredoxin 2 (Grx2) in the cells,and to investigate the protective effects of Grx2 on human LECs against UVB-induced apoptosis.Methods Human LECs (HLE-B3) were cultured and exposed to different energy of UVB (0,10,30,50 mJ/cm2) with the wavelength of 297 nm.The morphology of the cells was examined under the optical microscope 2,4,8,12 and 16 hours after irradiation of UVB.The survival rate of the cells was evaluated with cell counting kit 8 (CCK8).The apoptosis rate of the cells was detected by TUNEL assay.Real-time quantitative PCR and Western blot were employed to detect the expressions of Grx2 mRNA and Grx2 protein in the cells,respectively.The cells were transfected with pcDNA3.1-Grx2 plasmid by lipofectamine 2000 as the Grx2 transfected group,and pcDNA3.1 plasmid was transfected into cells as the empty plasmid group.The cells were irradiated by 50 mJ/cm2 for 4 hours,and the apoptosis rate of human LECs was detected by TUNEL assay.Results Cultured cells grew well with the green fluorescence for Grx2 expression in the non-UVB exposure group,and shrinkage and death of the cells were found after UVB irradiation.The survival rate of the cells was gradually reduced after irradiation of 10,30,50 mJ/cm2 UVB as the increase of UVB energy and lapse of time.The expression level of Grx2 mRNA were 2.53±0.48 and 3.53±0.14 in the 10 mJ/cm2 UVB group and 30 mJ/cm2 UVB group 4 hours after irradiation,which were significantly higher than 1.01±0.08 and 1.00±0.09 in the non-UVB exposure group (all at P＜0.05).The expression level of Grx2 mRNA in the cells was 15.30±3.01 at 1 hour after irradiation in the 50 mJ/cm2 UVB group,which was significantly higher than 1.00 ±0.07 in the non-UVB exposure group (P＜0.05).The expression levels of Grx2 protein showed the same tendency to Grx2 mRNA.The apoptosis rate of the cells in the Grx2 transfected group was (15.34± 1.71) ％,and that in the empty plasmid group was (22.11 ± 2.46) ％ at 4 hours after 50 mJ/cm2 UVB irradiation,with a significant difference between them (t =3.189,P ＜ 0.05).Conclusions UVB irradiation induced damage of human LECs in dose-and time-dependent manner.However,the expression of Grx2 in human LECs is up-regulated transiently after exposure of UVB,which has a protective effect on the cells against the UVB-induced apoptosis.
ABSTRACT
Background Ultraviolet B (UVB) is one of the main causes of cataract formation,and its mechanism is associated with the apoptosis of lens epithelial cells (LECs).MiroRNA-133b (miR-133b) can regulate oxidative stress-induced LECs apoptosis.However,whether miR-133b is associated with UVB-induced cataract is not elucidated.Objective This study was to observe the inhibitory effects of miR-133b on UVB-induced cataract and its regulating mechanism.Methods Twenty 8-week-old C57BL/6 mice were randomized into cataract model group and normal control group.The mouse eyes in the cataract model group exposed to 302 nm UVB for 5 minutes once per day for consecutive 1 week,with the irradiation intensity of 300 W/cm2,and the mice in the normal control group did not receive any intervention.Five mice in each group were sacrificed and 10 eyeball sections were prepared.Human LECs (SRA01/04) were exposed to UVB for 25 minutes and served as UVB-induced group,the cells in the normal control group did not receive any intervention.The UVB-induced cells were inoculated to 96-well plate and divided into 4 groups,and 50 nmol/L miR-133b mimic,miR-133b mimic control agent,miR-133b inhibitor and miR-133b inhibitor control agent were transfected into the cells with lipofectamine2000.The expression of miR-133b mRNA and a target gene BCL2L2,which was identified by online miRNA database (www.mirab.org) in the cells were detected by real-time quantitative PCR to evaluate the transfected efficacy,and the apoptosis of the cells in mouse lens tissue and different transfected groups were assayed by TUNEL.The use and care of the mice followed ARVO Statement.Results The arrangement of LECs was regular and no apoptotic cell was seen in the normal control group,and the apoptotic cells showed the red fluorescence in the cataract model group.The apoptotic rate of human LECs was (43.90±9.30) ％ in the UVB-induced group,and that in the normal control group was (1.08±0.49)％,showing a significant difference between the two groups (t =-7.963,P =0.015).The relative expression levels of miR-133b mRNA in the model mouse lens and UVB-induced human LECs were evidently lower and relative expression levels of BCL2L2 mRNA were higher than those in normal mice and normal LECs (miR-133b mRNA:t =-2.958,P =0.042;t =-6.195,P =0.003;BCL2L2 mRNA:t =3.761,P =0.020;t =12.437,P =0.000).The relative expression level of miR-133b mRNA was significantly increased and the relative expression level of BCL2L2 mRNA was reduced in the miR-133b mimic group in comparation with the miR-133b mimic control group (t=10.883,-5 927.617;both at P＜ 0.01);compared with the miR-133b inhibitor control group,the relative expression level of miR-133b mRNA was significantly decreased and that of BCL2L2 mRNA was evidently increased in the miR-133b inhibitor group (t =-1 606.622,17.556;both at P ＜ 0.01).The apoptotic rate of human LECs was (43.62 ± 9.19) ％ and (17.55 ± 4.24) ％ in the miR-133b mimic control group and miR-133b mimic group,with a significant difference between them (t =-4.462,P =0.011),and the apoptotic rate in the miR-133b inhibitor group was (78.23 ± 12.42) ％,which was significantly higher than (48.01 ±9.68) ％ in the miR-133b inhibitor control group (t =3.324,P =0.029).Conclusions miR-133b can prevent UVB-induced cataract probably by negatively targeting the BCL2L2 expression to regulate the apoptosis of LECs.