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Oxidative stress is one of the major risk factors for age-related cataract, methionine sulfoxide reductase B1(MsrB1) has been shown to play an important role for lens cell function, resistance to oxidative stress. However, the mechanism of MsrB1 gene silencing on H2O2-induced human lens epithelial cells (LECs) apoptosis remains virtually unknown.
This study attempted to investigate the role of MsrB1 gene in protecting human LECs against H2O2-induced damage.
Human LECs cell line SRA01/04 cells were cultured in DMEM medium. Different concentrations (200, 400, 600, 800 and 1 000 μmol/L) of H2O2 were added to medium treat the cells for 12, 24 and 36 hours respectively to select the optimum inducing dose and time. The cells were divided into 4 groups. Lipofectamine 2000 containing MsrB1 siRNA was transfected into the cells in the MsrB1 siRNA transfected group, 200 μmol/L H2O2 treated the cells for 24 hours as the H2O2-induced group, and MsrB1 siRNA was transfected into the cells before H2O2 treatment as the MsrB1 siRNA transfected combined H2O2-induced group. The routinely cultured cells were as normal control group. Cell viability was detected by MTT assay. The relative expression of MsrB1 mRNA in the cells was analyzed by real-time PCR. Morphological changes of nuclei in the cells of different groups were observed using Hoechst 33528 staining under the fluorescence microscope. Cell apoptosis and cell cycle were quantified using flow cytometry.
The relative expressing values of MsrB1 mRNA were 1.063±0.058, 1.013±0.049 and 0.235±0.024, and the expressing values of MsrB1 mRNA in the MsrB1 siRNA transfected group were significantly lower than those of the normal control group and H2O2-induced group (t=31.744, 35.807, both at P<0.001). The cell viability was lowest 24 hours after 200 μmol/L H2O2 induced. Hoechst 33528 staining showed less contractive decrescent nuclei in the MsrB1 siRNA transfected group, and those in the H2O2-induced group and MsrB1 siRNA transfected combined H2O2-induced group were more. Flow cytometry revealed that the apoptotic rates in the normal control group, MsrB1 siRNA transfected group, H2O2-induced group and MsrB1 siRNA transfected combined H2O2-induced group were (1.36±0.35)%, (3.26±0.31)%, (5.13±0.59)% and (8.73±0.48)%. The cells distributed mainly in the G2/M phase in the H2O2-induced group. The percentage of the cells in the G1 phase of the MsrB1 siRNA transfected combined H2O2-induced group was (52.78±1.68)%, which was significantly higher than (44.99±1.37)% in the H2O2-induced group (t=6.224, P<0.01), while the percentage of cells in the G2/M phase also was significantly lower than that in the H2O2-induced group ([34.97±2.31]% versus [42.39±1.41 ] %) (t=-6.213, P<0.01).
MsrB1 gene plays a protecting role on human LECs against oxidative damage by decreasing cell apoptosis through regulating the cell cycle.