Molecular mechanism of lutein protecting Müller cells against oxidative stress induced by H2O2 / 中华实验眼科杂志

ABSTRACT

Background Oxidative stress is a main cause of age-related macular degeneration (AMD).Lutein has a preventive role for AMD, but its antioxidant mechanism remains unclear.Objective Present study was to investigate the effect of lutein on oxidative stress of Müller cells and its signaling pathway.Methods Human Müller cells (human Müller cell strain) were cultured, and the cells at logarithmimic growth phase were incubated in 96 well plate overnightly.Oxidative stress cell models were established by adding 160 μmol/L H2O2, a median lethal dose for Müller cells.The models were divided into the model control group and 12.5,25.0,50.0 mg/L lutein groups,and the different concentrations of lutein were used to culture the cells for 24 hours, respectively.The routine cultured cells served as the blank control group.Growth of the cells was assayed by MTT method (absorbancy);the reactive oxygen species (ROS) content in the cells was assayed by flow cytometry;the mRNA and protein levels of nuclear factor-E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) in the cells were detected by quantitative real-time PCR and Western blot, respectively.Results The inhibitory effects on the cells were gradually enhanced with the increase of H2O2 concentrations,showing a significant difference among the groups (F =43.890,P＜0.01).A significant difference was found in apoptotic rate of the cells among the blank control group,model control group and 12.5,25.0,50.0 mg/L lutein groups (F =346.770, P =0.000) , and the apoptosis rate was significant elevated with the increase of lutein dose (all at P＜0.05).The ROS contents in the cells were 1.92±0.18,64.89±2.86,52.70±2.80,32.61 ±4.20 and 5.68 ± 1.35 in the blank control group, model control group and 12.5,25.0,50.0 mg/L group, respectively, with significant difference among the groups (F =324.900, P =0.000), and the ROS content was gradually reduced as the increase of lutein dose (all at P＜0.05).The relative mRNA and protein expressions of Nrf2 and HO-1 were remarkedly higher in the 12.5,25.0,50.0 mg/L lutein groups than those in the model control group (F =236.960,242.620,186.830,263.120, all at P =0.000) , and no significant difference was seen in the relative expression level of nuclear Nrf2 protein among the groups (F =1.790, P =0.210).Conclusions Lutein can induce the expression of antioxidant enzymes by inducing the expression of nuclear translocation of Nrf2 and consequently inhibit the oxidative stress status.