S-allyl-L-cysteine Protects Retinal Pigment Epithelium Cells From Hydroquinone-induced Oxidative Stress Apoptosis

Objectives:Age-related macular degeneration(ARMD)is a degenerative change in the macula of the retina.Retinal pigment epithelium(RPE)degenerative death is an evident hallmark of advanced ARMD.The present study aims to evaluate the protective effects of S-allyl-L-cysteine(SAC),a bioactive component from aged garlic extracts,on the oxidative stress-related apoptosis of RPE cells and investigate the potential underlying mechanisms.Methods:(1)ARPE-19 cells were stimulated with hydroquinone and cell viability was evaluated at different time intervals for 24 h,48 h and 72 h by using Cell counting kit-8(CCK-8)assay;(2)The cells were treated with SAC of escalating concentrations about 2 μM,10μM,50 μM,250 μM and 1250 μM for 48 h,followed by CCK-8 assessment of cell viability;(3)Divide the cells into three groups.Ctrl group:the cells were not treated.HQ group:the cells were stimulated with hydroquinone.Experimental group:The cells were pre-treated with SAC at the concentrations of 2 μM,10 μM,50 μM,250μM,and 1250 μM for 2 h respectively,and then incubated with hydroquinone for another 48 h.CCK-8 assay was performed to evaluate the optimal concentration of SAC;(4)Divide the cells into 4 groups.Ctrl group:the cells were not treated.HQ group:Hydroquinone induces apoptosis in ARPE-19 cells.SAC group:the cells were treated by SAC alone.Experimental group:ARPE-19 cells were exposed to hydroquinone for 24 h in the presence or absence of 2 h-pretreatment of SAC.The apoptosis rate of ARPE-19 cells was detected by flow cytometric analysis or terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling(TUNEL)staining assay;(5)As above,Divide the cells into 4 groups.Cell morphological changes were observed under an inverted phase-contrast microscope;(6)As above,Divide the cells into 4 groups.ROS production was otherwise assessed by flow cytometry and fluorescence microscope.(7)As above,Divide the cells into 4 groups.The expression levels of antioxidant-related protein Nrf2 was measured by Western blot analysis,Also,the expression patterns of other antioxidant factors,including NQO-1,SOD-1,SOD-2,and HO-1 were examined through immunoblottingResults:(1)CCK-8 revealed that the stimulation of HQ significantly reduced cell viability,which gradually decreased over time;(2)After the treatment of different concentrations of SAC,cell viability did not change significantly according to the results of CCK-8;(3)After pre-treatment of SAC,compared with the group treated with HQ alone,the reduction of cell viability was alleviated.The difference was statistically significant.According to the result of CCK-8,50 μM was the best protective concentration of SAC;(4)Flow cytometry and TUNEL staining demonstrated that HQ substantially enhanced cell apoptosis.SAC treatment alone has no influence on cell apoptosis.When cells were pretreated with SAC,HQ-induced apoptosis was alleviated,and the difference was statistically significant;(5)Inverted phase contrast microscope revealed that morphology of the cells in SAC group did not change compared with the CTRL group.After HQ treatment,the number of adherent cells reduced significantly compared with CTRL group.After SAC pre-treatment,cell morphology was improved and apoptotic cell number decreased compared with HQ group;(6)As indicated by flow cytometry and inverted fluorescence microscopy,ROS production in the HQ group was significantly higher than that in the CTRL group.There was no significant change in SAC group.Compared with HQ group,ROS production in SAC-pretreated group was reduced remarkably,and the difference was statistically significant;(7)Western blotting demonstrated that cells could enhance self-protection under the stimulation of HQ,resulting in the increase of protein levels of antioxidant factors.However,after SAC pre-treatment,the expression of antioxidant genes was significantly reduced and the difference was statistically significant.Conclusions:These data suggest that SAC can effectively attenuate hydroquinone-induced oxidative damage in human RPE cells.Our work is the first to demonstrate that SAC modulates oxidative stress-induced RPE apoptosis,thereby providing new insights into the prevention of ARMD.