| Purpose:The purpose of this study is to investigate the protection effects of17?-estradiol??E2?on hydrogen peroxide?H2O2?induced oxidative damage in retinal pigment epithelial cells?RPE?and blue LED light induced retinal damage in SD rats.To further improve the pathogenesis of how the?E2 effect on the age-related macular degeneration?AMD?progression,and to find a new way of clinical prevention and treatment of AMD.Methods:?1?Experiment in vitro:we first cultured cells of human RPE cell line ARPE-19 in vitro and we treated ARPE-19 cells with 0,20,30,40,50,60,70 or80?M of H2O2 for 24 h.Then cell viability was determined by CCK-8 to determine the most suitable concentration of H2O2.To evaluate the effect of?E2 on cell proliferation,the cells were treated with 0,1,10,40,60 and 80?M of?E2 for 2 h before exposure to 40?M of H2O2 for 24 h,after which cell viability was quantified.Cellular reactive oxygen species?ROS?were measured using DCF fluorescence.Apoptosis was evaluated by Annexin V/PI staining.Western Blot was used to detect the protein expression level of p-Akt and apoptosis related factors Bcl-2,Bax and Cleaved Caspase 3.What's more,Autophagosomes were examined by electron microscopy.The expression of autophagy related factors Beclin 1,LC3B or LC3-II/LC3-I mRNA and protein expression were detected by q-PCR and Western Blot.?2?Experiment in vivo:The rats were exposed to 3000 lux of blue LED?460±10 nm?for 2 h?9:00 AM to 11:00 AM?in a blue LED exposure box.To assessment the model of blue LED light induced SD rats retinal degeneration?RD?,we used HE staining and ERG.Also,to assessment the protection of?E2,the rats were intravitreally injected with 4?l of 10?M?E2.Then HE staining and ERG were detected.Apoptosis was evaluated by terminal dUTP nick-end labeling?TUNEL?.Immunohistochemistry and Western Blot were used to detect the expression level of p-Akt and apoptosis related factors Bcl-2,Bax and Cleaved Caspase 3.What's more,Autophagosomes were examined by electron microscopy.The expression of autophagy related factors Beclin 1,LC3B or LC3-II/LC3-I expression level were detected by Immunohistochemistry and Western Blot.Results:?1?Experiment in vitro:40?M H2O2 significantly decreased ARPE-19cell viability,and pretreatment with 10?M of?E2 significantly increased ARPE-19cell viability.The ROS levels were H2O2-treated group increased than control group,while?E2 pretreatment reduced the ROS levels than H2O2-treated ARPE-19 cells group.Also the apoptosis were H2O2-treated group increased than control group,but?E2 pretreatment reduced the apoptosis than H2O2-treated ARPE-19 cells group.Furthermore,H2O2 treatment decreased protein expression of p-Akt and Bcl-2 and increased protein expression of Cleaved Caspase 3 and Bax than control group.However,?E2 treatment increased protein expression of p-Akt and Bcl-2 and decreased protein expression of Cleaved Caspase 3 and Bax than H2O2-treated ARPE-19 cells.H2O2 treatment increased the number of autophagosomes and downregulated the expression of LC3-II/LC3-I and Beclin 1 and the effects were further enhanced with?E2 treatment.?2?Experiment in vivo:HE showed that the retina structure of rats was distinct.After the rats were exposed blue light LED,the retinal structure was obviously disordered,the thickness of ONL was thinner and reductions in the a-and b-wave ERG amplitudes.?E2 treatment significantly prevented blue LED exposure-induced reductions in the a-and b-wave ERG amplitudes,retinal structure disruption,photoreceptor loss and ONL thickness decreases.?E2 also decreased retinal cell apoptosis in blue LED-induced RD.Furthermore,?E2 treatment increased protein expression of p-Akt and Bcl-2 and decreased protein expression of Cleaved Caspase3 and Bax during blue LED-induced RD.?E2 treatment also increased the number of autophagosomes and upregulated the expression of LC3-II/LC3-I and Beclin 1.Conclusion:?1?H2O2 induced ARPE-19 cell oxidative damage model and blue light LED lamp induced SD rat retinal injury model were successfully established.?2??E2 protects against H2O2-induced oxidative stress in ARPE-19 cells and blue LED-induced RD in SD rats.?3??E2 protects against H2O2-induced oxidative stress in ARPE-19 cells and blue LED-induced RD in SD rats by decreasing apoptosis and nhancing autophagy. |
PDF Full Text Request