Study On The Protective Mechanism Of 17β-estradiol On Age-related Macular Degeneration

Purpose:The purpose of this study is to investigate the protection effects of17β-estradiol（βE₂）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 theβE₂ 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 or80μM 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 ofβE₂ on cell proliferation,the cells were treated with 0,1,10,40,60 and 80μM ofβE₂ for 2 h before exposure to 40μM 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 ofβE₂,the rats were intravitreally injected with 4μl of 10μMβE_2.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:40μM H₂O₂ significantly decreased ARPE-19cell viability,and pretreatment with 10μM ofβE₂ significantly increased ARPE-19cell viability.The ROS levels were H₂O₂-treated group increased than control group,whileβE₂ pretreatment reduced the ROS levels than H₂O₂-treated ARPE-19 cells group.Also the apoptosis were H₂O₂-treated group increased than control group,butβE₂ 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,βE₂ 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 withβE₂ 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.βE₂ 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.βE₂ also decreased retinal cell apoptosis in blue LED-induced RD.Furthermore,βE₂ treatment increased protein expression of p-Akt and Bcl-2 and decreased protein expression of Cleaved Caspase3 and Bax during blue LED-induced RD.βE₂ 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）βE₂ protects against H₂O₂-induced oxidative stress in ARPE-19 cells and blue LED-induced RD in SD rats.（3）βE₂ protects against H₂O₂-induced oxidative stress in ARPE-19 cells and blue LED-induced RD in SD rats by decreasing apoptosis and nhancing autophagy.