D-galactose Induces Retinal Aging Through Aβ Protein

ObjectiveExperiments were conducted to investigate the production of β-amyloid protein(Aβ protein)and damages in the process of mice retinal aging induced by D-galactose(D-Gal)In vitro and in vivo.Methods1.Animal Experiments30 C57BL/6j male mice at 6 weeks of age were randomly divided into 3groups,each with 10 mice: control group(subcutaneous injection of saline),experimental group(subcutaneous injection of 125mg/kg·d,350mg/kg·d D-Gal).After 12 weeks of continuous administration in the experimental group,electroretinogram(ERG)was used to evaluate changes of retinal function,and optical coherence tomography(OCT)to detect changes of retinal structure between control and experimental groups,and immunofluorescence staining was used to detect the expressions of Aβ protein,Aβ degrading enzyme Neprilysin(NEP),and Aβ synthase BACE-1 in epithelium(RPE).2.Cell experimentsAdult retinal pigment epithelium-19(ARPE-19)cells were treated with different concentration of D-Gal to establish aging model.The RPE cells were treated by 25,50,100,and 200 mmol/L D-Gal for 24 hours to induce aging.CCK-8 detection was used to evaluate cell viability,senescence-relatedβ-galactosidase(SA-β-Gal)staining was used to observe cell senescence,and immunofluorescence staining was used to detect Aβ protein and Aβ degrading enzymes NEP and Aβ synthase BACE in senescent cells-1 expression level,Western blot method was used to detect the expression levels of Aβ-degrading enzyme NEP,Aβ synthase BACE-1,Cyclin E(CCNE),and apoptosis-related protein Cleaved-caspase-3.Results1.Animal experiments1.The ERG results showed that compared with the control group,the D-Gal caused damages in the retinal function of mice,and the amplitude of a wave and b wave of ERG were all significantly reduced significantly compared with control(P<0.01).2.OCT test results showed that the structure of the retina in the control group was intact.The RPE layer’s structures of the retina in the D-Gal experimental group were obviously disorder,and it became more severe with increasing concentration of D-Gal.3.The immunofluorescence staining results showed that compared with the control group,the Aβ protein in the RPE layer of the retina in the D-Gal experimental group increased significantly(P<0.01).4.The immunofluorescence staining results showed that compared with the control group,the Aβ-degrading enzyme NEP in the RPE layer of the retina in the D-Gal experimental group was significantly reduced(P<0.01).5.The results of immunofluorescence staining showed that compared with the control group,the Aβ synthase BACE-1 in the RPE layer of the retina in the D-Gal experimental group increased significantly(P<0.01).2.Cell experiments1.The results of CCK-8 detection of cell viability showed that compared with the control group,the ARPE-19 cell viability of the D-Gal experimental group was significantly decreased(P<0.01)in dose-dependent.2.The staining results of aging-related β-galactosidase(SA-β-Gal)showed that compared with the control group,the positive rate of SA-β-Gal staining in ARPE-19 cells in the D-Gal experimental group was significantly increased(P<0.01),suggesting that the APPE-19 cell senescence model has been established successfully.3.The immunofluorescence staining results showed that compared with the control group,the Aβ protein expression in ARPE-19 cells in the D-Gal experimental group was significantly increased(P<0.01).4.The immunofluorescence staining results showed that compared with the control group,the expression of Aβ degrading enzyme NEP in ARPE-19 cells in the D-Gal experimental group was significantly reduced(P<0.01);Western blot results showed that compared with the control group,D The expression of Aβ-degrading enzyme NEP in ARPE-19 cells in the Gal experimental group was significantly reduced(P<0.01).5.The immunofluorescence staining results showed that compared with the control group,the expression of Aβ synthase BACE-1 in ARPE-19 cells in the D-Gal experimental group was significantly increased(P<0.01);Western blot results showed that compared with the control group,The expression of Aβsynthase BACE-1 in ARPE-19 cells in the D-Gal experimental group was significantly increased(P<0.01).6.Western blot results showed that compared with the control group,the expression of cyclin CCNE in ARPE-19 cells in the D-Gal experimental group was significantly reduced(P<0.01);the expression of apoptosis-related protein Cleaved-caspase-3 was significantly increased(P <0.01).Conclusions1.D-Gal promots the production of Aβ protein in the process of the retinal senescence of and ARPE-19 cells,that probably through inhibiting the Aβdegrading enzyme NEP and enhancing the Aβ synthase BACE-1,thereby promoting the increase of Aβ synthesis.2.During the ARPE-19 cells aging process induced by D-Gal,CCNE expression is down-regulated,and Cleaved-Capase-3 is activated,thus causing ARPE-19 cells damage eventually.This damage may be associated with the increased synthesis of Aβ protein in the cell aging process.