Effect Of A₂E On Ca²⁺-PKC Signaling Pathway In Human RPE Cells Irradiated By Blue Light

Objective:The effect of A₂E on Ca²⁺-PKC signaling pathway in blue light-irradiated human RPE was explored by establishing a model of human retinal pigment epithelial cell injury induced by blue light-loaded N-retinyl-N-retinyl-ethanolamine（A₂E）.Methods:Human RPE primary cells were cultured in the 4th to 6th generation cells for the experiment.The same batch and the same generation of cells were used to randomly divide the cells into five groups:control group（no A₂E,no blue light）,Light group,Light+chloroquine group,light+A₂E group,light+A₂E+chloroquine group,first treated with chloroquine（final concentration of 15uM）,then loaded with A₂E,the final concentration was 25μM（the most suitable concentration established in the previous period）,action time2 In hours,the blue light intensity was（2000±500）Lux,and after 6 hours of illumination,the culture was continued for 24 hours and the culture was terminated.（1）Using FLUO-3calcium ion fluorescent probe combined with calcium ion,each group of RPE cells were observed and photographed by laser scanning confocal microscopy（LSCM）.The fluorescence intensity of each group was obtained by software analysis.（2）The concentration of inositol triphosphate（IP3）and diacylglycerol（DAG）in each group was measured by enzyme-linked immunosorbent assay（ELISA）.（3）The PKC activity in each group was determined by non-radioactive nuclides.（4）The cells were divided into four groups:control group,light group,chloroquine group,light+chloroquine group,and apoptosis of RPE cells was detected by flow cytometry.Statistical analysis was performed on the experimental data using SPSS 18.0 statistical software.Results:Laser scanning confocal microscopy was used to detect the intracellular free calcium concentration in each group:control group（25.219±2.956）,light group（29.115±3.100）,light+chloroquine group（33.268±2.867）,light+A₂E group（40.907±2.531）Light+A₂E+chloroquine group（51.831±4.944）,the difference was statistically significant（F=135.845,P=0.000）,light group,light+chloroquine group,light+A₂E group,light+A₂E+chloroquine group,The fluorescence intensity was higher than control group（P=0.000,P=0.000,P=0.003,P=0.000）.The fluorescence intensity of light+A₂E group was higher than light group（P=0.002）,The fluorescence intensity of the light+chloroquine group was higher than light group（P=0.002）.（2）IP3 concentration in each group:control group（23.259±1.983）,light group（69.802±3.317）,light+chloroquine group（107.703±0.524）,light+A₂E group（145.975±1.848）,light+A₂E+chloroquine Group（197.21±5.455）;the difference between the groups was statistically significant（F=1563.031,P=0.000）,light group,light+chloroquine group,light+A₂E group,light+A₂E+chloroquine group,intracellular IP3 concentration was higher than control group（P=0.000,P=0.000,P=0.000,P=0.000）,the intracellular IP3 concentration in the light+A₂E group was higher than that in the light group,the difference was statistically significant（P=0.000）,the intracellular IP3 concentration in the light+chloroquine was higher than t light group,and the difference was statistically significant（P=0.000）.The intracellular DAG concentration of each group was:control group（1.341±0.140）,light group（2.972±0.118）,light+chloroquine group（4.815±0.120）,light+A₂E group（6.386±0.156）,light+A₂E+chloroquine group（8.043）.±0.105),the overall group compared with the mean,the difference was statistically significant（F=779.443,P=0.000）,light group,light+chloroquine group,light+A₂E group,light+A₂E+chloroquine group intracellular DAG concentration was higher than control group,The difference was statistically significant（P=0.000,P=0.000,P=0.000,P=0.000）.The intracellular DAG concentration in the light+A₂E group was higher than that in the light group,and the difference was statistically significant（P=0.000）,the intracellular DAG concentration in the light+chloroquine group was higher than that in the light group（P=0.000）.（3）The non-radioactive radionuclide method was used to determine the PKC activity in each group.The control group（0.220±0.044）and the light group（0.704±0.066）),light+chloroquine group（1.366±0.015）,light+A₂E group（1.394±0.257）,light+A₂E+chloroquine group（2.838±0.166）,the difference between the groups,the difference was statistically significant（F=97.301,P=0.000）,light group,light+chloroquine group,light+A₂E group,light+A₂E+chloroquine group PKC activity was higher than the control group,the difference was statistically significant（P=0.007,P=0.000,P=0.000,P=0.000）,PKC activity in the light+A₂E group was higher than that in the light group,the difference was statistically significant（P=0.001）,and the PKC activity in the light+chloroquine group was higher than light group.the difference was statistically significant（P=0.001）.（4）Flow cytometry was used to detect apoptosis of RPE cells:control group（0.90±4.19）,light group（0.61±12.63）,chloroquine group（2.80±20.73）,light+chloroquine group（1.69±48.78）,the difference between the groups,the difference was statistically significant（F=252.541,P=0.000）,the light group,chloroquine group,light+chloroquine group apoptotic rate were higher than the control group（P=0.001,P=0.000,P=0.000）,the apoptotic rate of light+chloroquine group was higher than chloroquine group and light group（P=0.000,P=0.000）.Conclusion:（1）Blue light illumination and A₂E loading can increase the intracellular calcium concentration of human RPE cells,increase PKC activity,increase the concentration of IP3 and DAG in the cytoplasm,and synergize the blue light and A2E load.（2）Chloroquine can further increase the intracellular calcium concentration,PKC activity and intracellular IP3,DAG concentration of human RPE cells exposed to blue light or human RPE cells loaded with A₂E.