The Effect Of A2E And Blue Light On The Uptake And Release Calcium In Lysosome And Mitochondria Of Human RPE Cells

Objective:To explore the effect of A2E and blue light on the uptake and release of calcium in lysosome and mitochondria by establishing a blue light induced injury model of human RPE cells loaded with A2EMethods:The primary human RPE cells were cultured,and the cells which were transferred to 4-6 generations were used for the experiment.The cells were randomly divided into five groups:control group,blue light group,blue light+nifedipine group,A2E loaded+blue light group,A2E loaded+blue light+nifedipine group;the intensity of blue light was(2000 ± 500)lux for 6 hours,and the culture continued for 24 hours after exposure to blue light;A2E with a final concentration of 25 μM was added 2 hours before the light;nifedipine with a final concentration of 10-4 M was added 1 hour before the light;(1)using the calcium fluorescence probe CaTM-2 AM combined with the calcium in the cytoplasm,the fluorescence of RPE cells in each group was observed and photographed by laser scanning confocal microscope,and the fluorescence intensity was analyzed(2)using the calcium fluorescence probe Rhod-2 AM combined with the calcium in the mitochondria,the fluorescence of RPE cells in each group was observed and photographed by laser scanning confocal microscope,and the fluorescence intensity was analyzed(3)the acid lysosome of human RPE cells was labeled by Lysotracker red,and Fluo-3 AM was used to bind intracellular calcium,laser scanning confocal microscopy was used to observe and take photos,and the fluorescence intensity in lysosome after colocalization was analyzed.(4)The cells were divided into four groups:control group,blue light group,A2E loaded group,A2E loaded+blue light group.The changes of mitochondrial membrane potential were detected by JC-1Results:(1)The fluorescence intensity of calcium in cytoplasm of human RPE cells was detected by laser scanning confocal microscope.It was found that the fluorescence intensity of calcium in blue light group,blue light+nifedipine group,A2E loaded+blue light group,A2E loaded+blue light+nifedipine group were higher than the control group,the difference was statistically significant(P<0.05);the fluorescence intensity in blue light+nifedipine group is lower than that in blue light group(P<0.05),A2E loaded+blue light group,A2E loaded+blue light+nifedipine group are higher than blue light group(P<0.05);A2E loaded+blue light group,A2E loaded+blue light+nifedipine group is higher than blue light+nifedipine group,the difference is statistically significant(P<0.05);the fluorescence intensity of A2E loaded+blue light group was higher than that of A2E loaded+blue light+nifedipine group(P<0.05).(2)The fluorescence intensity of calcium in the mitochondria of RPE cells in each group was detected by laser scanning confocal microscopy.It was found that the fluorescence intensity of calcium in blue light group,blue light+nifedipine group was higher than that in the control group,and the difference was statistically significant(P<0.05).A2E loaded+blue light group and A2E loaded+blue light+nifedipine group were lower than control group(P<0.05);the fluorescence intensity in blue light+nifedipine group,A2E loaded+blue light group,A2E loaded+blue light+nifedipine group was lower than the blue light group(P<0.05);A2E loaded+blue light group,A2E loaded+blue light+nifedipine group was lower than blue light+nifedipine group(P<0.05);The fluorescence intensity of the A2E loaded+blue light group was lower than that of A2E loaded+blue light+nifedipine group(P<0.05).(3)Laser scanning confocal microscopy was used to detect the fluorescence intensity of calcium in lysosomes of RPE cells.It was found that the fluorescence intensity of calcium in blue light group,blue light+nifedipine group was higher than that in control group,and the difference was statistically significant(P<0.05)A2E loaded+blue light group,A2E loaded+blue light+nifedipine group were lower than control group(P<0.05);the fluorescence intensity in blue light+nifedipine group,A2E loaded+blue light group,A2E loaded+blue light+nifedipine group was lower than blue light group(P<0.05);A2E loaded+blue light group,A2E loaded+blue light+nifedipine group is lower than blue light+nifedipine group,(P<0.05);the fluorescence intensity of A2E loaded+blue light group was lower than that of the A2E loaded+blue light+nifedipine group(P<0.05).(4)Flow cytometry was used to detect the mitochondrial membrane potential of RPE cells.The greater the degree of depolarization,the lower the mitochondrial membrane potential level.Therefore,the levels of mitochondrial membrane potentials in blue light group,A2E loaded group,A2E loaded+blue light group were lower than control group,and the difference was statistically significant(P<0.05).The blue light group and A2E loaded group were higher than A2E loaded+blue light group(P<0.05)Conclusion:(1)Blue light irradiation can cause calcium concentration in cytoplasm,lysosome and mitochondria of RPE cells to increase without affecting calcium uptake by lysosome and mitochondria.(2)A2E damages the lysosomal and mitochondrial membranes to release calcium into the cytoplasm.(3)Blue light and A2E can reduce mitochondrial membrane potential,and they have synergistic effect.