| Objective: This study is aimed to establish the A2 E and blue light induced RPE cells damage model to explore the effect of A2 E on lysosomal membrane permeability during the process of blue light induced RPE cells damage, and lays the foundation for following experiments.Methods:(1) MTT assay was used to detect RPE cells viability loaded by different concentration of A2 E after cultured for different times, and determined the optimum concentration of A2 E loaded in RPE cells.(2) Fluorescence microscope was used to observe fluorescence intensity change in RPE cells loaded with different concentration(0, 10, 25, 50μM) of A2 E.(3) The RPE cells were divided into four groups randomly: control group, blue light group, A2 E load group and blue light + A2 E load group. TUNEL、flow cytometry were used to detect RPE cells apoptosis.(4) Confocal laser scanning microscope was used to observe lysosomal membrane permeability change by acridine orange(AO) staining. Analyzed the fluorescence intensity by confocal analysis software.Results:(1) MTT assay showed a downward trend of cells proliferation rate which RPE cells loaded with increasing concentration(10μM, 25μM, 50μM) of A2 E and cultured for 12 h, 24 h, 48 h, 72 h. When RPE cells loaded with A2 E concentration of 10μM after cultured for 12 h, 24 h, 48 h, 72 h, the differences were not significant compared with the control group(P=0.821, 0.641, 0.406, 0.572). When RPE cells loaded with A2 E concentration of 25μM and 50μM after cultured for 12 h, the differences were not significant compared with the control group(P=0.169, 0.058). Compared with the OD value of control group(0.508±0.035), the OD value decreased(0.423±0.035),(0.349±0.010) when RPE cells loaded with A2 E concentration of 25μM and 50μM after cultured for 24 h, the differences were significant(P=0.000, 0.000). Compared with the OD value of control group(0.688±0.064), the OD value decreased(0.544±0.042),(0.439±0.086) when RPE cells loaded with A2 E concentration of 25μM and 50μM after cultured for 48 h, the differences were significant(P=0.000, 0.000). Compared with the OD value of control group(0.878±0.032), the OD value decreased(0.661±0.021),(0.430±0.082) when RPE cells loaded with A2 E concentration of 25μM and 50μM after cultured for 72 h, the differences were significant(P=0.000, 0.000). Therefore, we determined 25μM as the optimum concentration of A2 E loaded in RPE cells for subsequent experiment.(2) With different loading concentration of A2 E in RPE cells, the intensity of cytoplasm fluorescence in RPE cells changed from green to yellow gradually as A2 E loading concentrations increased when observed by fluorescence microscopy.(3) The RPE cells apoptosis rate of control group(3.39±0.15%), A2 E load group(4.61±1.44%), blue light group(8.21±0.52%), blue light+A2E load group(24.77±1.49%) was detected by flow cytometry, and the difference was significant among groups(F=130.292,P=0.000). The apoptosis rate of blue light group, blue light+A2E load group were higher than control group, the differences were significant(P=0.004,0.000). However, the difference was not significant when A2 E load group compared with control group(P=0.348). Compared with blue light group and A2 E load group, the apoptosis rate of blue light+A2E load group was higher, the difference was significant(P=0.000, 0.000). Compared with A2 E load group, the apoptosis rate of blue light group was higher, the difference was significant(P=0.019).(4) The RPE cells apoptosis rate of control group(10.40±2.46%), A2 E load group(24.07±1.17%), blue light group(43.00±4.41%), blue light+A2E load group(53.00±4.50%) was detected by TUNEL, and the difference was significant among groups(F=92.423,P=0.000). The apoptosis rate of A2 E load group, blue light group, blue light+A2E load group were higher than control group, the differences were significant(P=0.001, 0.000, 0.000). Compared with blue light group and A2 E load group, the apoptosis rate of blue light+A2E load group was higher, the difference was significant(P=0.007, 0.000). Compared with A2 E load group, the apoptosis rate of blue light group was higher, the difference was significant(P=0.000).(5) The lysosomal red granule fluorescence intensity weakened while cytoplasm green fluorescence intensity enhanced in light group, A2 E load group, blue light+A2E load group when detected by confocal laser scanning microscope. The mean fluorescence intensity of control group(37.00±1.30), blue light group(29.48±0.62), A2 E load group(32.37±0.72) and blue light+A2E load group(22.16±0.73) were analyzed by Leica Confocal Software(LCS Lite), and the differences between these groups were significant(F=246.955, P=0.000). The mean fluorescence intensity of light group, A2 E load group and blue light+A2E load group were lower than control group, the difference was significant(P=0.000, 0.000, 0.000). The blue light group was lower than A2 E load group, the difference was significant(P=0.000). And compared with blue light group and A2 E load group, the mean fluorescence intensity of blue light+A2E load group was lower, the difference was significant(P=0.000, 0.000).Conclusion:(1) The optimum concentration of A2 E loaded in RPE cells is 25μM.(2) Blue light and A2 E joint blue light can lead to RPE cells apoptosis, and both have a synergistic effect.(3) Blue light, A2 E and A2 E joint blue light can increase the permeability of lysosomal membrane in RPE cells. |
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