Effect of rapamycin on autophagy of retinal pigment epithelial cells under light induction
Objective:To investigate the effect of rapamycin on autophagy in retinal pigment epithelial cells under light induc-tion.Methods:The autophagy double labeled mRFP-eGFP-LC3 plasmid was transfected into ARPE-19 cells to obtain a stable ex-pression of transfected cell line,and then randomly divided into the 6 h control group,the 6 h model group and the 6 h rapamycin group;the 12 h control group,the 12 h model group,the 12 h rapamycin group and the 24 h control group,the 24 h model group,the 24 h rapamycin group.The control groups were incubated in tinfoil and protected from light;the model groups received light stimulation;the rapamycin groups received light stimulation after the addition of 10 μmol/L rapamycin,and the cells were irradiat-ed at(16 500±500)lx light intensity for 6 h,12 h and 24 h using a LED elektrolumineszenz as the light source.The changes in autophagic flow were analyzed qualitatively and the expression of Beclin 1,LC3 and P62 autophagy-related proteins was quantified by Western blot assay.Results:Compared with the control group,the cell survival rate of the model group decreased significantly after 6 h,12 h and 24 h of light exposure(P<0.001).The rapamycin group had a significantly higher cell survival rate after light exposure than the model group(P<0.05).After 6 h,12 h,and 24 h of light exposure,the number of red fluorescent spots in the model group gradually increased compared with the control group,and the number of green fluorescent spots also increased.The number of yellow spots in the merge map started to increased significantly at 12 h of light exposure.The red fluorescent spots in the rapamycin group were stronger than those in the model group at 6 h,12 h and 24 h of light exposure,and the green fluores-cence was weaker than that in the model group.Transmission electron microscopy showed that more autophagic vesicles were ob-served in the model group,and a large number of autophagic vesicles were observed in the rapamycin group.Western blot results showed that the relative expression levels of autophagy-related proteins Beclin1 and LC3Ⅱ/LC3Ⅰ in the model group increased after 6 h,12 h and 24 h of light exposure,and the expression of P62 protein was low.After rapamycin intervention,compared with the model group,the expression of Beclin 1 protein in the rapamycin group was not significantly different at 6 h of light expo-sure,and gradually increased at 12 h and 24 h of light exposure.The ratio of LC3Ⅱ/LC3Ⅰ was higher than that of the model group;The difference in P62 protein expression was not significant.Conclusion:Light irradiation can induce autophagy in ARPE-19 cells,and rapamycin can up-regulate the activity of autophagy.
国家科技期刊平台
NSTL
万方数据
维普
