自噬激活剂雷帕霉素对A2E诱导视网膜色素上皮细胞自噬调节的影响

Regulation of autophagy activator rapamycin on the autophagy of retinal pigment epithelium cells induced by N-retinylidene-N-retinylethanolamine

目的探讨自噬激活剂雷帕霉素对N-亚视黄基-N-视黄基乙醇胺(A2E)诱导视网膜色素上皮(RPE)细胞自噬调节的影响。方法取人RPE细胞(ARPE-19细胞系)进行培养。将细胞分为4组,其中,对照组:使用DMEM-F12完全培养基孵育ARPE-19细胞;A2E组:使用含20μmol·L^(-1) A2E的DMEM-F12完全培养基孵育ARPE-19细胞;雷帕霉素组:使用含100 nmol·L^(-1)雷帕霉素的DMEM-F12完全培养基预处理细胞1 h后,再用DMEM-F12完全培养基孵育ARPE-19细胞;雷帕霉素联合A2E组:使用100 nmol·L^(-1)雷帕霉素预处理细胞1 h后,再用含20μmol·L^(-1) A2E的DMEM-F12完全培养基孵育ARPE-19细胞。使用CCK-8法检测各组细胞增殖情况,Procarta细胞因子分析试剂盒检测各组细胞培养上清液中细胞因子表达情况。透射电镜观察各组细胞超微结构,Western blot法检测各组细胞自噬相关蛋白Beclin-1和P62的表达,免疫荧光染色法检测各组细胞自噬标志物LC3蛋白的表达。结果与对照组相比,A2E组细胞生存率显著下降(P<0.001),而雷帕霉素联合A2E组细胞生存率得到了部分恢复,与A2E组相比差异有统计学意义(P<0.01)。与A2E组相比,雷帕霉素联合A2E组中10种细胞因子含量均有所下降(均为P<0.01)。与对照组相比,雷帕霉素组细胞内出现散在自噬囊泡,A2E组细胞内出现较多的自噬囊泡,而雷帕霉素联合A2E组细胞内出现大量聚集的大体积的自噬囊泡。与对照组相比,A2E组中Beclin-1蛋白相对表达量显著升高(P<0.001),P62蛋白相对表达量显著降低(P<0.001);与A2E组相比,雷帕霉素联合A2E组中Beclin-1蛋白相对表达量明显升高(P<0.01),而P62蛋白相对表达量明显降低(P<0.001)。雷帕霉素联合A2E组ARPE-19细胞内LC3蛋白的绿色荧光斑点大量聚集,与A2E组相比荧光强度增加,斑点体积更大,数量也更多。结论雷帕霉素能够进一步激活A2E诱导的ARPE-19细胞自噬活性,一定程度上降低了A2E对RPE细胞造成的损伤,并抑制了ARPE-19细胞中炎症因子和血管生成因子的分泌。

Objective To investigate the regulation action of autophagy activator rapamycin on the N-retinylidene-N-retinylethanolamine(A2E)induced autophagy of retinal pigment epithelium(RPE)cells.Methods The adult RPE cells(ARPE-19 cell line)were cultured and divided into the control group(the ARPE-19 cells were cultured in DMEM-F12 complete medium),A2E group(the ARPE-19 cells were cultured in DMEM-F12 complete medium containing 20μmol·L^(-1) A2E),rapamycin group(the ARPE-19 cells were pre-treated in DMEM-F12 complete medium containing 100 nmol·L^(-1) rapamycin for 1 h and then cultured in normal DMEM-F12 complete medium),and rapamycin+A2E group(the ARPE-19 cells were pre-treated in DMEM-F12 complete medium containing 100 nmol·L^(-1) rapamycin for 1 h and then cultured in DMEM-F12 complete medium containing 20μmol·L^(-1) A2E).Cell Counting Kit-8 was used to determine the proliferation of ARPE-19 cells in each group.Procarta Cytokine Assay Kit was used to measure the expression of cytokines in the cell culture supernatant of each group.The transmission electron microscope was used to observe the ultrastructure of ARPE-19 cells in each group.The expressions of autophagy-related proteins Beclin-1 and P62 were detected by Western blot,and the expression of autophagy marker protein LC3 was detected by immunofluorescence staining.Results Compared with the control group,the survival rate of ARPE-19 cells in the A2E group decreased significantly(P<0.001),while the survival rate in the rapamycin+A2E group partially recovered compared to the A2E group(P<0.01).Compared with the A2E group,the levels of 10 cytokines decreased in the rapamycin+A2E group(all P<0.01).Compared with the control group,there were scattered autophagic vesicles in the ARPE-19 cells in the rapamycin group,more autophagic vesicles in the A2E group,and a large number of aggregated autophagic vesicles with large sizes in the rapamycin+A2E group.Compared with the control group,the expression of protein Beclin-1 in the A2E group was significantly increased(P<0.001),while the expression of P62 was significantly decreased(P<0.001).Compared with the A2E group,the expression of protein Beclin-1 in the rapamycin+A2E group was significantly increased(P<0.01),while the expression of P62 declined considerably(P<0.001).Compared with the A2E group,the green fluorescence spots of protein LC3 in ARPE-19 cells in the rapamycin+A2E group were clustered significantly,with higher fluorescent intensity,larger size and quantity.Conclusion Rapamycin can further activate the A2E-induced ARPE-19 cell autophagy activity,reduce the injury caused by A2E to RPE cells,and inhibit the secretion of inflammatory factors and angiogenic factors in ARPE-19 cells.