脂褐素成分A2E诱导人视网膜色素上皮细胞自噬及损伤的研究

目的 探讨N-亚视黄基-N-视黄基-乙醇胺(A2 E)能否诱导人视网膜色素上皮细胞(ARPE-19细胞)的自噬及损伤反应,并从自噬的角度探索其与年龄相关性黄斑变性(AMD)发病相关的分子机制.方法 CCK-8法筛选A2E作用于ARPE-19细胞的最佳浓度用于后续实验.采用多重细胞因子检测技术检测A2E作用于ARPE-1...

Research on autophagy and injury of human retinal pigment epithelial cells induced by lipofuscincomponent A2E

Objective　To investigate whether N-retinylidene-N-retinylethanolamine (A2E) can induce autophagy and injury response of human adult retinal pigment epithelium-19 (ARPE-19) cells, and to study the molecular mechanism of A2E related to age-related macular degeneration (AMD) from the perspective of autophagy.Methods　CCK-8 methods was used to screen the optimal concentration of A2E on ARPE-19 cells for subsequent experiments. The effect of A2E on the expression levels of intercellular adhesion molecule (ICAM), interleukin (IL)-1β, IL-6, IL-8, IL-10, angiopoietin-2 (Ang-2), placental growth factor (PIGF), insulin-like growth factor-1 (IGF-1), transforming growth factor-β (TGF-β) and vascular endothelial growth factor A (VEGFA) in ARPE-19 cells were detected by multiple cytokine assay. The ultrastructural changes in ARPE-19 cells were observed by transmission electron microscopy, the expression of autophagy related protein LC3 was detected by immunofluorescence labeling, and the expression of autophagy related protein Beclin-1 and p62 was detected by Western blot.Results　According to the CCK-8 results, 20 μmol·L^-1 A2E on ARPE-19 cells was selected in the following experiments. After treated with 20 μmol·L^-1 A2E on ARPE-19 cells, the expression levels of ICAM, IL-1β, IL-6, IL-8, IL-10, Ang-2, IGF-1 and TGF-β were increased from 6 h, and the expression of PIGF and VEGFA were also increased after 12 h. The differences were statistically significant between ARPE-19 cells treated with A2E and the control without A2E treatment (all P<0.05). By transmission electron microscopy, after 20 μmol·L^-1 A2E acting on ARPE-19 cells for 1 h, a bowl-shaped, unclosed double-layer membrane structure began to appear in the cytoplasm called “phagophores”. Autophagosomes and autophagolysosomes appeared at 3 h, 6 h and 12 h, the content of autophagic vesicles decreased at 24 h and the volume of autophagic vesicles decreased gradually, indicating the degeneration and degradation of autophagic lysosomes. By fluorescence microscope, compared with the controls without A2E treatment, when ARPE-19 cells were treated with 20 μmol·L^-1 A2E for 6 h, the fluorescence of LC3 in the cytoplasm showed punctate aggregation. At 12 h, the number of fluorescent spots and granules reached the maximum, and the difference was most obvious. At 24 h, the number of green fluorescent spots began to decrease, and the fluorescence intensity was also weakened. Western blot analysis showed that, when compared with cells treated with A2E, the expression levels of Beclin-1 protein in ARPE-19 cells was significantly increased at 1 h, 3 h, 6 h, 12 h and 24 h after A2E treatment, while the expression levels of p62 protein was decreased at 1 h, 3 h, 6 h, 12 h and 24 h after treatment with A2E (all P<0.05).Conclusion　A2E can stimulate ARPE-19 cells to secrete a variety of AMD related inflammatory factors and neovascular factors, and it can also activate autophagy, which provides an insight for further research on the role of autophagy in the pathogenesis of AMD.