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

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

目的探讨N-亚视黄基-N-视黄基-乙醇胺(A2E)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-19细胞后对细胞间黏附分子(ICAM)、白细胞介素(IL)-1β、IL-6、IL-8、IL-10、促血管生成素-2(Ang-2)、胎盘生长因子(PIGF)、胰岛素样生长因子1(IGF-1)、转化生长因子-β(TGF-β)以及血管内皮生长因子A(VEGFA)表达的影响,透射电子显微镜观察ARPE-19细胞超微结构的变化,免疫荧光标记检测细胞内自噬相关蛋白LC3的表达变化,Western blot检测自噬相关蛋白Beclin-1和p62的表达变化。结果依据CCK-8法检测结果,选择20μmol·L^(-1)的A2E作用于ARPE-19细胞用于后续实验。20μmol·L^(-1)的A2E作用于ARPE-19细胞后,细胞因子ICAM、IL-1β、IL-6、IL-8、IL-10、Ang-2、IGF-1、TGF-β的表达水平自6 h起均有所升高,PIGF和VEGFA的表达水平在A2E作用12 h后也明显升高,与未给予A2E处理的对照相比差异均有统计学意义(均为P<0.05)。透射电子显微镜下可见,20μmol·L^(-1)的A2E作用于ARPE-19细胞1 h后细胞质内可见碗状、尚未收口的双层膜结构的“吞噬泡”,作用3 h、6 h、12 h时出现自噬体结构以及自噬溶酶体结构,自噬囊泡在A2E作用24 h时开始出现内容物减少,体积逐渐缩小。荧光显微镜下可见,与未给予A2E处理的对照相比,20μmol·L^(-1)的A2E作用于ARPE-19细胞6 h起即可见细胞质内LC3荧光呈现点状聚集,至12 h时荧光斑点数量达到最多、颗粒也最大、差异最明显,24 h时绿色荧光斑点数量开始下降,荧光强度也较前减弱。Western blot检测结果显示,与未给予A2E处理的对照相比,ARPE-19细胞中Beclin-1蛋白的表达在A2E作用1 h、3 h、6 h、12 h及24 h时均显著升高,p62蛋白的表达在A2E作用1 h、3 h、6 h、12 h及24 h时均下降(均为P<0.05)。结论A2E能够刺激ARPE-19细胞分泌多种AMD相关炎症因子和新生血管因子,同时也能激活自噬潮,这为进一步研究自噬在AMD发病中扮演的角色提供了新的思路。

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,20μmol·L^(-1) A2E on ARPE-19 cells was selected in the following experiments.After treated with 20μmol·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-βwere 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 20μmol·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 20μmol·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.