自噬抑制剂3-甲基腺嘌呤(3-MA)对A2E诱导视网膜色素上皮细胞自噬的调节以及相关细胞因子表达的影响

目的 探讨自噬抑制剂3-甲基腺嘌呤(3-MA)对N-亚视黄基-N-视黄基乙醇胺(A2 E)诱导视网膜色素上皮(RPE)细胞自噬的调节以及相关细胞因子表达的影响.方法 取人RPE细胞(ARPE-19细胞系)进行培养.将细胞分为4组.对照组:仅使用正常DMEM-F12完全培养基孵育ARPE-19细胞;A2E组:将ARPE-...

Regulation of autophagy inhibitor 3-methyladenine on the autophagy of retinal pigment epithelial cells induced by N-retinyl-N-retinylidene ethanolamine and its impact on the expression of related cytokines

Objective　To investigate the regulation of autophagy inhibitor 3-methyladenine (3-MA) on the autophagy of retinal pigment epithelial (RPE) cells induced by N-retinyl-N-retinylidene ethanolamine (A2E) and its impact on the expression of related cytokines.Methods　Human RPE cells (ARPE-19 cell line) were divided into the control group (ARPE-19 cells were cultured in normal DMEM-F12 complete medium), A2E group (ARPE-19 cells were cultured in DMEM-F12 complete medium containing 20 μmol·L^-1 A2E for 12 h), 3-MA group ［ARPE-19 cells were first pretreated in DMEM-F12 complete medium containing 10 mmol·L^-1 3-MA (American Sigma-Aldrich Company) for 1 h and then cultured in normal DMEM-F12 complete medium for 12 h］, and 3-MA+A2E group (ARPE-19 cells were first pretreated in DMEM-F12 complete medium containing 10 mmol·L^-1 3-MA for 1 h and then cultured in DMEM-F12 complete medium containing 20 μmol·L^-1 A2E for 12 h). CCK-8 assay was used to determine the proliferation of ARPE-19 cells in each group. The transmission electron microscope was used to observe the ultrastructural changes of ARPE-19 cells in each group. The expression of autophagy-related proteins Beclin-1 and P62 in ARPE-19 cells was detected by Western blot, and the expression of autophagy marker protein LC3 was detected by immunofluorescence assay. Procarta cytokine assay kit was used to measure the expression of 10 cytokines in the cell culture supernatant of each group, including intercellular adhesion molecules, interleukin (IL)-1β, IL-6, IL-8, IL-10, angiopoietin 2, placental growth factor, insulin-like growth factor-1, transforming growth factor, and vascular endothelial growth factor A.Results　Compared with the A2E group, the survival rate of ARPE-19 cells significantly reduced by 27.16 % and the levels of 10 cytokines in the cell culture supernatant significantly increased in the 3-MA+A2E group (all P<0.01). Compared with the control group, the ultrastructure of ARPE-19 cells in the 3-MA group did not change significantly, while a great number of autophagic vacuoles were found in the cytoplasm of ARPE-19 cells in the A2E group. The number of autophagic vacuoles in the 3-MA+A2E group was significantly lower than that in the A2E group. The fluorescence intensity in the cytoplasm of ARPE-19 cells in the 3-MA+A2E group was weaker than that in the A2E group, and the number of green fluorescence spots was also significantly reduced. Compared with the control group, the expression of 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 Beclin-1 in the 3-MA+A2E group was significantly reduced (P<0.01), while the expression of P62 was significantly increased (P<0.001).Conclusion　Under the joint action of 3-MA and A2E, the expression level of inflammatory factors and vascular endothelial growth factors is significantly increased, while the number level of autophagic lysosomes and the expression level of autophagic proteins are decreased, suggesting that autophagy plays an important role in the occurrence and development of age-related macular degeneration.