Nguyen Hoang Anh Thu, Lee Haesung, Lee Sook-Jeong
Department of Bioactive Material Sciences and Research Center of Bioactive Materials, Jeonbuk National University, Jeonju, Jeollabuk-do, 54896, South Korea.
Department of Bioactive Material Sciences and Research Center of Bioactive Materials, Jeonbuk National University, Jeonju, Jeollabuk-do, 54896, South Korea.
Exp Eye Res. 2022 May;218:109004. doi: 10.1016/j.exer.2022.109004. Epub 2022 Feb 24.
The antimalarial drug chloroquine (CQ) induces retinopathy, a disorder characterized by lysosomotropic alteration. In this study, we examined whether D4476 (4-(4-(2,3-dihydrobenzo [1,4] dioxin-6-yl)-5-pyridin-2-yl-1H-imidazole-2-yl) benzamide), a specific casein kinase 1 inhibitor, alleviate CQ-induced retinopathy in adult retinal pigment epithelial (ARPE-19) cells. Cultured ARPE-19 cells were exposed to CQ with or without D4476 and cell death was quantified using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. To examine autophagy flux, ARPE-19 cells were transfected with green fluorescence protein light chain 3 (GFP-LC3)-red fluorescence protein (RFP)-LC3ΔG plasmid DNA and co-stained with the lysosomal-associated membrane protein (LAMP)-1 antibody. Western blotting and fluorescence-activated cell sorting (FACS) showed apoptosis, whereas the fluorescence intensity of 2'-7'-dichlorofluorescein diacetate revealed levels of cellular oxidative stress. We then confirmed the effect of D4476 on the interaction between Beclin 1 and B-cell lymphoma-2 (Bcl-2) through immunoprecipitation with an anti-Bcl-2 antibody. Following CQ exposure, ARPE-19 cells accumulated autophagosomes because of defective lysosomal degradation. Furthermore, CQ trapped Beclin 1 with Bcl-2, disturbing autophagy initiation and autolysosome formation. However, D4476 alleviated CQ-induced effects by rescuing ARPE-19 cells from CQ-induced toxicity by modulating the association between Beclin 1 and Bcl-2. Therefore, D4476 controls autophagy and apoptosis simultaneously by upregulating autophagy flux, decreasing ROS formation, and triggering the expression of anti-apoptotic proteins through inhibition of mTOR, JNK, and p38 MAPK signals. We conclude that D4476 is a promising treatment strategy for CQ-mediated retinopathy.
抗疟药物氯喹（CQ）可诱发视网膜病变，这是一种以溶酶体亲和性改变为特征的病症。在本研究中，我们检测了特异性酪蛋白激酶1抑制剂D4476（4-(4-(2,3-二氢苯并[1,4]二恶英-6-基)-5-吡啶-2-基-1H-咪唑-2-基)苯甲酰胺）是否能减轻成年视网膜色素上皮（ARPE-19）细胞中CQ诱导的视网膜病变。将培养的ARPE-19细胞暴露于含或不含D4476的CQ中，并使用3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）法对细胞死亡进行定量。为检测自噬通量，用绿色荧光蛋白轻链3（GFP-LC3）-红色荧光蛋白（RFP）-LC3ΔG质粒DNA转染ARPE-19细胞，并用溶酶体相关膜蛋白（LAMP）-1抗体进行共染色。蛋白质免疫印迹法和荧光激活细胞分选（FACS）显示细胞凋亡，而二氯荧光素二乙酸酯的荧光强度揭示了细胞氧化应激水平。然后，我们通过用抗Bcl-2抗体进行免疫沉淀，证实了D4476对Beclin 1与B细胞淋巴瘤-2（Bcl-2）之间相互作用的影响。CQ暴露后，由于溶酶体降解缺陷，ARPE-19细胞中自噬体积累。此外，CQ使Beclin 1与Bcl-2结合，干扰自噬起始和自噬溶酶体形成。然而，D4476通过调节Beclin 1与Bcl-2之间的关联，将ARPE-19细胞从CQ诱导的毒性中拯救出来，从而减轻了CQ诱导的效应。因此，D4476通过上调自噬通量、减少活性氧生成并通过抑制mTOR、JNK和p38丝裂原活化蛋白激酶信号触发抗凋亡蛋白的表达，同时控制自噬和细胞凋亡。我们得出结论，D4476是CQ介导的视网膜病变的一种有前景的治疗策略。
