Qin Tingyu, Gao Shasha
Department of Ophthalmology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450000, China.
J Ophthalmol. 2018 Jul 12;2018:5392432. doi: 10.1155/2018/5392432. eCollection 2018.
As far as we know, during the development of age-related macular degeneration (AMD), the activity of proteasome in retinal pigment epithelium cells (RPE) gradually decreases. And a lot of research has shown that age-related macular degeneration is closely related to inflammation and autoimmune. Moreover, there are many cytokines (CKs) involved in the course of inflammation. In this study, we are going to investigate how the decrease of proteasome activity affects the production of interleukin-6 (IL-6) in human retinal pigment epithelium cells (ARPE-19).
Cultured ARPE-19 was treated with or without MG132, a proteasome inhibitor, and the levels of IL-6 mRNA (messenger ribonucleic acid) in RPE at 1 h, 4 h, 8 h, and IL-6 protein in the culture medium at 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h were measured by real-time polymerase chain reaction (real-time PCR) and enzyme-linked immunosorbent assay (ELISA). The protein levels of MCP-1 (monocyte chemoattractant protein-1) in the culture medium at 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h were also measured by ELISA. Then we tested which of cell signal pathways regulating the production of IL-6 were activated when we added MG132 into the medium by Western blot and electrophoretic mobility shift assays (EMSA). After that, we put the inhibitors of these activated cell signal pathways into the medium individually to see which inhibitor can counteract the effect of upregulating the levels of IL-6 in the culture medium of RPE.
MG132 decreased the secretion of MCP-1 in the culture medium of RPE, but it increased the expression of IL-6 mRNA in RPE and IL-6 protein level in the culture medium of RPE. MG132 treatment was also found to enhance the level of phosphorylated p38 mitogen-activated protein kinases (MAPKs) and c-Jun N-terminal Kinase (JNK) by Western blotting. More importantly, the effect of MG132 on upregulating the levels of IL-6 was inhibited by SB203580, an inhibitor of P38 MAP kinases. But the JNK inhibitor, SP600125, cannot prevent the effect of upregulating the levels of IL-6 by MG132 in the RPE culture medium.
We concluded that the proteasome inhibitor, MG132, upregulates IL-6 production in RPE cells through the activation of P38 MAPKs.
据我们所知，在年龄相关性黄斑变性（AMD）的发展过程中，视网膜色素上皮细胞（RPE）中蛋白酶体的活性逐渐降低。许多研究表明，年龄相关性黄斑变性与炎症和自身免疫密切相关。此外，有许多细胞因子（CKs）参与炎症过程。在本研究中，我们将研究蛋白酶体活性的降低如何影响人视网膜色素上皮细胞（ARPE - 19）中白细胞介素 - 6（IL - 6）的产生。
用蛋白酶体抑制剂MG132处理培养的ARPE - 19细胞，或不进行处理，通过实时聚合酶链反应（real - time PCR）和酶联免疫吸附测定（ELISA）分别检测1小时、4小时、8小时时RPE中IL - 6信使核糖核酸（mRNA）的水平，以及2小时、4小时、6小时、8小时、10小时和12小时时培养基中IL - 6蛋白的水平。还用ELISA检测2小时、4小时、6小时、8小时、10小时和12小时时培养基中单核细胞趋化蛋白 - 1（MCP - 1）的蛋白水平。然后通过蛋白质印迹法和电泳迁移率变动分析（EMSA）检测当向培养基中加入MG132时，哪些调节IL - 6产生的细胞信号通路被激活。之后，我们将这些激活的细胞信号通路的抑制剂分别加入培养基中，观察哪种抑制剂可以抵消上调RPE培养基中IL - 6水平的作用。
MG132降低了RPE培养基中MCP - 1的分泌，但增加了RPE中IL - 6 mRNA的表达以及RPE培养基中IL - 6蛋白的水平。通过蛋白质印迹法还发现，MG132处理可提高磷酸化的p38丝裂原活化蛋白激酶（MAPKs）和c - Jun氨基末端激酶（JNK）的水平。更重要的是，P38 MAP激酶抑制剂SB203580可抑制MG132上调IL - 6水平的作用。但JNK抑制剂SP600125不能阻止MG132上调RPE培养基中IL - 6水平的作用。
我们得出结论，蛋白酶体抑制剂MG132通过激活P38 MAPKs上调RPE细胞中IL - 6的产生。
