Department of Ophthalmology, Beijing Shijitan Hospital, Capital Medical University, Beijing, People’s Republic of China.
Invest Ophthalmol Vis Sci. 2012 Nov 9;53(12):7567-75. doi: 10.1167/iovs.12-9446.
To evaluate the expression of monocyte chemoattractant protein-1 (MCP-1) in the rodent model of diabetic retinopathy (DR) and to study the stimulation of microglial activation by retinal neuronal MCP-1 in vitro.
Diabetes mellitus was induced by streptozotocin (STZ) injection. The expression of MCP-1 was determined using immunohistochemical methods, Western blotting and RT-PCR analyses. Retinal neurons and microglia were separated and co-cultured in a Transwell apparatus. The levels of soluble MCP-1 that were produced after stimulation of retinal neurons by adding advanced glycation end products (AGEs) to the medium were measured by ELISA. The degree of microglial activation was measured by testing microglial migration and the level of soluble TNF-α in the medium by ELISA. The ability of neuronal MCP-1 to stimulate microglia activation was examined by pre-exposing the retinal neurons to AGEs and an MCP-1 antibody or to AGEs and SiRNA specific to MCP-1.
A marked increase in the expression of MCP-1 was detected 4 weeks after STZ injection, and the expression was consistently upregulated at 3 and 5 months in the rodent DR model. Stimulation with AGEs significantly increased the expression of MCP-1 in retinal neurons, which activated microglial cells, including increased microglial migration and upregulated secretion of TNF-α. Retinal neurons that were pre-exposed to AGEs and an MCP-1 antibody or MCP-1 knockdown displayed greatly reduced microglial migration and TNF-α secretion.
Upregulation of MCP-1 began during the early stage of DR and increased with the development of the disease. Retinal neurons are the main source of MCP-1, and they play an important role in retinal microglial activation, which may be an important link in the pathogenesis of DM.
评估单核细胞趋化蛋白-1（MCP-1）在糖尿病性视网膜病变（DR）啮齿动物模型中的表达，并研究视网膜神经元 MCP-1 对体外小胶质细胞激活的刺激作用。
通过链脲佐菌素（STZ）注射诱导糖尿病。使用免疫组织化学方法、Western blot 和 RT-PCR 分析来确定 MCP-1 的表达。通过 Transwell 装置分离和共培养视网膜神经元和小胶质细胞。通过 ELISA 测量加入晚期糖基化终产物（AGEs）刺激培养基中后产生的可溶性 MCP-1 的水平。通过 ELISA 测试小胶质细胞迁移和培养基中可溶性 TNF-α的水平来测量小胶质细胞的激活程度。通过预先用 AGEs 和 MCP-1 抗体或 AGEs 和针对 MCP-1 的 SiRNA 处理视网膜神经元来检查神经元 MCP-1 刺激小胶质细胞激活的能力。
在 STZ 注射后 4 周检测到 MCP-1 的表达明显增加，并且在 DR 啮齿动物模型中 3 和 5 个月持续上调。AGEs 的刺激显著增加了视网膜神经元中 MCP-1 的表达，从而激活了小胶质细胞，包括增加了小胶质细胞迁移和上调了 TNF-α的分泌。预先用 AGEs 和 MCP-1 抗体或 MCP-1 敲低处理的视网膜神经元显示小胶质细胞迁移和 TNF-α分泌大大减少。
MCP-1 的上调始于 DR 的早期阶段，并随着疾病的发展而增加。视网膜神经元是 MCP-1 的主要来源，它们在视网膜小胶质细胞激活中起重要作用，这可能是 DM 发病机制中的一个重要环节。
