米诺环素在L-谷氨酸诱导的视网膜神经节细胞损伤中的保护作用及分子机制

目的 探讨米诺环素在L-谷氨酸诱导的视网膜神经节细胞(RGC)毒性中的保护作用和分子机制.方法 实验研究.原代小鼠RGCs体外培养24 h后,随机分为3组:对照组,L-谷氨酸组(100 μmol/L、500 μmol/L、1 mmol/L和2 mmol/L)及L-谷氨酸+米诺环素组(30 μmol),观察不同浓度L-谷氨酸对RGC的存活率与轴突生长的损伤作用及米诺环素的保护作用.体内实验,将雌性B6小鼠随机分为实验组(30只)和对照组(30只).两组小鼠腹腔内分别注射米诺环素(实验组,60 mg/kg)或生理盐水(对照组),每天1次,连续7 d.第2天时,两组小鼠玻璃体腔内注射2μl L-谷氨酸(2 mmol/L),诱导RGC损伤.免疫组化染色分析β-Ⅲ-tubulin阳性细胞数目变化及视网膜神经胶质酸性蛋白(GFAP)表达情况,Real-time PCR和免疫印迹法分别检测小鼠视网膜组织中干扰素γ(IFN-γ)、白细胞介素(IL-1)、肿瘤坏死因子α(TNF-α)、GFAP与波形蛋白(Vimentin)的mRNA及蛋白表达水平.结果 体外实验显示,与对照组相比,L-谷氨酸降低RGC的存活率,与剂量和干预时间呈负相关.同时L-谷氨酸可明显抑制RGC轴突的生长,RGC轴突长度>2BL、1～2 BL、<1 BL占总细胞数比例分别从50.38%、7.83%和3.72%降至31.43%、5.05%和1.29%.而米诺环素能明显减轻L-谷氨酸对RGC的毒性作用,改善RGC轴突生长,各组细胞比例回升至51.00%、8.10%和2.43%,谷氨酸与对照组相比、米诺环素组与谷氨酸相比,差异有统计学意义(F=18.87,P<0.01).体内实验结果显示,与对照组相比,L-谷氨酸组小鼠RGC数目显著减少(45.00±10.21和68.50±2.86),而米诺环素治疗后可明显改善L-谷氨酸诱导的RGC损伤,RGC数目恢复至62.00±11.65,(F=7.6,P<0.01).谷氨酸处理后视网膜组织中GFAP的表达水平明显增高,而米诺环素明显降低视网膜组织中GFAP的表达.同时,L-谷氨酸显著提高小鼠视网膜组织中炎症相关因子IFN-γ、IL-1、TNF-α及胶质细胞相关蛋白Vimentin和GFAP的基因及蛋白表达水平,而米诺环素可显著抑制这些因子的表达.结论 L-谷氨酸损伤可诱导RGC凋亡、抑制RGC轴突生长,并上调炎症因子及视网膜相关胶质蛋白的基因与蛋白表达水平,米诺环素对L-谷氨酸所导致的视网膜神经节细胞损伤具有明显的保护作用.

Protective effect of minocycline on glutamate-induced retinal ganglion cell injury and molecular mechanism

Objective To investigate the protective effect and molecular mechanism of minocycline on toxicity of retinal ganglion cells induced by L-glutamate. Methods Primary mouse retinal ganglion cells (RGC) were isolated from mouse retinalin vitro. RGC were divided into control group, L-glutamate group,and L-glutamate + minocycline group, the cell survival rate and nerve axon growth length were observed. In vivo study, B6 mice was intravitreal injected with 2 μl L-glutamate (2 mmol/L) to construct a toxic damage to retinal ganglion cells animal model. One day before the beginning of the experiment, mice were daily intraperitoneal injected with minocycline (60 mg/kg, saline injected in the control group) till day 7, β3 tubulin positive cells and retinal GFAP protein expression were evaluated by tissue i mmol/L unofluorescence assay. Real-time PCR and Western blot assay were used to detect IFN-γ, IL-1, TNF-α and GFAP and vimentin mRNA and protein expression level in retinal tissues. Results Compared with control group, RGC survival rate in L-glutamate group was significantly reduced with a dose-and time-dependent. In addition,axon growth was inhibited with the treatment of glutamic acid, while these effects were abolished in the minocycline group ( F = 18.87, P < 0. 01 ). Animal study showed that the number of RGC dramatically decreased, however, expression of GFAP in retinal tissue significantly increased in L-glutamate treated mice,compared to control (F = 7.6, P < 0.01 ). Minocycline treatment significantly improved L-glutamate-induced ganglion cell damage and significantly reduced their GFAP expression. Both mRNA and protein expression levels of IFN-γ, IL-1, TNF-α and GFAP and Vimentin in retinal tissue of L-glutamic acid group significantly upregulated compared to control, while the minocycline significantly reduced the expression of these factors.Conclusions L-glutamic acid can induce retinal ganglion cells damage, inhibit axon growth and increase inflammatory and glial-cell related genes and proteins expression. Minocycline could significantly protect retinal ganglion cells from the injury caused by L-glutamate.