Background: The demonstrated role of mitogen-activated protein kinase (MAPK) in both cell apoptosis and the inflammation pathway makes it an attractive target for photoreceptor protection. The aim of this study was to investigate the protective effects of MAPK antagonists against photoreceptor degeneration and retinal inflammation in a rat model of light-induced retinal degeneration.
Methods: Sprague Dawley rats were treated with intravitreal injections of MAPK antagonists, inhibitors of p-P38, phosphorylated-extracellular regulated kinase (p-ERK) 1/2, and p-c-Jun N-terminal kinase (JNK) just before they were assigned to dark adaptation. After dark adaptation for 24 h, rats were exposed to blue light (2500 lux) in a light box for 24 h, and then returned to the normal 12-h light/12-h dark cycle. Samples were collected at different time points. MAPK expression during light exposure was examined with immunofluorescence. Photoreceptor death was detected with histopathology and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The expression of retinal p-ERK1/2, caspase 3, activated caspase 3, tumor necrosis factor (TNF)-α, and interleukin (IL)-1β was examined by Western blotting. Differences between groups were evaluated using unpaired one-way analysis of variance and least significant difference post hoc tests.
Results: MAPKs (P38, ERK1/2, and p-JNK) were phosphorylated and activated in the light injury groups, compared with normal group, and their expressions were mainly elevated in the outer nuclear layer (ONL). Among the selected MAPK antagonists, only the p-ERK1/2 inhibitor attenuated the loss of photoreceptors and the thinning of ONL in light injury groups. Besides, p-ERK1/2 inhibitor refrained light-induced photoreceptor apoptosis, which was presented by TUNEL positive cells. Light injury significantly increased the expression of p-ERK1/2 (1.12 ± 0.06 vs. 0.57 ± 0.08, t = 9.99, P < 0.05; 1.23 ± 0.03 vs. 0.57 ± 0.08, t = 11.90, P < 0.05; and 1.12 ± 0.12 vs. 0.57 ± 0.08, t = 9.86, P < 0.05; F = 49.55, P < 0.001), and induced caspase 3 activating (0.63 ± 0.06 vs. 0.14 ± 0.05, t = 13.67, P < 0.05; 0.74 ± 0.05 vs. 0.14 ± 0.05, t = 16.87, P < 0.05; and 0.80 ± 0.05 vs. 0.14 ± 0.05, t = 18.57, P < 0.05; F = 100.15, P < 0.001), compared with normal group. The p-ERK1/2 inhibitor significantly reduced p-ERK1/2 overexpression (0.61 ± 0.06 vs. 1.12 ± 0.06, t = -9.26, P < 0.05; 0.77 ± 0.06 vs. 1.23 ± 0.03, t = -8.29, P < 0.05; and 0.68 ± 0.03 vs. 1.12 ± 0.12, t = -7.83, P < 0.05; F = 49.55, P < 0.001) and downregulated caspase 3 activating (0.23 ± 0.04 vs. 0.63 ± 0.06, t = -11.24, P < 0.05; 0.43 ± 0.03 vs. 0.74 ± 0.05, t = -8.86, P < 0.05; and 0.58 ± 0.03 vs. 0.80 ± 0.05, t = -6.17, P < 0.05; F = 100.15, P < 0.001), compared with light injury group. No significant change in the total level of caspase 3 was seen in different groups (F = 0.56, P = 0.75). As for inflammation, light injury significantly increased the expression of TNF-α (0.42 ± 0.04 vs. 0.25 ± 0.05, t = 5.99, P < 0.05; 0.65 ± 0.03 vs. 0.25 ± 0.05, t = 14.87, P < 0.05; and 0.86 ± 0.04 vs. 0.25 ± 0.05, t = 22.58, P < 0.05; F = 160.27, P < 0.001) and IL-1β (0.24 ± 0.01 vs. 0.19 ± 0.02, t = 2.33, P < 0.05; 0.35 ± 0.02 vs. 0.19 ± 0.02, t = 7.97, P < 0.05; and 0.48 ± 0.04 vs. 0.19 ± 0.02, t = 14.69, P < 0.05; F = 77.29, P < 0.001), compared with normal group. P-ERK1/2 inhibitor significantly decreased the overexpression of TNF-α (0.22 ± 0.02 vs. 0.42 ± 0.04, t = -7.40, P < 0.05; 0.27 ± 0.02 vs. 0.65 ± 0.03, t = -14.27, P < 0.05; and 0.33 ± 0.03 vs. 0.86 ± 0.04, t = -19.58, P < 0.05; F = 160.27, P < 0.001) and IL-1β (0.13 ± 0.03 vs. 0.24 ± 0.01, t = -5.77, P < 0.05; 0.17 ± 0.01 vs. 0.22 ± 0.02, t = -9.18, P < 0.05; and 0.76 ± 0.05 vs. 0.48 ± 0.04, t = -13.12, P < 0.05; F = 77.29, P < 0.001), compared with light injury group.
Conclusion: The p-ERK1/2 inhibitor might protect the retina from light-induced photoreceptor degeneration and retinal inflammation.
P- ERK1/2抑制剂对光损伤致视网膜感光细胞退化的保护作用 摘要 背景：丝裂原活化蛋白激酶（MAPK）在细胞凋亡和炎症途径中的作用使其成为光感受器保护的可能重要靶点。本研究旨在探讨MAPK拮抗剂对光诱导大鼠视网膜变性模型中光感受器变性和视网膜炎症的保护作用。 方法：SD大鼠在暗适应前给与玻璃体腔内注射MAPK拮抗剂，包括p-P38抑制剂、p-细胞外调节激酶（ERK）1/2抑制剂和p-c-Jun N末端激酶（JNK）抑制剂。暗适应24小时后，大鼠在照光箱蓝光中暴露（2500勒克斯）24小时，然后恢复到正常的12小时光/12小时暗循环，随后在不同时间点采集样品。本研究用免疫荧光法检测光暴露后细胞MAPK的表达，用组织病理学和末端脱氧核苷酸转移酶dUTP缺口末端标记（TUNEL）染色检测光感受器凋亡，并用免疫印迹法检测视网膜p-ERK1/2、caspase 3、活化caspase 3、肿瘤坏死因子（TNF-α）、白细胞介素（IL-β）的蛋白表达。各组之间的差异采用非配对单因素方差分析和LSD事后分析测算。 结果：MAPK在光诱导视网膜变性模型中被磷酸化和激活，其表达上调主要表现在视网膜外核层（ONL），提示MAPK途径参与了光诱导的视网膜变性。在我们所选择的MAPK拮抗剂中，只有p-ERK1/2抑制剂显著减轻了光损伤带来的感光细胞丢失和视网膜外核层（ONL）的厚度变薄。此外，P-ERK1/2抑制剂减少了光损伤诱导的视网膜外核层（ONL）细胞TUNEL染色阳性率。与对照组相比，光损伤1天、3天、5天组p-ERK1/2（1.12 ± 0.06 vs. 0.57 ± 0.08, t=9.99, P <0.05; 1.23 ± 0.03 vs. 0.57 ± 0.08, t=11.90, P <0.05; and 1.12 ± 0.12 vs. 0.57 ± 0.08, t=9.86, P <0.05；F=49.55, P <0.001）和活化caspase 3（0.63 ± 0.06 vs. 0.14 ± 0.05, t=13.67, P <0.05; 0.74 ± 0.05 vs. 0.14 ± 0.05, t=16.87, P <0.05; and 0.80 ± 0.05 vs. 0.14 ± 0.05, t=18.57, P <0.05；F=100.15, P <0.001）的表达显著增加。p-ERK1/2 抑制剂显著降低光损伤组相应时间点p-ERK1/2 的表达（0.61 ± 0.06 vs. 1.12 ± 0.06, t=-9.26, P <0.05; 0.77 ± 0.06 vs. 1.23 ± 0.03, t=-8.29, P <0.05; and 0.68 ± 0.03 vs. 1.12 ± 0.12, t=-7.83, P <0.05; F=49.55, P <0.001）和活化caspase 3的水平（0.23 ± 0.04 vs. 0.63 ± 0.06, t=-11.24, P <0.05; 0.43 ± 0.03 vs. 0.74 ± 0.05, t=-8.86, P <0.05; and 0.58 ± 0.03 vs. 0.80 ± 0.05, t=-6.17, P <0.05; F=100.15, P <0.001）. 所有组间总caspase 3的表达无显著变化（F=0.56, P =0.75）。视网膜炎症指标的检测显示，与对照组相比，光损伤1天、3天、5天组TNF-α（0.42 ± 0.04 vs. 0.25 ± 0.05, t=5.99, P <0.05; 0.65 ± 0.03 vs. 0.25 ± 0.05, t=14.87, P <0.05; and 0.86 ± 0.04 vs. 0.25 ± 0.05, t=22.58, P <0.05; F=160.27, P <0.001）和 IL-1β（0.24 ± 0.01 vs. 0.19 ± 0.02, t=2.33, P <0.05; 0.35 ± 0.02 vs. 0.19 ± 0.02, t=7.97, P <0.05; and 0.48 ± 0.04 vs. 0.19 ± 0.02, t=14.69, P <0.05; F=77.29, P <0.001）的表达显著增加。P-ERK1/2抑制剂显著降低光损伤组相应时间点TNF-α（0.22 ± 0.02 vs. 0.42 ± 0.04, t=-7.40, P <0.05; 0.27 ± 0.02 vs. 0.65 ± 0.03, t=-14.27, P <0.05; and 0.33 ± 0.03 vs. 0.86 ± 0.04, t=-19.58, P <0.05; F=160.27, P <0.001）和 IL-1β（0.13 ± 0.03 vs. 0.24 ± 0.01, t=-5.77, P <0.05; 0.17 ± 0.01 vs. 0.22 ± 0.02, t=-9.18, P <0.05; and 0.76 ± 0.05 vs. 0.48 ± 0.04, t=-13.12, P <0.05; F=77.29, P <0.001）的表达。 结论：p-ERK抑制剂可能对光诱导的感光细胞变性和视网膜炎症反应有保护作用。.
Keywords: Apoptosis; Inflammation; Retinal Degeneration.