Zhang Xi, Liao Yanhong, Ye Ting
Departmental Health Check Section, Ningbo Eye Hospital Ningbo, Zhejiang, China.
Int J Clin Exp Pathol. 2020 Aug 1;13(8):2058-2064. eCollection 2020.
This study explores the correlations of stromal cell-derived factor-1 (SDF-1) and CXC chemokine receptor 4 (CXCR4) levels with caspase-3 expression in the retina of rats after optic nerve injury.
A total of 24 adult healthy specific pathogen-free Sprague-Dawley rats were selected and randomly divided into an optic nerve injury group (n=16) and an optic nerve sham-injury group (n=8). The optic nerve injury group was further sub-divided into a 3 d group (n=8) and a 7 d group (n=8) after their injuries. In the optic nerve injury group, the left eye of each rat was removed and prepared for the optic nerve injury model using the optic nerve clamping method. In the sham-injury group, the optic nerve in the left eye was only exposed without being clamped. The rats were sacrificed at 3 d and 7 d after their optic nerve injuries, and the retina was isolated. The expressions of SDF-1, CXCR4, and caspase-3 in the retina of the rats in each group were measured using an immunohistochemical method. Messenger ribonucleic acid (mRNA) and the protein expressions of SDF-1, CXCR4, and caspase-3 (cleaved caspase-3) in the retinas of rats were measured using quantitative real-time polymerase chain reaction (qPCR) and western blotting, respectively. Moreover, the correlations of the expression of SDF-1 and CXCR4 with caspase-3 expression were analyzed using the Spearman method. The apoptosis of retinal ganglion cells of rats in each group was observed using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining.
Immunohistochemistry of the retinas revealed that, compared with those in the sham-injury group, the expressions of SDF-1 and CXCR4 in the retina in the 3 d group and the 7 d group were gradually increased. Caspase-3 expression was significantly elevated at 3 d after the injuries, but obviously decreased at 7 d after the injuries. The results of qPCR showed that the relative expression levels of SDF-1 and CXCR4 mRNA in the retina at 3 d and 7 d after optic nerve injuries were also significantly higher than those in the sham-injury group (<0.01), and the caspase-3 mRNA expression was initially increased at 3 d but reduced at 7 d after the injuries (<0.01). Western blotting for the detection of the SDF-1, CXCR4 and caspase-3 proteins indicated changes similar to those of the qPCR. Spearman analysis results demonstrated that there was a positive correlation between the SDF-1 and CXCR4 expressions, but the expressions of SDF-1 and CXCR4 had negative correlations with caspase-3 expression. TUNEL staining showed that apoptosis of the retinal cells was increased in the 3 d group but significantly decreased in the 7 d group.
After optic nerve injury, the continuous increase of the SDF-1 and CXCR4 levels suppresses the apoptosis of retinal cells and repairs the retina by inhibiting the cleavage activation of caspase-3. This provides new insights for the ongoing treatment of optic nerve injury.
本研究探讨基质细胞衍生因子-1（SDF-1）和CXC趋化因子受体4（CXCR4）水平与视神经损伤后大鼠视网膜中半胱天冬酶-3（caspase-3）表达的相关性。
选取24只成年健康无特定病原体的Sprague-Dawley大鼠，随机分为视神经损伤组（n = 16）和视神经假损伤组（n = 8）。视神经损伤组在损伤后进一步分为3 d组（n = 8）和7 d组（n = 8）。在视神经损伤组，每只大鼠的左眼摘除并采用视神经夹闭法制备视神经损伤模型。在假损伤组，仅暴露左眼视神经但不夹闭。在视神经损伤后3 d和7 d处死大鼠，分离视网膜。采用免疫组织化学方法检测各组大鼠视网膜中SDF-1、CXCR4和caspase-3的表达。分别采用定量实时聚合酶链反应（qPCR）和蛋白质印迹法检测大鼠视网膜中SDF-1、CXCR4和caspase-3（裂解的caspase-3）的信使核糖核酸（mRNA）和蛋白质表达。此外，采用Spearman法分析SDF-1和CXCR4表达与caspase-3表达的相关性。采用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）染色观察各组大鼠视网膜神经节细胞的凋亡情况。
视网膜免疫组织化学显示，与假损伤组相比，3 d组和7 d组视网膜中SDF-1和CXCR4的表达逐渐增加。损伤后3 d时caspase-3表达显著升高，但损伤后7 d时明显降低。qPCR结果显示，视神经损伤后3 d和7 d时视网膜中SDF-1和CXCR4 mRNA的相对表达水平也显著高于假损伤组（<0.01），损伤后3 d时caspase-3 mRNA表达最初增加，但损伤后7 d时降低（<0.01）。检测SDF-1、CXCR-4和caspase-3蛋白的蛋白质印迹显示变化与qPCR相似。Spearman分析结果表明，SDF-1和CXCR4表达之间呈正相关，但SDF-1和CXCR4的表达与caspase-3表达呈负相关。TUNEL染色显示，3 d组视网膜细胞凋亡增加，但7 d组显著减少。
视神经损伤后，SDF-1和CXCR4水平持续升高，通过抑制caspase-3的裂解激活来抑制视网膜细胞凋亡并修复视网膜。这为视神经损伤的现行治疗提供了新的见解。
