半乳糖凝集素-1抑制剂OTX008对氧诱导视网膜病变小鼠视网膜新生血管的抑制作用

OTX008 inhibits retinal neovascularization in oxygen-induced retinopathy mice

目的 观察半乳糖凝集素-1（Galectin-1）特异性抑制剂OTX008对氧诱导视网膜病变小鼠视网膜新生血管（RNV）的抑制作用,并探讨其作用机制.方法 7日龄C57BL/6J小鼠128只采用随机数字表法随机分为正常组、单纯模型组、OTX008干预组和磷酸盐缓冲液（PBS）空白对照组,每组32只.正常组小鼠在正常氧环境下饲养;其余3组小鼠建立氧诱导视网膜病变模型.小鼠12日龄时,OTX008干预组小鼠双眼按0.25μg/μl的剂量玻璃体腔注射OTX008 1 μl,PBS空白对照组注射等体积PBS.正常组及单纯模型组小鼠不做其他任何干预和处理.小鼠17日龄时,各组作视网膜冰冻切片并行免疫荧光染色,定性观察Galectin-1蛋白的分布情况;作视网膜组织石蜡切片并行苏木精-伊红染色,计数突破内界膜的血管内皮细胞核;作全视网膜铺片并行免疫荧光染色,观察视网膜血管变化并统计RNV区、无灌注区、缺氧区面积;采用蛋白免疫印迹法（Westernblot）检测Galectin-1、神经纤毛蛋白质-1（Neuropilin-1）、磷酸化血管内皮生长因子受体2（pVEGFR2）的蛋白相对表达量.结果 与正常组比较,单纯模型组、PBS空白对照组小鼠视网膜Galectin-1蛋白荧光在视网膜神经节细胞层、内丛状层及内核层明显增强;OTX008干预组小鼠视网膜Galectin-1蛋白荧光较单纯模型组、PBS空白对照组明显减弱.单纯模型组、PBS空白对照组小鼠视网膜中突破内界膜的血管内皮细胞核计数较正常组明显增加,差异有统计学意义（t=9.314,P＜0.05）;OTX008干预组小鼠视网膜中突破内界膜的血管内皮细胞核计数较单纯模型组、PBS空白对照组明显下降,差异有统计学意义（t=8.038、7.774,P＜0.05）.与单纯模型组、PBS空白对照组比较,OTX008干预组小鼠RNV区（t=13.250、12.570）、无灌注区（t=15.590、12.430）和缺氧区（t=9.542、9.928）面积明显减小,差异有统计学意义（P＜0.05）.Western blot检测结果显示,与单纯模型组及PBS空白对照组相比,OTX008干预组小鼠视网膜Galectin-1（t=24.800、23.060）、Neuropilin-1（t=4.120、3.530）、pVEGFR2（t=25.880、15.480）蛋白相对表达量明显下调,差异有统计学意义（P＜0.05）.结论 Galectin-1特异性抑制剂OTX008可抑制氧诱导RNV形成,降低视网膜组织缺氧.其机制可能与抑制Galectin-1表达,减少Galectin-1与Neuropilin-1结合,降低pVEGFR2表达有关.

Objective To investigate the inhibitory effects and possible related mechanism of OTX008 [a selective inhibitor of galectin-1 （Galectin-1）] on retinal neovascularization （RNV） in mouse model of oxygeninduced retinopathy （OIR）.Methods 7-day-old （P7） C57BL/6J mice were randomly （according to random number table） divided into 4 groups including normal group,OIR group,OIR-OTX008 group and OIRphosphate buffered saline （PBS） group.To establish the OIR mouse model,mice from all groups except normal group were expose to （75±2）% oxygen for 5 days and then to room air.OIR-OTX008 group received an intravitreal injection of 1 μl （0.25 μg/μl） OTX008 at P12,OIR-PBS group received the equal volume （1 μl） of PBS injection.Mice from 4 groups were euthanized at P17,and retinas were collected for molecular biological analysis and morphological study.RNV was evaluated by counting the number ofpre-retinal neovascular nuclei and the whole-mount immunofluorescent staining of mouse retina.Cyrosections of retinas were imaged via confocal microscopy to observe the enrichment of staining of Galectin-1.Protein levels of Galectin-1,Neuropilin-1 and phosphorylation of vascular endothelial growth factor receptor 2 （pVEGFR2） were determined with Western blot.Results At P17,Galectin-1 expressed higher in retinal ganglion cell layer,inner plexiform layer and inner nuclear layer from OIR group and OIR-PBS group than normal group.Galectin-1 expressed less in cryosection retinas from OIR-OTX008 group than OIR group and OIR-PBS group.The numbers ofpre-retinal neovascular cell nuclei from OIR group and OIR-pBS group were obviously more than that from normal group （t=9.314,P〈0.05）.The number of pre-retinal neovascular cell nuclei from OIR-OTX008 group were obviously lower than those from OIR group and OIR-PBS group （t=8.038,7.774;P〈0.05）.The RNV tufts area （t=13.250,12.570）,non-perfusion area （t=15.590,12.430） and hypoxic area （t=9.542,9.928） from OIR-OTX008 group were significantly smaller than those in OIR group and OIR-PBS group （P〈 0.05）.Protein levels of Galectin-1 （t=24.800,23.060）,Neuropilin-1 （t=4.120,3.530） and pVEGFR2 （t=25.880,15.480） in the OIR-OTX008 group were significantly down-regulated than those from OIR group and OIR-PBS group （P〈0.05）.Conclusion Intravitreal injection of OTX008 inhibits RNV and ameliorates retinal hypoxia in mice model of OIR possibly through down-regulating Galectin-1,Neurolinpin-1 and pVEGFR2.