5-脂氧合酶对缺氧诱导的视网膜新生血管形成的抑制作用

Inhibitory effects of 5-lipoxygenase on oxygen-induced retinal neovascularization in mice

目的 观察5-脂氧合酶（5-LOX）对缺氧诱导的视网膜新生血管形成的抑制作用,探讨视网膜新生血管形成的可能机制.方法 鼠龄为7d的C57BL/6J幼鼠随机分为正常组、氧诱导视网膜病变（OIR）模型组、大剂量干预组、小剂量干预组、干预对照组.各组分别为12只.除正常组外,其余各组小鼠与哺乳母鼠共同置于氧浓度为（75±2）％的氧箱内饲养5d后转移至正常环境中饲养5d,建立OIR模型.12～17日龄时,大剂量干预组、小剂量干预组小鼠腹腔每天分别注射100、50 mg/kg 5-LOX抑制剂去甲二氢愈创木酸;干预对照组小鼠腹腔每天注射等体积1％二甲基亚砜;OIR模型组小鼠出氧箱后不作任何处理.石蜡切片苏木精-伊红染色并计数突破视网膜内界膜的血管内皮细胞核,CD34标记鼠新生血管内皮细胞,逆转录-聚合酶链反应（RT PCR）检测视网膜中5-LOX、血管内皮生长因子（VEGF）-a、VEGF受体-2（VEGFR-2）的mRNA含量.蛋白质免疫印迹法（Western blot）检测视网膜中5-LOX、VEGF-a、VEGFR-2、细胞外磷酸化信号调节蛋白激酶（P-ERK） 1/2的蛋白表达含量.结果 不同剂量干预组突破内界膜的视网膜新生血管内皮细胞核数较OIR模型组、干预对照组明显减少,差异有统计学意义（F=73.390,P＜0.05）.RT-PCR、Western blot检测结果显示,不同剂量干预组小鼠视网膜5-LOX、VEGF-a、VEGFR-2的mRNA和蛋白表达均较OIR模型组、干预对照组减少,差异有统计学意义（F=92.668,P＜0.05）;大剂量干预、小剂量干预组之间VEGFR-2蛋白相对表达量比较,差异无统计学意义（F=2.118,P＞0.05）;5-LOX、VEGF-a、P-ERK 1/2相对蛋白表达比较,差异均有统计学意义（F=86.490、165.128、139.424,P＜0.05）.结论 缺氧可以诱导视网膜中5-LOX的高表达;5 LOX选择性抑制可以降低其表达并减少缺氧诱导的新生血管形成.

Objective To investigate the inhibitory effects of 5-lipoxygenase （5-LOX） on oxygeninduced retinal neovascularization in mice and to explore its possible mechanisms.Methods 7-day-old C57BL/6J mice were randomly divided into normal group,oxygen induced retinopathy （OIR） model group,large-dose group,small-dose group and control group with 12 mice in each group.The mice with their mothers were kept in （75±2）％ of oxygen environment for 5 days and then returned to normoxia for 5 days to establish the OIR model except for normal group.From postnatal day 12 to 17,the large-dose group and small-dose group received intravitreous injection of 5-LOX at dose of 100 mg/kg and 50 mg/kg respectively,while the control group received the same volume of 1 ％ dimethyl sulfoxide.The mice in the OIR group received no treatment.The number of endothelium cell nuclei breaking through the inner limiting membrane （ILM） was counted on hematoxylin and eosin-stained retinal section.The mRNA expression of 5 LOX,vascular endothelial growth factor （VEGF） a,VEGF receptor 2 （VEGFR-2） on retinal tissue were detected by reverse transcription polymerase chain reaction （RT-PCR）.The protein expression of 5 LOX,VEGF a,VEGFR-2 and phosphorylation extracellular signal-regulated kinase （P-ERK） 1/2 on retinal tissue were detected by Western blot.Results The number of vascular cell nuclei breaking through the ILM in the large-dose group and small dose group decreased significantly compared with the OIR group and control group （F=73.390,P＜0.05）.The mRNA expression and protein expression of 5-LOX,VEGFa,VEGFR 2 on retinal tissue were decreased significantly in the large-dose group and small dose group as compared with the OIR group and control group （F =92.668,P＜ 0.05）.The difference of VEGFR-2 protein expression between large-dose group and small dose group was not significant （F=2.118,P＞0.05）.The differences of 5-LOX,VEGF a,P-ERK 1/2 protein expression between large-dose group and small-dose group were significant （F=86.490,165.128,139.424; P＜0.05）.Conclusion Hypoxia may induce 5 LOX expression in the retina.Retinal neovascularization was significantly inhibited by selective inhibition of 5 LOX.