ω-3多不饱和脂肪酸抑制小鼠视网膜新生血管的实验研究

Regulation of diet containing omega-3 polyunsaturated fatty acids on retinal neovascularization in mice

目的:探讨膳食途径摄入ω-3多不饱和脂肪酸(polyunsaturated fatty acids,PUFAs)抑制小鼠视网膜新生血管的作用及其机制。方法:选取60只7日龄C57BL/6J小鼠随机分为3组:A组在正常环境中给予普通膳食饲养;B、C组是将小鼠与哺乳母鼠共同置于氧浓度为75%±2%的氧箱内饲养5d后转移至正常氧环境中饲养5d建立氧诱导视网膜病变模型,其中C组按体质量给予ω-3PUFAs 7.5mg/(kg·d)饲养,B组给予普通膳食饲养。分别于出生后17d时处死小鼠,铺片法计算视网膜新生血管相对面积,HE染色检测突破内界膜的血管内皮细胞核数目,GC-MS检测视网膜组织中ω-3PUFAs/ω-6PUFAs相对含量和比例,实时荧光定量聚合酶链反应(Real-Time PCR)分析视网膜组织中过氧化物酶增殖活化受体-γ(preoxisome proliferatoractivated receptorγ,PPAR-γ)、血管内皮生长因子-A(VEGF-A)和血管内皮生长因子受体2(VEGFR-2)的mRNA表达。结果:出生后17d三组小鼠间新生血管面积及突破视网膜内界膜的血管内皮细胞核数比较差异有统计学意义(F_(新生血管面积)=20.45,P<0.05;F(内皮细胞核数)=48.66,P<0.05),新生血管面积A组与B组之间比较,差异有统计学意义(t=8.64,P<0.05),C组与B组之间比较,差异有统计学意义(t=8.91,P<0.05)。HE染色法观察内皮细胞核数,A组与B组比较差异有统计学意义(t=38.51,P<0.05);C组与B组比较差异有统计学意义(t=19.86,P<0.05)。三组小鼠视网膜ω-3PUFAs和ω-6PUFAs相对含量比较,差异有统计学意义(F=129.86、112.44,均P<0.05),C组含量分别与A、B组比较,差异均有统计学意义(t=23.15、25.42;t=16.43、11.95,均P<0.05);三组小鼠视网膜ω-3PUFAs/ω-6PUFAs比例、视网膜PPAR-γmRNA表达、视网膜VEGF-A mRNA表达、视网膜VEGFR-2 mRNA表达比较,差异有统计学意义(F比例=10.30,FPPAR-γ=138.24,FVEGF-A=69.12,FVEGFR-2=52.45,均P<0.05),C组ω-3PUFAs/ω-6PUFAs比例与B组比较提高,差异均有统计学意义(P<0.05);C组PPAR-γmRNA表达水平明显高于B组,C组VEGF-A mRNA表达水平及VEGFR-2mRNA表达水平低于B组,差异均有统计学意义(P<0.05)。结论:通过膳食途径摄入一定量的ω-3PUFAs,可能通过上调ω-3PUFAs/ω-6PUFAs比例,激活PPAR-γ,进而减少VEGF-A和VEGFR-2表达,抑制视网膜新生血管形成。

AIM:To study the regulation mechanism of diet containing omega-3polyunsaturated fatty acids(co-3PUFAs)on retinal neovascularization in an oxygen-induced retinopathy(OIR)mouse model.METHODS:Sixty C57BL/6J mice,seven-day-old,were classified into3groups:A the normal control group,B the OIR model group,C the ca-3PUFAs diet group.Each group has twenty mice and separated fed by their lactating mice.The normal control group was fed in a standard atmosphere environment,B,C groups were first fed in a hyper-oxygen atmosphere of(75±2)%oxygen percentage for5d,then continue fed in a standard atmosphere.The co-3PUFAs diet group was fed with dose base on their weight by7.5mg/kg/d.All mice were sacrificed when they were seventeen-day-old,the relative neovascularization areas(NA)were calculated by fluorescein angiography on flat-mounted retina.The number of endothelial cell nuclei breaking through the inner linmiting membrane(ILM)was counted on hematoxylin and eosin-stained retinal section.The co-3PUFAs/co-6PUFAs relative amount and ratio was measured by GC-MS in the retina.A real-time PCR and Western Blot method were used to detect the mRNA,peroxisome pro I iferator-avtivated receptor-y(RPAR-y),vascular endothelial growth factor-A(VEGF-A)and vascular endothelial growth factor receptor2(VEGFR-2)in the retina.?RESULTS:There was a significant different in all groupson the relative neovascularization areas and the number of endothelial cell nuclei breaking through the ILM(FNA=20.45,P<0.05;F,lm=48.66,P<0.05).NA between Group A and B had a significant difference(f=8.64,P<0.05),the same between Group C and B(f=8.91,P<0.05).The cell nuclei breaking through ILM in Group A and B was significantly different(f=38.51,P<0.05),the same in Group C and B(f=19.86,P<0.05).For the relative contain in retina of co-3PUFAs and co-6PUFAs,there was a significant different among all groups(F=129.86,F=112.44;ail P<0.05).That of Group C was significant different than other two groups(f=23.15,25.42;f=16.43,11.95;P<0.05).There were significant different among all groups on co-3PUFAs/a>-6PUFAs ratio,retinal RPAR-ymRNA expression,retinal VEGF-A mRNA expression and VEGFR-2mRNA expression(=10.30%FRPAR Y=138.24,=69.12,=52.45;P<0.05).Thea>-3PUFAs/co-6PUFAs ratio of Group C was higher than that of Group B(P<0.05).Compared to Group B,on one hand Group C had a higher expression(P<0.05〉,on other hand Group C had a lower expression on VEGF-A mRNA and VEGFR-2mRNA(P<0.05).?CONCLUSION:The diet rich with o>-3PUFAs uplifts the a>-3PUFAs/a>-6PUFAs ratio and activates RPAR-y to lower expression of VEGF-A and VEGFR-2to inhabit oxygen induced retinal neovascularization.