EPA治疗后对糖尿病性视网膜病变大鼠视网膜组织VEGF、flk-1、bax、bcl-2表达的影响

Effect of EPA therapy on the expression of VEGF,Flk-1,Bax and Bcl-2 in retina of diabetic retinopathy rats

目的观察二十碳五烯酸(eicosapentaenoic acid,EPA)治疗后对糖尿病性视网膜病变(diabetic retinopathy,DR)大鼠视网膜组织血管内皮生长因子(vascular endothelial growth factor,VEGF)、flk-1、bax、bcl-2表达的影响,以阐明EPA治疗DR的机制。方法链脲佐菌素(streptozocin,STZ)60mg/kg腹腔注射制备SD大鼠糖尿病模型共36只,随机分为4组:C组12只成模后1个月VEGF(0.05μg)双眼玻璃体注射;D组12只成模后1个月VEGF(0.05μg)+EPA(0.5μg)双眼玻璃体注射;E组8只成模后1个月VEGF(0.05μg)双眼玻璃体注射,2周后EPA(0.5μg)双眼玻璃体注射;F组4只成模后1个月VEGF(0.05μg)双眼玻璃体注射,4周后EPA(0.5μg)双眼玻璃体注射。采用完全随机设计实验方法和免疫组织化学方法对联合注药法建立的大鼠增殖型糖尿病性视网膜病变(proliferative diabetic rentinopathy,PDR)模型及EPA治疗后视网膜组织中VEGF、flk-1、bax、bcl-2的表达进行观察,应用MIAS 1998型图像分析系统测定其在视网膜节细胞层和内网层的平均光密度值。结果背景期和增殖前期给药组VEGF、flk-1在视网膜内层的表达减弱,而增殖期给药对抑制VEGF表达无效。EPA可通过减弱bax在视网膜的表达抑制DR进展,背景期和增殖前期疗效优于增殖期。EPA可抑制DR进展,使bcl-2在视网膜的代偿性高表达减弱,8周时减弱消失,bcl-2继续发挥保护神经细胞作用。结论 EPA在背景期和增殖前期体内应用可通过封闭flk-1受体、抑制VEGF发挥作用延缓DR进展;还可下调凋亡促进基因bax的表达抑制视网膜神经细胞的凋亡减轻DR进展;早期可抑制机体的保护性bcl-2高表达,但在8周时消失,bax/bcl-2比值降低,对周细胞的缺失有保护作用。

Objective To identify the effect of eicosapentaenoic acid(EPA)on expression of vascular endothelial growth factor(VEGF)、flk-1、bax、bcl-2 in retinas of diabetic rats and to elucidate the mechanism of EPA on diabetic retinopathy(DR)treatment.Methods Thirty-six diabetic models of SD rats were prepared by intraperitoneal injection of streptozotocin(STZ)60 mg/kg.One month later the rats were divided into 4 groups randomly.C:12 rats were received intravitreal injection of VEGF(0.05μg)both eyes.D:12 rats were received intravitreal injection of VEGF(0.05μg)and EPA(0.5μg)both eyes.E:8 rats were received intravitreal injection of VEGF(0.05μg)both eyes,and 2 weeks later received intravitreal injection of EPA(0.5μg)both eyes.F:4 rats were received intravitreal injection of VEGF(0.05μg)both eyes,and 4 weeks later received intravitreal injection of EPA(0.5μg)both eyes.Using complete random design empirical method,we observed the variation of VEGF、flk-1、bax、bcl-2 expression in diabetic rat retina established by symphysial injection and those treated by EPA by immunohistochemistry and measured the average optical density value of them in retinal ganglion cell layer and inner plexiform layer by MIAS 1998 type image analysis system.Results VEGF、flk-1 expression was weakened in inner retina layer after EPA injection during background stage and preproliferation stage.But it is ineffective as given EPA during proliferation stage.EPA can weaken expression of bax in retinas and then inhibit advancement of DR after injection intravitreously.Therapeutic efficacy was better as given during background and preproliferation stage than during proliferation stage.EPA can weaken the higher expression of bcl-2 in retinas by inhibiting advancement of DR,as time went to 8 weeks,the weakening effect disappeared and bcl-2 continued to protect the nerve cells.Conclusion EPA can inhibit advancement of DR not only by suppressing VEGF effect through blocking flk-1 receptor as given during background and preproliferation stages in vivo,but also by inhibiting apoptosis of retinal nerve cells through downregulating apoptosis trigger gene bax expression.At early stage,it can lessen compensatory higher bcl-2 expression.At 8 weeks,this effect disappeared and bax/bcl-2 ratio was decreased,which had the protection to pericyte deficiency.