Objective To investigate the mechanisms of quercetin in retinal angiogenesis via in vitro and in vivo studies.Methods Human umbilical vein endothelial cells (HUVECs) was used in in vitro study,and oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (CNV) animal models were used in in vivo study.In the in vitro cell study,normal control group,VEGF-165 treatment group and VEGF-165 combined with quercetin treatment group were used.Cell counting kit-8 (CCK8) and Transwell were used to measure HUVECs cell viability,including proliferation and migration.OIR and CNV animal models were administrated through intraperitoneal injection of quercetin,and the non-perfusion area and CNV aera were evaluated.Western blot assay was used to detect the expression of integrin α5 and integrin β3 expression.In the animal study,18 breastfeeding mice and 18 infant mice were randomly divided into normal control group,model control group and quercetin treatment group,6 for each group.The animal feeding and use was in accordance with the standards set by the ARVO,and the experiment was approved by the Ethic Committee for Experimental Animal of Fu Xing Hospital,Capital Medical University (2016-KY-0036).Results In the in vitro study,VEGF-165 (20 ng/ml) promoted the proliferation and migration of HUVECs,while quercetin (50 μmol/L) inhibited HUVECs proliferation and migration significantly,comparing to the VEGF-165 treatment group (proliferation:Fgroups =18.51,P =0.00;migration:F =85.74,P =0.00).In the in vivo studies,quercetin 20 mg/(kg·day) decreased the non-perfusion area and CNV area comparing to the untreated groups,with significant differences between them (t =6.02,P =0.00;t =5.79,P =0.00).Besides,quercetin down-regulated the expression of integrin α5 and integrin β3 significantly both in the in vitro and in vivo studies.Western blot test showed that integrin α5 and integrin β3 in VEGF-165+quercetin processing 24 hours group were significantly decreased than those in the VEGF-165 group,with significant differences between them (t =4.46,P＜0.05;t =5.18,P＜0.01).Compared with the VEGF-165 +quercetin 24 hours group,the integrin α5 and integrin β3 in VEGF-165 +quercetin processing 48 hours group were increased,but they were still significantly increased than those in the VEGF-165 group,with significant differences between them (t =6.54,P ＜ 0.05;t =7.17,P ＜ 0.01).For the animal studies,quercetin also inhibited the levels of integrin α5 and integrin β3 in OIR and CNV models (t =5.44,13.52;both at P=0.00).Conclusions Quercetin can inhibit the retinal and choroidal neovascularization through integrin pathway,which provides a new treatment strategy for clinical therapy.%目的 探讨槲皮素体外、体内抑制视网膜新生血管及脉络膜新生血管(CNV)生成及发展的作用机制.方法 体外研究采用人脐静脉内皮细胞(HUVECs),体内研究采用氧诱导视网膜病变(OIR)动物模型及激光诱导的CNV动物模型.细胞学研究中分为正常对照组、血管内皮生长因子165(VEGF-165)处理组和VEGF-165+槲皮素处理组.利用细胞计数试剂盒8(CCK8)及Transwell法检测HUVECs的增生及迁移能力,采用Western blot法检测整合素α5及整合素β3蛋白的表达.体内研究中利用OIR动物模型及激光诱导的CNV动物模型评估腹腔内注射槲皮素在抑制视网膜新生血管及CNV中的效果,C57乳母鼠和6～8周龄C57小鼠各18只,均采用随机数字表法随机分为正常对照组、模型对照组和槲皮素处理组,每组6只,采用Western blot法检测各组视网膜中整合素α5和整合素β3蛋白的表达. 结果 在体外研究中,20 ng/mlVEGF-165可以促进HUVECs的增生及迁移,而50 μmol/L槲皮素可以抑制HUVECs的增生及迁移,正常对照组、VEGF-165处理组以及VEGF-165+槲皮素处理组细胞增生值和穿过Boyden小室的细胞数量整体比较,差异均有统计学意义(F分组=18.51,P=0.00;F=85.74,P=0.00).在体内研究中,槲皮素连续腹腔内注射20 mg/(kg·d)可以减少OIR模型视网膜无血管灌注区面积,并减少激光诱导的CNV面积,与模型对照组相比,差异均有统计学意义(t=6.02,P=0.00;t =5.79,P=0.00).Western blot检测显示,VEGF-165+槲皮素处理24 h组整合素α5和整合素β3蛋白表达量均较VEGF-165处理组明显降低,差异均有统计学意义(￡=4.46,P＜0.05;t=5.18,P＜0.01).VEGF-165+槲皮素处理48 h组整合素α5和整合素β3蛋白的表达量较VEGF-165+槲皮素处理24 h组有所升高,但与VEGF-165处理组相比仍降低,差异均有统计学意义(t=6.54,P＜0.05;t=7.17,P＜0.01).在OIR及CNV模型中,槲皮素可以抑制模型对照组中整合素α5及整合素β3的表达,差异均有统计学意义(t=5.44、13.52,均P=0.00).结论 槲皮素可以通过调控整合素抑制视网膜新生血管的形成,为新生血管的治疗提供新的途径.
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