熊果酸对糖尿病视网膜病变小鼠视网膜新生血管形成及氧化应激的抑制作用

Inhibitory effects of ursolic acid on diabetic retinopathy in mice

目的 探讨玻璃体内注射熊果酸（UA）对糖尿病视网膜病变（DR）小鼠视网膜损伤的保护作用及其机制.方法 使用四氧嘧啶建立糖尿病小鼠模型.12周龄清洁级健康C57BL/6雄性小鼠60只,随机分为6组：正常对照组、糖尿病模型组、阳性对照组（曲安奈德）和UA干预（低、中、高剂量）组,每组10只小鼠.药物干预1周后处死小鼠,取眼球制作石蜡切片,采用组织病理学方法检测各组小鼠视网膜新生血管情况;实时PCR检测视网膜血管内皮生长因子（VEGF）、环氧化酶2（COX-2）、基质金属蛋白酶2（MMP-2） mRNA的表达水平;Western印迹法检测视网膜VEGF、COX-2、MMP-2蛋白的表达水平;试剂盒检测超氧化物歧化酶（SOD）活力、丙二醛含量.结果 HE染色和过碘酸雪夫（PAS）染色结果显示,正常对照组内界膜平整,血管内皮细胞排列整齐,几乎不见新生血管;糖尿病模型组可见大量新生血管;低剂量UA干预组与糖尿病模型组结果相似;高剂量UA干预组与阳性对照组结果相似,内界膜平整,基本不见新生血管.VEGF、COX-2、MMP-2 mRNA表达水平糖尿病模型组显著高于正常对照组和中、高剂量UA干预组（均P＜0.001）;VEGF、COX-2、MMP-2蛋白表达水平糖尿病模型组显著高于正常对照组（P＜0.001）和低、中、高剂量UA干预组（均P＜0.05）.糖尿病模型组视网膜组织丙二醛含量显著高于正常对照组（P =0.001）和中、高剂量UA干预组（P=0.026、0.007）,SOD活性显著低于正常对照组（P ＜0.001）和低、中、高剂量UA干预组（P=0.040、0.021、0.003）.结论 熊果酸可能通过抑制DR小鼠视网膜VEGF、COX-2、MMP-2的表达抑制视网膜新生血管形成,并通过明显的抗氧化应激作用缓解DR.

Objective To explore the protective effect and its mechanisms of intravitreal injection of ursolic acid （UA） on retinal injury in diabetic retinopathy （DR） mice.Methods DR model was set up by using alloxan.60 C57BL/6 male mice （clean animal,aged 12 weeks） were randomly and evenly divided into 6 groups：blank control group,DR model group,positive control group （triamcinolone acetonide）,UA intervention groups （low,moderate and high dose）.Mice were killed one week after drug intervention and the eyeballs were removed to prepare paraffin section.Retinal new blood vessel was examined by histopathological methods.The mRNA and protein expressions of vascular endothelial growth factor （VEGF）,cyclooxygenase 2 （COX-2）,and Matrix Metalloproteinase 2 （MMP-2） in retinal tissues were detected by real-time PCR and Western blotting,respectively.The superoxide dismutase （SOD） activity and malondialdehyde （MDA） content were detected by related kits.Results The HE and PAS staining showed uniform inner limiting membrane structure and orderly vascular endothelial ceils in blank control group,and neovascularization was seldom detected.In DR model group,numerous new blood vessels were formed beyond inner limiting membrane.UA intervention group with low dose had almost the same results with DR group,while the results of UA intervention group with high dose were similar with positive control group,in which the inner limiting membrane structure was uniform and new blood vessels were seldom seen.The mRNA expressions of VEGF,COX-2,and MMP-2 in DR model group were significantly higher than those of blank control group （P 〈0.001） and UA intervention group with moderate and high dose （P 〈0.001）.The protein expressions of VEGF,COX-2 and MMP-2 in DR model group were significantly higher than those of blank control group （P 〈 0.001） and UA intervention group with low,moderate and high dose （all P 〈 0.05）.The MDA content of retinal tissue in DR model group was significantly higher than those of blank control group （P =0.001） and UA intervention groups with moderate and high dose （P =0.026,0.007）.The activity of SOD was significantly lower in DR model group than those of blank control group （P 〈 0.001） and UA intervention groups with low,moderate and high dose （P =0.040,0.021,0.003）.Conclusion UA may inhibit the formation of new blood vessels through reducing the expressions of VEGF,COX-2 and MMP-2 in retinal tissues,and promote a remission from DR by obvious resistance to oxidative stress.