玻璃体腔注射熊果酸对小鼠氧诱导视网膜新生血管的抑制作用

Inhibitory effect of ursolic acid on high oxygen-induced mouse retinal neovascularization by intravitreal injection

背景 视网膜新生血管疾病严重影响视功能,目前虽然有较多的抗新生血管药物,但多针对单一的干预靶点,疗效有限.研究证实,熊果酸具有多种生物学效应,其中包括抗血管新生作用,但其对眼科血管疾病的疗效尚不清楚. 目的 观察熊果酸对小鼠氧诱导视网膜新生血管形成的抑制作用. 方法 采用随机数字表法将7日龄清洁级C57BL/6J小鼠60只随机分为6个组,即空白对照组、PBS对照组、阳性对照组和低、中、高剂量熊果酸组.空白对照组小鼠在正常环境中喂养,其他各组小鼠与哺乳的母鼠置于体积分数（75±2）％的高氧环境中连续饲养5d,小鼠12日龄时将模型小鼠及其哺乳母鼠返回正常空气环境中,以诱导视网膜新生血管产生.造模成功后按照分组不同分别于小鼠玻璃体腔注射无菌PBS 3μl、曲安奈德注射液（1 ml∶40 mg）3μl或1.5、3.0、6.0μg熊果酸各3μl.小鼠17日龄时过量麻醉法处死,摘除双侧眼球制备视网膜组织切片.采用组织病理学方法检查各组小鼠视网膜血管新生情况,计数突破视网膜内界膜的新生血管内皮细胞核数目;采用逆转录PCR（RT-PCR）法分别检测和比较各组小鼠视网膜组织中血管内皮生长因子（VEGF） mRNA、环氧合酶-2（COX-2）mRNA及基质金属蛋白酶-2（MMP-2）mRNA的相对表达量. 结果 视网膜切片后苏木精-伊红染色显示,PBS对照组小鼠视网膜中突破内界膜的新生血管内皮细胞核数为（18.65±3.24）个/视野,明显高于空白对照组的（0.78±0.11）个/视野,差异有统计学意义（t=2.24,P＜0.05）;中剂量熊果酸组、高剂量熊果酸组小鼠视网膜突破内界膜的新生血管内皮细胞核数分别为（13.32±1.87）个/视野和（8.93±1.09）个/视野,明显少于PBS对照组和低剂量熊果酸组的（18.65±3.24）个/视野和（15.44±2.02）个/视野,差异均有统计学意义（均P＜0.05）,而高剂量熊果酸组小鼠视网膜突破内界膜的新生血管内皮细胞核数与阳性对照组的（9.14±1.13）个/视野接近,差异无统计学意义（t=1.17,P＞0.05）.RT-PCR检测表明,PBS对照组小鼠视网膜组织中COX-2 mRNA、VEGF mRNA和MMP-2 mRNA相对表达量均明显高于空白对照组,差异均有统计学意义（t=13.45、12.49、14.32,均P＜0.05）,且高剂量熊果酸组小鼠视网膜组织中COX-2mRNA、VEGF mRNA和MMP-2 mRNA相对表达量均明显低于PBS对照组和低剂量熊果酸组,差异均有统计学意义（均P＜0.05）,而高剂量熊果酸组与阳性对照组间的差异均无统计学意义（均P＞0.05）.结论 熊果酸以剂量依赖的方式下调氧诱导的缺血小鼠视网膜组织中VEGF、COX-2及MMP-2的表达,从而抑制视网膜新生血管的产生.

Background Retinal neovascular diseases affect visual function.Although many drugs have been used to manage the visual diseases,their effectiveness is less than satisfactory.Studies showed that ursolic acid has multiple biological effects including anti-vascularization.However,the effect of ursolic acid on retinal neovascular diseases is unclear now.Objective This study was to observe the inhibitory effect of ursolic acid on the high oxygen-induced mouse retinal neovascularization after intravitreal injection.Methods Sixty clean 7-day-old C57BL/6J mice were divided into the blank control group,PBS control group,positive control group （triamcinolone） and low,moderate and high dose （1.5,3.0 and 6.0 μg） ursolic acid groups randomly.The blank control group mice were raised in normal environment,and the mice from other groups were fed in the environment with O2 concentration at （75±2）% for 5 days together with the maternal mice.The mice then were back to the normal air environment to induce retinal neovascularization.Then,the drugs were intravitreally immediately injected in the mice of the different groups.The mice were sacrificed at the 17-day old for the preparation of retinal sections.Retinal new blood vessel was examined by haematoxylin and eosin stain under the light microscope,and the number of vascular endothelial cell nucleus breaking the inner limiting membrane was counted.The gene expressions of vascular endothelial growth factor （VEGF）,cyclooxygenase-2 （COX-2） and matrix metalloproteinase-2 （MMP-2） in the mouse retinas were quantitatively assayed using reverse transcription PCR.Results The number of endothelial nuclei newly-generated vessel breaking internal limiting membrane in the mice of PBS control group was （18.65±3.24）/field,which was more than （0.78±0.11）/field of the blank control group obviously （t =2.24,P〈0.05）.The number of endothelial nuclei newly-generated breaking internal limiting membrane in the moderate-or high-dose ursolic acid group was less than that of moderate group obviously,it was statistically significant（P〈0.05）.The number of vascular endothelial cell nuclei breaking internal limiting membrane in high high-dose group was （13.32 ± 1.87）/field and （8.93 ± 1.09） /field,showing significant decreases in comparison with the PBS control group and low-dose ursolic acid group （18.65±3.24）/field （15.44±2.02）/field （all at P〈0.05）.However,no significant difference were seen in the number of new vascular endothelial cell nucleus between the high-dose ursolic acid group and the positive control group（9.14±1.13）/field （t=1.17,P〉0.05）.The relative expressions of COX-2 mRNA,VEGF mRNA and MMP-2 mRNA in the mouse retinas were higher in the PBS control group than those in the blank control group （t =13.45,12.49,14.32,all at P〈0.05）,and those in the moderate-dose or high-dose ursolic acid group were lowed in comparison with the PBS control group and the low-dose ursolic acid group （all at P〈0.05）,but there were no significant differences between the high-dose ursolic acid group and the positive control group （all at P〉0.05）.Conclusions Ursolic acid can suppress retinal neovascularization by down-regulating the expressions of VEGF,COX-2 and MMP-2 in oxygen-induced retinopathy of mouse in dose-dependent manner.