熊果酸对氧诱导新生小鼠视网膜新生血管形态的影响

Effect of ursolic acid on retinal vascular form in oxygen-induced mouse retinopathy

目的探讨熊果酸（uA）对氧诱导新生小鼠视网膜病变模型新生血管形态学的影响。方法将7日龄清洁级C57BL／6J小鼠60只采用随机数字表法随机分为6组：空白对照组、模型对照组（PBS）、阳性对照组（曲安奈德）和uA干预组（低、中、高剂量），每组10只小鼠（均取右眼，各10只眼）。空白对照组小鼠在空气中喂养，其他各组均置于体积分数（750±20）mL／L氧气（O2）的高体积分数氧环境中饲养，连续5d。至小鼠出生12d时将模型对照组小鼠及其哺乳母鼠返回正常空气环境（210mL／LO2）中，以诱导小鼠视网膜新生血管产生。模型成功后，立即给予各组相应的药物治疗：模型对照组小鼠玻璃体腔注射无菌PBS3止，uA干预组（低、中、高剂量）小鼠分别玻璃体腔注射1．5、3．0、6．0斗guA各3I,zL，阳性对照组小鼠玻璃体腔曲安奈德注射液（1mL：40mg）3止。17日龄时过量麻醉处死小鼠，摘除眼球制备标本。制作视网膜组织病理切片，HE染色，计数突破视网膜内界膜的新生血管内皮细胞核数目；并采用视网膜铺片技术观察各组小鼠视网膜新生血管形态改变。结果组织病理切片观察结果：空白对照组视网膜内界膜结构均一，血管内皮细胞排列整齐，偶见血管内皮细胞突破内界膜。模型对照组可见大量血管内皮细胞团突破内界膜，形成新生血管腔。不l司剂量uA干预组比较，高剂量uA干预组与阳性对照组接近，可见少量血管内皮细胞突破内界膜，内界膜结构较均一，未形成或者形成新生血管腔。突破小鼠视网膜内界膜的新生血管内皮细胞核数比较：模型对照组明显高于空白对照组（P〈0．05）；高剂量UA干预组明显低于模型对照组（P〈0．05）；高剂量uA干预组明显低于低剂量uA干预组（P〈0．05）。视网膜铺片显示：空白对照组视网膜血管自视盘向四周呈放射状均匀分布，管径较粗，分支良好，周边视网膜血管结构清晰。模型对照组视网膜大量新生血管，管径纤细，走行僵直，结构紊乱，分布不均匀，中央可见大片无灌注区。不同剂量uA干预组比较：高剂量uA干预组视网膜血管分布和走形与阳性对照组接近，较模型对照组新生血管分布明显好转，且明显较低剂量干预组的视网膜新生血管分布好，无明显的无灌注区。结论uA抑制氧诱导小鼠视网膜新生血管的形成，且抑制效果呈剂量依赖性。

Objective To investigate the effect of ursolic acid （UA） on retinal vascular form in oxygen-in- duced mouse retinopathy. Methods The 60 clean 7-day C57BL/6J mice were divided into 6 groups randomly：blank control group, model control group（ PBS）, positive control group（ triamcinolone）, UA intervention group（ low dose, meta dose, high dose） ,with 10 mice in each group（ right eye as experimental subject, 10 eyes each group）. The blank control group mice were raised in air, with other groups of mice in （750 ＋ 20） mL/L 02 high-oxygen environment for 5 conse- cutive days. The model control group mice and breastfeeding mice were put back in air enviroment（210 mL/L 02 ） on the 12 day after birth to induce the generation of retinal neovascularization. The drug treatment was applied to the cor- responding groups immediately when models were successful：3 p^L sterile PBS was injected in＇ model control group, 3 p.L 1.5,3.0 and 6.0 p,g UA in UA intervention group,3 p.L triamcinolone（ 1 mL ： 40 mg） in positive control group. All mice were killed after overdose of anesthesia on the 17th day, and their eyeballs were made into retinal tissue sec- tions, HE dying, counting the neovaseulanzation endothelial nuclei number which broke the retinal internal limiting membrane. The morphologic changes of retinal vessels were estimated by observing the vascular pattern with the technol- ogy of stretched preparation of retina. Results From the observation of tissue pathology slice in blank control group, the structure of internal limiting membrane was even, and the vascular endothelial cells lined up evenly, while some vascular endothelial cells broke the internal limiting membrane occasionally. In model control group, a lot of vascular endothelial cells broke the internal limiting membrane and new vessel lumen was formed. The result of low UA intervention group was basically similar to that of model control group. The result of high-dose UA intervention group was close to that of positive control group, though some vascular endothelial cells broke the internal limiting membrane, and internal limiting membrane structure was even, and new vessel lumen was not formed. The endothelial nuclei number of newly-generated internal limiting membrane vessel in model control group was obviously higher than that of blank control group, while the high-dose UA intervention group was obviously lower than that of model control group, and the high-dose UA inter- vention group was lower than that of the low-dose UA intervention group obviously, and all the differences were statisti-cally significant（ P 〈 0.05）. Retinal flatmounts showed that in the blank control group retinal vascular appeared with u- niformly radiated distribution from optic disc in all directions, with bigger pipe diameter, proper branch and clear pe- ripheral retinal vascular structure. However,lots of retinal neovascularization was seen in model control group,with slen- der retinal diameter, line rigidity, structure disorder, and uneven distribution, and there was large non-peffused areas in the centeral area. Through comparison with different doses UA intervention group, retinal vascular distribution and pat- tern in the high-dose UA intervention group were close to the positive group. The distribution of retinal neovasculariza- tion was much better than the model control group and the low-dose UA intervention group, with no obvious non-per- fused areas. Conclusions UA can inhibit the formation of neovascularization in oxygen-induced mice retinal ischemia model. It has a positively correlated relationship with UA dose.