中药复脉饮对实验性大鼠缺血视网膜超微结构的影响

背景血清超氧化物岐化酶和一氧化氮水平的高低能够反映药物抗缺血性损害的效用,中药复脉饮能显著改善缺血性眼底病变患者的视功能预后.目的观察复脉饮对实验性大鼠缺血视网膜超微结构及血清超氧化物岐化酶和一氧化氮活性的影响.设计随机对照单一样本观察.单位山东中医药大学附属医院眼科.材料实验于2002-11/2003-07在山东中医药大学附属医院实验室和山东大学超微结构室完成.选取雄性Wistar大鼠60只,随机分为复脉饮治疗组、复明片对照组和生理盐水对照组,20只/组.中药复脉饮主要由枸杞子、黄精、丹参、五味子、黄芪等经水煮浓缩为含生药2g/mL.方法3组大鼠均采用升高眼压的方法制作视神经网膜缺血模型.O.5%戊巴比妥10 mL/kg腹腔注射麻醉加眼球表面麻醉后,将连接生理盐水输液管的7号输液针头直接刺人大鼠双侧前房,输液瓶距大鼠双眼高度为145 cm,此时在眼内形成约14 kPa眼压,可观察到球结膜苍白,眼底血流中断,视网膜缺血,加压1.5 h后,拔出针头,恢复灌注.复脉饮治疗组、复明片对照组均按10 mL/kg剂量给药(药物浓度均为2g/mL),生理盐水对照组以等量生理盐水喂养,分别于造模后连续灌胃3周.超氧化物岐化酶活性采用比色法测定,一氧化氮活性采用硝酸还原酶法测定.主要观察指标①各组血清超氧化物岐化酶及一氧化氮活性的比较.②运用透射电镜观察造模3周后各组大鼠缺血视网膜超微结构的改变.结果实验纳入大鼠60只,喂养过程中,生理盐水对照组因灌胃死亡2只,嘶咬死亡1只,剩余57只大鼠进入结果分析.①各组血清超氧化物岐化酶及一氧化氮活性的比较与复明片对照组比较,复脉饮治疗组超氧化物岐化酶活性无明显变化(t=-1.165,P＞0.05),但一氧化氮活性显著降低[(41.06±13.54),(27.39±7.45)imol/L,t=3.958,P＜0.01];与生理盐水对照组比较,复脉饮治疗组超氧化物岐化酶活性明显升高(t=4.628,P＜0.01),一氧化氮活性显著降低(t=-3.767,P＜0.01).②造模3周后各组大鼠缺血视网膜超微结构的变化复脉饮治疗组视网膜层次清晰,新生膜盘排列整齐,膜结构正常,视网膜神经节细胞数量及其细胞器结构基本正常,神经纤维层无明显空泡.复明片对照组视网膜层次清晰,新生膜盘排列稍显紊乱,膜结构基本正常,视网膜神经节细胞数量及各种细胞器的结构大部分正常,神经纤维层无明显空泡.生理盐水对照组视网膜层次欠清晰,新生膜盘排列紊乱,结构异常,视网膜神经节细胞数量少,细胞器结构不完整,神经纤维层出现明显的大空泡.结论复脉饮组大鼠血清超氧化物岐化酶水平显著升高,一氧化氮水平则明显下降,说明复脉饮对缺血性视网膜损伤具有良好的保护作用,可能与影响血清超氧化物岐化酶和一氧化氮水平、减轻缺血引起的视网膜超微结构损害有一定关系.

Effect of fumai yin on ultrastructure of ischemic retina in experimental rats

BACKGROUND: The levels of superoxide dismutase (SOD) and nitric oxide (NO) in serum reflect the efficacy of drugs on anti-ischemic damage.Fumai yin improves significantly the prognosis of visual function in ischemic hemorrhagic fundus disease.OBJECTIVE: To observe the effects of f umaiyin on ultrastructure of ischemic retina and serum SOD and NO activity in experimental rats.DESIGN: Randomized controlled observation on single sample was designed.SETTING: Department of Ophthalmology, Affiliated Hospital of Shandong University of Traditional Chinese Medicine,MATERIALS: The experiment was performed in Experiment Room of affiliated hospital of Shandong University of Chinese Medicine and Ultrastructure Room of Shandong University from November 2002 to July 2003.60 male Wistar rats were employed and randomized into fumaiyin group,fuming pian control group (fuming pian group) and the control group with physiological saline (PS group), 20 rats in each. Fumai yin is the concentrated decoction 2g/ml, composed of gouqizi (Fructus Lycii), huangjing (Rhizoma Polygonati), danshen (Radix Salviae Miltiorrhizae), wuweizi(Fructus Schisandrae), huangqi (Radix Astragali seu Hedysari), etc.METHODS: The model of retina ischemia was prepared by increasing in traocular pressure in 3 groups of rats. 0.5% pentobarbital 10 mL/kg was injected abdominally to anesthetize ocular surface. No.7 needle of tube with PS transfusion was inserted directly to bilateral anterior chambers of rats, and the infusion bottle was 145 cm above both eyes of rats, by which, 14kPa intraocular pressure was formed. It was observed that bulbar conjunctiva was pale, fundus blood flow was broken off and retina was is chemic. 1.5 hours later, the needle was withdrawn and perfusion was restored. In fumai yin group and fuming pian group, the drug was adminis trated 10 mL/kg (medicine concentration was 2 g/mL) and in PS group, PS of equal amount was applied. Gastric perfusion was done with different materials successively after modeling for 3 weeks. Colorimetry was used for assay of SOD activity and nitrate reductase method was used for assay of NO activity.MAIN OUTCOME MEASURES: ① Comparison of serum SOD and NO activities in each group. ② Alternation of ultrastructure of ischemic retina in 3 weeks of modeling observed with transmission electron microscope (TEM) in each group.RESULTS: Totally 60 rats were employed in the experiment. During breeding, in PS group, 2 rats were died of gastric perfusion and1 rat was died of biting, the rest 57 rats all entered result analysis. ① Comparison of serum SOD and NO activities in each group: Compared with fuming pian group, in fumai yin group, SOD activity was not changed remarkably (t=-1.165, P ＞ 0.05), but, NO activity was reduced significantly [(41.06±13.54), (27.39±7.45)μmol/L,t=3.958,P ＜ 0.01]. Compared with PS group, infumaiyin group, SOD activity was increased remarkably (t=4.628,P ＜ 0.01) and NO activity was reduced significantly (t=-3.767, P ＜ 0.01). ② Alternation of ultrastructure of ischemic retina in 3 weeks of modeling in each group: In fumaiyin group, retina was distinct in layer, newlyborne membrane discs were orderly in arrangement, membrane structure was normal, the numbers of retinal ganglion cells (RGCs) and organelle structure was basically normal and there was no obvious vacuole in neu rofibrous layer. In fuming pian group, retina was distinct in layer, newly borne membrane discs were slightly disturbed in arrangement, the numbers of RGCs and each organelle structure were mostly normal and there was no obvious vacuole in neurofibrous layer. In PS group, retina was not so distinct in layers, newly-borne membrane discs were disturbed in arrangement, the structure was abnormal, the numbers of RGCs was less,organalle structure was not integrative and large vacuole presented remarkably in neurofibrous layer.CONCLUSION: In fumaiyin group, SOD in serum was increased significantly and NO was reduced remarkably, explaining that fumaiyin provides good protection on injury of ischemic retina, which is probably related to that fumaiyin affects SOD and NO levels in serum and alleviates the damage of retinal ultrastructure induced by ischemia.