柚皮素β-环糊精包合物对大鼠实验性脉络膜新生血管的抑制作用

摘要

Background Choroidal neovascularization (CNV) leads to blindness in many fundus diseases.Study showed that naringenin suppresses CNV,but it presents with poor bioavailability because of its poor solubility in water.β-cyclodextrin (β-CD) can increase the water-solubility of drugs, however, whether the inhibitory effect of naringenin on CNV can be improved after clathrated with β-CD remains unclear.Objective This study was to compare the inhibitory effects of naringenin with naringenin/β-CD compounds on CNV in rats.Methods Naringenin/β-CD clathrate compounds were prepared with saturated solution,the solubility of naringenin in water was calculated based on standard curve.Thirty-two male Brown Norway rats were randomized into normal control group, model control group, naringenin group and naringenin/β-CD group.Laser-induced CNV models were created in the right eyes of rats from the model control group, naringenin group and naringenin/β-CD group.Naringenin and naringenin/β-CD clathrate compounds were intraperitoneally injected at a dose of 20 mg/kg in the rats of naringenin group and naringenin/β-CD group since the day after modeling, respectively, once per day for 4 weeks, and equal volume of DMSO was injected in the same way in the model control group.Fluorescein isothiocyanate dextran (FITC-D) was injected via rat hypoglossal vein for the preparation of flatmounts of choroid in the fourth week,and the areas of CNV were measured and compared among the groups.The retinal pigment epithelium (RPE)-choroid-sclera tissues were isolated from the rats, and the relative expression levels of vascular endothelial growth factor (VEGF) , cyclooxygenase-2 (COX-2),phosphatidylinositol-3-kinase (PI3K),p38mitogen-activated protein kinase (p38MAPK), matrix metalloproteinase (MMP)-2, MMP-9 mRNA and their proteins in RPE-choroid-sclera tissue were detected using real-time PCR and Western blot.Results The solubility of naringenin in water increased by 11.8 folds after encapsulated with β-CD.The CNV areas in the model control group, naringenin group and naringenin/β-CD group were (34.56± 1.67), (20.90± 1.47) and (13.20± 1.38) × 103 μm2 , respectively, showing significant reduces in the naringenin group and naringenin/β-CD group compared with the model control group (t =3.973 ,P＜O.05;t =5.532, P＜0.01) ,and the CNV area in the naringenin/β-CD group was significantly smaller than that in the naringenin group (t =3.605,P＜0.05).The relative expression levels of VEGF, COX-2, PI3K, p38MAPK, MMP-2, MMP-9 mRNA and their proteins were significantly declined in the normal control group,naringenin group and naringenin/β-CD group in comparison with the model control group (all at P＜0.05).In addition, the expression levels of VEGF mRNA and COX-2 mRNA and their proteins were significantly lower in the naringenin/β-CD group than those in the naringenin group (all at P＜0.05).Conclusions The naringenin/β-CD clathrate compounds can improve the water solubility of naringenin and enhance their inhibitory effect on rats CNV.The inhibitory effect of naringenin on rats CNV probably is associated with anti-inflammatory pathway.%背景 脉络膜新生血管(CNV)是导致多种眼底疾病致盲的主要原因,目前仍无有效的治疗方法.研究表明柚皮素能够抑制CNV的发生和发展,但由于其水溶性差而影响其生物利用度.β-环糊精(β-CD)可提高药物的水溶解度,但其与柚皮素包合后是否能改善柚皮素抑制CNV的作用尚不清楚. 目的 制备柚皮素β-CD包合物,比较其与柚皮素对大鼠CNV的抑制作用.方法 采用饱和溶液法制备柚皮素β-CD包合物,依据标准曲线法测定包合物中柚皮素的水溶解度.采用随机数字表法将32只SPF级健康雄性Brown-Norway大鼠分为正常对照组、模型对照组、柚皮素组和柚皮素β-CD组,每组8只.采用氪激光法在模型对照组、柚皮素组及柚皮素β-CD组大鼠右眼制作CNV模型.于视网膜光凝当天起柚皮素组和柚皮素β-CD组大鼠腹腔内分别注射20 mg/kg柚皮素和相同质量浓度柚皮素的β-CD包合物,每日1次,连续4周,模型对照组大鼠腹腔注射等容积的DMSO.给药后第4周经大鼠食下静脉注射荧光素异硫氰酸酯葡聚糖(FITC-D),制备脉络膜铺片,测量大鼠CNV面积;从各组大鼠实验眼分离出视网膜色素上皮(RPE)-脉络膜-巩膜复合体组织标本,分别采用实时荧光定量PCR及Western blot法检测标本组织中血管内皮生长因子(VEGF)、环氧化酶-2(COX-2)、磷脂酰肌醇(-3)激酶(PI3K)、磷酸化丝裂原活化蛋白激酶(p38MAPK)、基质金属蛋白酶(MMP)-2、MMP-9 mRNA及其蛋白的相对表达量. 结果 柚皮素经β-CD包合后在水中的溶解度较原药提高了11.8倍.模型对照组、柚皮素组和柚皮素β-CD组CNV面积分别为(34.56±1.67)、(20.90±1.47)和(13.20±1.38) ×103 μm2,柚皮素组、柚皮素β-CD组大鼠CNV面积明显小于模型对照组,差异均有统计学意义(t=3.973,P＜0.05;=5.532,P＜0.01),柚皮素β-CD组大鼠CNV面积小于柚皮素组,差异有统计学意义(t=3.605,P＜0.05).正常对照组、柚皮素组和柚皮素β-CD组标本织中VEGF、COX-2、PI3K、p38MAPK、MMP-2、MMP-9 mRNA及其蛋白的相对表达量明显低于模型对照组,差异均有统计学意义(均P＜0.05);柚皮素β-CD组标本组织中VEGF和COX-2 mRNA及其蛋白的相对表达量明显低于柚皮素组,差异均有统计学意义(均P＜0.05).结论 柚皮素经β-CD包合后水溶性显著增加,其对大鼠CNV的抑制作用较原药显著增强,柚皮素对CNV的抑制可能是通过其对炎症信号通路的抑制作用来实现的.