枸杞提取物及其成分叶黄素/玉米黄质在体内外对年龄相关性黄斑变性防治的影响  

Prevention and Treatment of Age-related Macular Degeneration by Extract of Fructus lycii and Its Constituents Lutein/Zeaxanthin: an in vivo and in vitro Experimental Research

作　　者：黄冰林[1,2] 丁淑华[3] 杭丽[3] 郑仕中[4] 李伟 徐新荣[3] 

机构地区：[1]南京中医药大学研究生院,南京210046 [2]江西中医学院附属医院五官科,南昌330006 [3]南京中医药大学附属江苏省中医院眼科,南京210029 [4]南京中医药大学药学院临床药学教研室,南京210023 [5]南京中医药大学药学院药学系,南京210022

出　　处：《中国中西医结合杂志》2013年第4期531-537,共7页Chinese Journal of Integrated Traditional and Western Medicine

基　　金：江苏省自然科学基金资助项目(No.BK2008494);江苏省中医药领军人才资助项目(No.LJ200911);江苏省中医药局科技项目(No.LZ11041)

摘　　要：目的研究枸杞提取物在体内对高脂饮食及氢醌诱发的年龄相关性黄斑变性(age-related macular degeneration,AMD)模型小鼠的半胱氨酸组织蛋白酶B(cathepsin B,Cat B)、半胱氨酸蛋白酶抑制剂C(cystatin C,Cys C)表达的抑制作用,并探讨叶黄素和玉米黄质在体外对过氧化氢(H2O2)诱导的人视网膜色素上皮(ARPE-19)细胞的基质金属蛋白激酶(MMP)2、金属蛋白激酶组织抑制因子(TIMP)2表达的影响。方法 8个月龄的雌性C57BL/6小鼠50只。10只正常饮食作为年龄对照组;40只高脂饮食6个月后,在饮水中加入氢醌(0.8%)继续3个月,随机分为模型对照组(10只)、高、中、低剂量治疗组(每组10只)分别以3.75、2.50、1.25g/(kg·d)枸杞浸膏灌胃,每日1次,持续3个月。观察期满处死动物,摘取眼球。免疫组化观察Cat B、Cys C的表达,RT-PCR、Western blot检测Cat B、Cys C mRNA及蛋白表达;细胞增殖活性的检测筛选H2O2、叶黄素和玉米黄质的药物浓度,Western blot检测细胞中MMP-2、TIMP-2蛋白表达。结果在体内模型对照组的Cat B、Cys C mRNA及蛋白的表达显著强于年龄对照组(P<0.05,P<0.01),而治疗各组Cat B、Cys C mRNA弱于模型对照组(P<0.01),中、高剂量组Cat B及各剂量组CysC蛋白表达弱于模型对照组(P<0.05,P<0.01)。在体外细胞中叶黄素和玉米黄质能够下调受H2O2诱导的ARPE-19细胞的MMP-2、TIMP-2的蛋白表达(P<0.05,P<0.01)。结论枸杞提取物能下调高脂饮食及氢醌诱发的模型小鼠Cat B、Cys C的高表达,叶黄素和玉米黄质对H2O2诱导的ARPE-19细胞MMP-2、TIMP-2的蛋白表达有下调作用。Objective To investigate the in vivo inhibition of extract of Fructus lycii （FL） on the expressions of cathepsin B （Cat B） and cystatin C （Cys C） in high-fat diet and hydroquinone （HQ） induced model mice with age-related macular degeneration （AMD）, and to explore the in vitro effects of lutein and zeaxanthin on hydrogen peroxide （H2O2） induced expressions of matrix metalloproteinase 2 （MMP-2） and tissue inhibitor of metalloproteinase 2 （TIMP-2） on ARPE-19 cells. Methods Fifty female 8-month-old C57BL/ 6 mice were recruited in this research. Ten mice fed with regular diet was taken as the age control group. The rest 40 mice were fed with high fat diet for 6 months, followed by adding HQ （0.8%） in the drinking water for 3 consecutive months. Then the modeled mice were randomly divided into the model control group （n=10）, the high （at the daily dose of 3.75 g/kg）, middle （at the daily dose of 2.50 g/kg）, and low dose （at the daily dose of 1.25 g/kg） FL groups, 10 in each group. The extract of FL at each dose was respectively administered to mice by gastrogavage for 3 successive months. By the end of the experiment, the mice were killed and their eyeballs were removed. The protein expressions of Cat B and Cys C were observed by immunohistochemical assay. The mRNA and protein expressions of Cat B and Cys C were detected by real-time PCR and Western blot respectively. The drug concentrations of H2O2, lutein, and zeaxanthin were screened and detected using the activity of cell proliferation. The protein expressions of MMP-2 and TIMP-2 were detected using Western blot. Results Compared with the age control group, the mRNA and protein expressions of Cat B and Cys C were significantly higher in the in vivo model control group （P0.05, P0.01）. The mRNA expressions of Cat B and Cys C were weaker in the middle and high dose FL groups than in the model control group （P0.05, P0.01）. In in vitro cells, lutein and zeaxanthin could down-regulate the protein expressions of

关 键 词：半胱氨酸组织蛋白酶B 半胱氨酸蛋白酶抑制剂C 枸杞提取物 基质金属蛋白激酶2 金属蛋白激酶组织抑制因子2 叶黄素 玉米黄质 

分 类 号：R276.7[医药卫生—中医五官科学]