【作者】 黄冰林；

【导师】 丁淑华；

【作者基本信息】 南京中医药大学 ， 中医五官科学， 2013， 博士

【摘要】 目的：体内实验,研究枸杞醇提物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)模型小鼠视网膜色素上皮(retinal pigment epithelium, RPE)下沉积物和Bruch膜超微结构的影响,以及对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) mRNA的转录和蛋白表达的影响；体外实验,探讨枸杞子的主要成分叶黄素和玉米黄质对过氧化氢(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(matrix metalloproteinases, MMP)2和金属蛋白激酶组织抑制因子(tissue inhibitors of metalloproteinases, TIMP)2蛋白表达的影响。方法：体内实验,选取8个月龄的雌性C57BL/6小鼠100只。按体重随机分5组,其中空白组、模型对照组、高剂量组、中剂量组、低剂量组,每组20只。空白组20只：正常饮食；余4组高脂饮食喂养6个月后,于饮水中加入氢醌(0.8%)继续饲养3个月；治疗组再予枸杞醇提物以灌胃法给药(高剂量组3.75g/kg/d、中剂量组2.50g/kg/d、低剂量组1.25g/kg/d),每日一次,继续持续3个月。观察期满处死动物,摘取眼球。电镜观察RPE下沉积物及Bruch膜；免疫组化观察Cat B、Cys C的表达;Real time-PCR检测Cat B、Cys C mRNA的表达；Western blot检测Cat B、Cys C蛋白的表达。体外实验,选用ARPE-19细胞常规培养后,细胞增殖活性的方法检测筛选H202及叶黄素和玉米黄质的最佳药物浓度,Western blot方法检测细胞中MMP-2, TIMP-2蛋白酶体表达水平。结果：体内实验,RPE下沉积物形成：模型对照组RPE下沉积物的形成与空白组相比明显增多(P<0.01),治疗组中：高剂量组与模型对照组相比有极明显减轻(P<0.01),中剂量组、低剂量组则有明显减轻(P<0.05)。Bruch膜厚度：模型对照组的Bruch膜厚度明显比空白组增厚(P<0.01),治疗各组的厚度则明显小于模型对照组(P<0.01)。Cat B、Cys C mRNA的表达：两者在模型对照组的表达显著高于空白组(P<0.01),治疗各组Cat B、Cys C mRNA的表达与模型对照组相比明显降低(P<0.01)。Cat B、Cys C的蛋白表达：Cat B蛋白在模型对照组的表达明显高于空白组(P<0.05),治疗组与模型对照组相比：高、中剂量组有显著降低(P<0.05),低剂量组无明显差异(P>0.05)；Cys C的蛋白表达在空白组与模型对照组相比有显著降低(P<0.05),治疗各组与模型对照组比均显著降低(P<0.01)。体内实验,浓度为1、10、100μmol/L的H202溶液与空白组(不加H202溶液的空白对照组)相比无明显统计学差异(P>0.05),浓度为200μmol/L的H202溶液存在统计学差异(P<0.05),浓度为300、400、500μmol/L的H202溶液则存在极显著性差异(P<0.01)。叶黄素及玉米黄质对ARPE-19细胞活性的影响：当药物浓度在1、10μmol/L时,与模型对照组相比无明显差异(P>0.05),浓度为30、50、70μmol/L时存在显著性差异(P<0.05),浓度为100μmol/L时存在极显著性差异(P<0.01)。MMP-2的蛋白表达：模型对照组MMP-2的蛋白较空白组有明显的高表达(P<0.05),药物浓度为30、50μmol/L时,MMP-2的蛋白表达与模型对照组比有显著性差异(P<0.05),当浓度到达70μmol/L时有极显著性差异(P<0.01)。TIMP-2的蛋白表达：模型对照组TIMP-2的蛋白较空白组有明显的高表达(P<0.05)。治疗组中当药物浓度为30μmol/L时TIMP-2的蛋白表达与模型对照组比并无统计学差异(P>0.05),浓度为50μmol/L时有统计学差异(P<0.05),浓度为70μmol/L时差异有极显著性(P<0.01)。结论：枸杞醇提物能下调Cat B、Cys C mRNA和蛋白的高表达。抑制高脂饮食和氢醌引起的模型小鼠RPE下沉积物形成及Bruch膜的增厚,机制可能与下调Cat B、Cys C的表达有关。枸杞子的主要成分叶黄素和玉米黄质对H202诱导的ARPE-19中的MMP-2、 TIMP-2蛋白酶体的高表达均有下调作用。更多还原

【Abstract】 Objective The aim of this study was to investigate the inhabiting effect of lyceum barbarum extracts on sub-RPE deposit formation in high-fat diet and oral hydroquinone (HQ) model mice in vivo, and the effect of an extract of Fructus lycii (gou qi zi) on Cathepsin B (Cat B)、Cystatin C (Cys C) expression in model mice in vivo, And to investigate the effect of Lutein and Zeaxanthin to hydrogen peroxide on MMP-2, TIMP-2proteasome expression induced by oxidative stress in ARPE-19cells in vitro.Methods In vivo:100eight-month-old C57BL/6female mice, Then mice were divided randomly into aging control model aging control, control (20mice) and3treatment groups (60mice,20mice each group).20were fed regular diet as aging control,80were fed a high-fat diet for6months followed by HQ (0.8%) in the drinking water for3months. Ethanol extract of Fructus lycii were given once-daily through oral administration in different dose (high:3.75g/kg/d, middle:2.50g/kg/d, low:1.25g/kg/d) for3months. At the end of the experimental period, the mice were killed and the eyes immediately removed. Transmission electron microscopy was used to observe sub-RPE deposit formation and Bruch membrane (BrM) thickness, semiquanttative grading of deposit severity was performed, The expression and location of Cat B and Cys C were examed by immunohistochemistry, The mRNA expression Cat B and Cys C were detected by real-time PCR and and the protein expression Cat B and Cys C were detected by Western blot, respectively. In vitro:Methylthiazolyltetrazolium was used to detect the effects of different concentrations of Hydrogen peroxide and Lutein and Zeaxanthin for ARPE-19cells, then the expression and distribution of MMP-2, TIMP-2proteasome in cells were detected by Western blot.Results In vivo:sub-RPE deposit formation:Compared to the aging control group, the sub-RPE deposit in the model control group mice significantly higher (P＜0.01), but Fructus lycii ethanol extract decreased the score in a dose-dependent fashion and at high and middle dose also led to a significant effect, at low dose led to a significant effect compared to the model mice (P＜0.05). The Bruch membrane of the model control was significantly thicker than that of aging control mice (P<0.01), Fructus lycii ethanol extract led to significantly reduced the thickness of Bruch membrane in a dose-dependent manner compared to the model mice (all P<0.01). Compared to the aging control, the mRNA expression of Cat B and Cys C were significantly higher in the model control mice (P<0.01), the mRNA expression of Cat B and Cys C were down-regulated by the extract of Fructus lycii (P<0.01). Compared to the aging control, the protein expression of Cat B and Cys C were significantly higher in the model control mice (P<0.05), At middle and high dose led to a significant effect compared to the model mice (P＜0.05), But at low dosen’t led to a significant effect compared to the model mice (P＞0.05). In vitro:cell viability assay showed that H2O2reduced ARPE-19cell proliferation dose-dependently and at1、10、100μM have no ignificant effect compared to the control cells (P>0.05), and produced a significant effect at200μM compared to the control cells (P<0.05), and also produced a significant effect at300、400、500μM compared to the control cells (P<0.01). Lutein/zeaxanthin at30、50、70μM produced a significant effect compared to the cells treated with H2O2alone (P<0.05). Compared to the control, the protein expression of MMP-2and TIMP-2were significantly higher in the model control (P<0.05).The protein expression of MMP-2also produced a significant effect at30、50、70μM compared to the model control(P<0.05at30、50, P＜0.01at70μM). The protein expression of TIMP-2at30μM led to no significant effect compared to the model mice (P>0.05), and have significant effect at50、70μM(P<0.05at50μM, P<0.01at70μM).Conclusion Fructus lycii ethanol extract could reduce RPE sediment and restore Bruch membrane in vivo leading to the improvement in AMD histopathology, and could down-regulated the expression of Cat B and Cys C in high-fat diet and oral HQ model mice. Lutein and Zeaxanthin could down-regulated the expression of MMP-2, TIMP-2proteasome in ARPE-19cells which treated by hydrogen peroxide first.更多还原