枸杞提取物对小鼠视网膜色素上皮细胞下沉积物形成的抑制作用
Inhibiting Effect of Lycium Barbarum Extracts on Sub-RPE Deposit Formation in High-fat Diet and Oral Hydroquinone Model Mice
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摘要: 目的 研究枸杞提取物对高脂饮食及氢醌诱发的小鼠视网膜色素上皮（RPE）细胞下沉积物形成的抑制作用。方法 8个月龄的雌性C57BL/6小鼠40只。8只予正常饮食，作为年龄对照组；32只高脂肪饮食喂养6个月后，饮水中加入氢醌（0.8%）继续饲养3个月，随机分为模型对照组（8只）、治疗组（分高剂量、中剂量、低剂量组，每组8只），治疗组以枸杞浸膏灌胃，高剂量3.75 g/（kg·d）、中剂量2.50 g/（kg·d）、低剂量1.25 g/（kg·d），每日1次，持续3个月。观察期满处死动物，摘取眼球。电镜观察RPE细胞下沉积物及Bruch膜；real time-PCR、western blot检测半胱氨酸组织蛋白酶B（Cat B）、半胱氨酸蛋白酶抑制剂C（Cys C）mRNA转录及蛋白表达。结果 造模后RPE细胞损害、RPE细胞下沉积物形成均较年龄对照组明显增加，模型对照组Bruch膜较年龄对照组明显增厚，枸杞提取物能减轻RPE细胞损害、减少沉积物的形成、抑制Bruch膜的增厚。Cat B、Cys C mRNA及蛋白表达模型对照组较年龄对照组增强，枸杞提取物能下调模型小鼠视网膜Cat B、Cys C的表达，随剂量的增加而作用加强，具剂量依赖关系，以Cys C表达下调更显著。结论 枸杞提取物能减轻高脂饮食和氢醌引起的模型小鼠RPE细胞损害，抑制RPE细胞下沉积物形成及Bruch膜增厚，机制可能是减少活性氧介质的产生，下调Cat B、Cys C的表达，并使这2种酶的综合作用向Cat B作用倾斜有关。Abstract: OBJECTIVE To investigate the inhabiting effect of lycium barbarum extracts on sub-RPE deposit formation in high-fat diet and oral hydroquinone (HQ) model mice. METHODS 40 eight-month-old C57BL/6 female mice， 8 were fed normal diet as aging control group; 32 were fed a high-fat diet for 6 months followed by HQ (0.8%) in the drinking water for 3 months. Model mice were randomly divided into model control group (8 mice) and 3 treatment groups (8 mice each group). Lycium barbarum extracts were given through different dose lavage one time every day(high:3.75g/kg/d， middle:2.50g/kg/d， low:1.25g/kg/d) for 3 months. At the end of the experimental period， the mice were killed and the eyes were immediately removed. Transmission electron microscopy was used to observe sub-RPE deposit formation and Bruch membrane (BrM) thickness， semiquantitative grading of deposit severity was performed; The mRNA and protein expression of cathepsin B（Cat B）and cystatin C（Cys C）were detected by real-time PCR and western blot respectively. RESULTS Mice fed a high-fat diet and HQ showed RPE damage was severe， formation of sub-RPE deposits was increasing， in addition， BrM was typically thickened，the severity in model control group was statistically greater than in the aging control group. Lycium barbarum extracts could reduce RPE damage， inhibit sub-RPE deposit formation and thickening of BrM. Compared with the aging control group， the mRNA and protein expression of Cat B and Cys C were significantly higher in the model control group. Expression of Cat B and Cys C were down regulated by the lycium barbarum extracts， and the effect was enhanced as the dosage was increased in a dose dependent manner， there was an especially significant reduction of Cys C expression.CONCLUSION Lycium barbarum extracts could reduce RPE damage， and inhibit sub-RPE deposit formation and thickening of BrM in high-fat diet and oral HQ mice model. The mechanism may be to reduce generation of ROS， down regulate the expression of Cat B and Cys C， and comprehensive actions of Cat B and Cys C shift to the function of Cat B.
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