补肾益气活血方对干性年龄相关性黄斑变性模型小鼠视网膜氧化损伤的保护作用
Protective Effect of Bushen Yiqi Huoxue Fang on Retinal Oxidative Damage in Model Mice of Dry Age-Related Macular Degeneration
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摘要: 研究补肾益气活血方对干性年龄相关性黄斑变性(AMD)模型小鼠视网膜氧化损伤的保护作用及机制。方法 6月龄的雌性C57BL/6小鼠60只，其中10只作为年龄对照组，另50只高脂肪饮食喂养6月后，饮水中加入氢醌（0.8%）继续饲养3月构建干性AMD小鼠模型，并随机分为溶媒对照组、阳性药物(乐盯)组、补肾益气活血方组（低、中、高剂量组），灌胃给药，每日1次，持续3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；眼静脉取血分离血清，摘取眼球分离视网膜并匀浆，检测血清和视网膜SOD、GSH-Px、CAT酶活性及ROS、MDA含量；qPCR、Western blot检测Nrf2、HO-1、NQO-1、GCL mRNA转录及蛋白表达。结果 与年龄组对照组比，溶媒对照组小鼠RPE下沉积物显著增加，Bruch膜明显增厚；与溶媒对照组比，补肾益气活血方组小鼠RPE下沉积物减少，对Bruch膜增厚有抑制作用；补肾益气活血方可提高血清及视网膜组织SOD、GSH-Px、CAT酶活性，降低血清及视网膜组织ROS、MDA含量；补肾益气活血方中、高剂量可显著升高Nrf2、HO-1、NQO-1、GCL mRNA转录水平，上调胞核Nrf2及HO-1、NQO-1、GCL蛋白表达水平。结论 补肾益气活血方对干性AMD模型小鼠视网膜氧化损伤有保护作用，机制可能是激活Nrf2通路，提高SOD、GSH-Px、CAT酶活性，上调下游靶基因酶HO-1、NQO-1、GCL表达水平，增强对模型动物体内活性氧的清除作用。Abstract: OBJECTIVE The study was aimed at evaluating protective effect of Bushen Yiqi Huoxue Fang on retinal oxidative damage in model mice of dry age-related macular degeneration (AMD) and to elucidate the underlying mechanism. METHODS 60 six-month-old C57BL/6 female mice， 10 were fed normal diet as aging control， 50 were fed a high-fat diet for 6 months followed by HQ (0.8%) in the drinking water for 3 months to established dry AMD model mice . Model mice were divided randomly into vehical control， positive drug(lutein) and 3 treatment groups (dosage:low，middle，high) .Drugs were given once-daily through gastrogavage for 3 months. At the end of the experimental period， the mice were killed and the eyes immediately removed. Transmission electron microscopy were used to evaluate sub-RPE deposit formation and Bruch membrane (BrM ) thickness; The serum were separated from the blood sampling of mice eye vein， mice retina were detached and homogenated， the serum and retina SOD， GSH-PX， CAT enzyme activity and ROS， MDA content were detected; The mRNA and protein expression of Nrf2， HO-1， NQO-1， GCL were detected by qPCR and Western blot， respectively. RESULTS Compared with aging control mice， the sub-RPE deposits was significantly increased， and the Bruch membrane was significantly thickened in vehical control mice; Bushen Yiqi Huoxue Fang can significantly inhibit sub-RPE deposit formation and reduce thickening of BrM in model mice compared to the vehical control mice， the sub-RPE deposits was decreased， BrM thickness was inhibited in treatment mice. Bushen Yiqi Huoxue Fang could increase SOD， GSH-PX and CAT enzyme activity， reduce ROS， MDA content of retina and serum in mice. mRNA and protein expressions of Nrf2， HO-1，NQO-1， GCL of HO-1， NQO-1 were up-regulated in mice treated with Bushen Yiqi Huoxue Fang(middle， high dosage) . CONCLUSION Bushen Yiqi Huoxue Fang could protect the retina against oxidative damage in model mice of dry AMD， the underlying mechanism may be to increase SOD， GSH-PX and CAT enzyme activity and up-regulate expressions of Nrf2， HO-1，NQO-1， GCL through activation of Nrf2 pathway， which may strengthen the clearance of reactive oxygen species in model mice.
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