徐赵钕　章仕淼　刘玲玲

[摘要] 目的 探讨明目地黄丸对糖尿病视网膜病变（DR）大鼠视网膜组织中细胞自噬及对Akt-mTOR信号通路的影响。 方法 采用高脂高糖饲料饲养联合腹腔注射链脲佐菌素（40 mg/kg）法制备DR大鼠模型，采用随机数字表法分为模型组、羟苯磺酸钙组（150 mg/kg）和明目地黄丸低（150 mg/kg）、中（300 mg/kg）、高（600 mg/kg）剂量组，每组10只，另设正常对照组10只，连续用药10周，监测各组大鼠体重及血糖。给药10周后，透射电子显微镜观察视网膜组织中细胞自噬小体数量；蛋白印迹法检测视网膜组织中自噬相关蛋白Beclin1、微管相关蛋白1轻链3-Ⅱ（LC3-Ⅱ）、Akt、p-Akt、mTOR、p-mTOR蛋白水平。 结果 与正常对照组比较，模型组大鼠视网膜组织现空泡样变化、细胞排列稀疏紊乱等病理变化，自噬小体数目明显减少（P < 0.05），Beclin、LC3-Ⅱ蛋白表达明显下调（P < 0.05），p-Akt、p-mTOR蛋白表达显著上调（P < 0.05）；与模型组比较，明目地黄丸低、中、高剂量组大鼠视网膜组织病理变化均显著改善（P < 0.05），自噬小体数目显著增多（P < 0.05），Beclin、LC3-Ⅱ蛋白表达显著上调（P < 0.05），p-Akt、p-mTOR蛋白表达明显下调（P < 0.05），呈剂量依赖效应。 结论 明目地黄丸能抑制DR大鼠视网膜组织中细胞自噬过程，其機制可能通过Akt-mTOR信号通路调控自噬相关蛋白表达而实现。

[关键词] 明目地黄丸；糖尿病视网膜病变；细胞自噬；Akt-mTOR信号通路

[中图分类号] R587.2 [文献标识码] A [文章编号] 1673-7210（2018）07（b）-0016-06

Effects of Mingmu Dihuang Pills on autophagy and Akt-mTOR signaling pathway in retinal tissue of diabetic retinopathy rats

XU Zhaonü ZHANG Shimiao LIU Lingling

Department of Endocrinology， Wenzhou Central Hospital， Zhejiang Province， Wenzhou 325000， China

[Abstract] Objective To investigate the effects of Mingmu Dihuang Pills on autophagy and Akt-mTOR signaling pathway in retinal tissue of diabetic retinopathy （DR） rats. Methods The DR rat model was prepared by high fat and high sugar feed combined with intraperitoneal injection of Streptozotocin （40 mg/kg） method， and they were divided into model group， Calcium Dobesilate group （150 mg/kg）， low （150 mg/kg）， medium （300 mg/kg）， high （600 mg/kg） dose of Mingmu Dihuang Pills groups by random number table method， with 10 rats in each group， and 10 rats were selected as normal control group， all the rats were given the medicine continuously for 10 weeks， the weight and blood sugar of the rats in each group were monitored. After 10 weeks of administration， the number of autophagic corpuscles in the retinal tissue was observed by transmission electron microscope； Western blot was used to detect autophagy related protein Beclin1， microtubule associated protein 1 light chain 3-Ⅱ （LC3-Ⅱ）， Akt， p-Akt， mTOR and p-mTOR protein in retinal tissue. Results Compared with the normal control group， the retina tissue of rats in the model group showed the changes of vacuoles， the cells arranged sparsely and disorder and so on， the number of autophagic corpuscles decreased significantly （P < 0.05）， the expression of Beclin and LC3-Ⅱ protein was obviously down-regulated （P < 0.05）， the expression of p-Akt and p-mTOR protein was significantly up-regulated （P < 0.05）； compared with the model group， the pathological changes of rats retina tissue in the low， medium and high dose of Mingmu Dihuang Pills groups were significantly improved （P < 0.05）， the number of autophagic corpuscles increased significantly （P < 0.05）， the expression of Beclin and LC3-Ⅱ protein was significantly up-regulated （P < 0.05）， the expression of p-Akt and p-mTOR protein was obviously down-regulated （P < 0.05）， in dose-dependent manners. Conclusion Mingmu Dihuang Pills can inhibit the process of autophagy in retinal tissue of diabetic retinopathy rats， and its mechanism may be achieved by the regulating the expression of autophagy related proteins through the Akt-mTOR signaling pathway.

[Key words] Mingmu Dihuang Pills； Diabetic retinopathy； Autophagy of cells； Akt-mTOR signal pathway

糖尿病视网膜病变（diabetic retinopathy，DR）是由于视网膜长期处于高糖环境，导致血-视网膜屏障被破坏，视网膜发生损伤的一种疾病，是糖尿病常见并发症之一[1]。随着糖尿病患者病程的增加和年龄的增长，DR的发生率逐渐增加。研究发现，自噬功能紊乱在糖尿病及其并发症的发生过程中具有重要作用，高血糖环境可抑制视网膜组织中细胞自噬作用，导致活性氧、晚期糖化终末产物等代谢产物累积，造成视网膜损伤[2]。中医认为，DR是由血瘀络阻、气阴双虚引起。明目地黄丸为我国特色中药制剂，以山茱萸、熟地黄、茯苓、山药、白芍等多味中药组成，具有养肝、滋肾、明目等药理活性[3-4]。……