破淤汤干预糖尿病缺血性足溃疡的AMPK/mTOR信号通路机制研究

Study on the mechanism of AMPK/mTOR signaling pathway in the intervention of diabetic ischemic foot ulcer by Poyu Decoction

关键词:  糖尿病  缺血性溃疡  破淤汤  自噬  AMPK/mTOR信号通路

Key words:  diabetes mellitus  ischemic ulcer  Poyu Decoction  autophagy  AMPK/mTOR signaling pathway

摘要:

目的 基于AMPK/mTOR信号通路探讨破淤汤对促进糖尿病缺血性足溃疡创面伤口愈合的作用机制。方法 按照随机数字表法取10只大鼠构建普通创面模型作为空白对照组，其余各组予高糖高脂饲料喂养，8周后腹腔注射链脲佐菌素构建糖尿病溃疡大鼠模型50只，随机分为糖尿病对照组、西药干预组和破淤汤低、中、高剂量组，每组10只。给药4周后，观察记录溃疡创面愈合情况，并计算创面愈合率，分别在治疗后第7天和第14天取材。苏木精-伊红（HE）染色观察溃疡创面病理学结构；CD31免疫组织化学染色观察血管新生；采用免疫组织化学法检测创面肉芽组织中自噬相关蛋白p62、微管相关蛋白轻链3（LC3）表达；采用蛋白免疫印记法（Western blot）检测腺苷酸活化蛋白激酶（AMPK）/哺乳动物雷帕霉素靶蛋白（mTOR）信号通路关键蛋白表达情况。结果 与空白对照组相比，糖尿病对照组的创面愈合率较低，微血管密度（MVD）减小，LC3蛋白表达量降低，P62蛋白表达量升高，磷酸化腺苷酸活化蛋白激酶（p-AMPK）/AMPK蛋白表达量降低、磷酸化乳动物雷帕霉素靶蛋白（p-mTOR）/mTOR表达量升高（P＜0.05）；与糖尿病对照组相比，西药干预组及破淤汤低、中、高剂量干预组的创面愈合率升高，MVD增加，LC3蛋白表达量升高，P62蛋白表达量降低，p-AMPK/AMPK蛋白表达量升高、p-mTOR/mTOR表达量降低（P＜0.05）。结论 破淤汤可能通过激活AMPK/mTOR信号通路关键蛋白的表达促进自噬、减轻炎症反应来加速糖尿病缺血性足溃疡的愈合。

Abstract:

Objective The mechanism of promoting wound healing of diabetic ischemic foot ulcers by Poyu Decoction based on the AMPK/mTOR signaling pathway was explored.Methods The 10 rats were selected according to random number table method to construct a common wound model as a blank control group, and the other groups were fed with high-sugar and high-fat diet. After 8 weeks, 50 rats with diabetic ulcer were constructed by intrabitoneal injection of streptozotocin, and randomly divided into diabetes control group, cilostat group and Poyu Decoction low-dose, medium-dose and high-dose groups with 10 rats in each group. After 4 weeks of administration, the ulcer wound healing was observed and recorded, and the wound healing rate was calculated. Samples were taken on the 7th and 14th day after treatment, respectively. The pathological structure of ulcer wound was observed by HE staining. Angiogenesis was observed by CD31 immunohistochemical staining. The expression of p62 protein and LC3 protein in wound granulation tissue was detected by immunohistochemical method. The expression of key proteins in AMPK/mTOR signaling pathway was detected by Western blot.Results Compared with blank control group, the wound healing rate of diabetic control group was lower, the number of MVD was less, the expression of LC3 protein was decreased, the expression of p62 protein was increased, the expression of p-AMPK/AMPK protein was decreased, and the expression of p-mTOR/mTOR was increased(P < 0.05). Compared with the diabetic control group, the wound healing rate in the cilostazole intervention group and the low-dose, medium-dose and high-dose intervention groups of Poyu Decoction was increased, the MVD was higher; the expression level of LC3 protein was increased; the expression level of p62 protein was decreased; the expression level of P-AMPK/AMPK protein was increased, and the expression level of p-mTOR/mTOR was decreased(P < 0.05).Conclusion The Poyu Decoction may accelerate the healing of diabetic ischemic foot ulcers by promoting autophagy and reducing inflammatory responses through activating the expression of key proteins in the AMPK/mTOR signaling pathway.