丹黄消炎液对糖尿病溃疡大鼠创面TGF-β1、Twist1、Clusterin表达及EMT过程的影响

Danhuang Anti-inflammatory Liquid’s Effect on the Expression of TGF-β1,Twist1,Clusterin and the EMT Process of Diabetic Ulcer Rat’s Wound

目的探究丹黄消炎液对糖尿病性溃疡愈合过程中上皮-间质转化(epithelial-mesenchymal transition,EMT)过程的影响及可能的调控机制。方法复制糖尿病性溃疡大鼠模型。随机分成空白组(普通创面组)、模型组(糖尿病性创面组)、对照组(碘伏组)、实验组(丹黄消炎液组),观察各组创面愈合情况并计算愈合率;采用及蛋白免疫印迹法Western Blot)和实时荧光定量聚合酶链式反应法(RT-qPCR)检测干预第7天、14天时各组创面组织内转化生长因子-β1(TGF-β1)、人Twist相关蛋白1(Twist1)、聚集蛋白(Clusterin)、E-钙黏蛋白(E-cadherin)、纤连蛋白(Fibronectin)的蛋白和mRNA表达情况。结果干预第7天,空白组和实验组的创面愈合率显著高于对模型和组照组(P<0.05);与模型组,空白组和实验组E-cadherin表达显著降低,Fibronectin表达显著升高,TGF-β1、Twist1、Clusterin蛋白和mRNA表达显著升高,以上差异均具有统计学意义(P<0.05);与对照组相比,实验组E-cadherin表达显著降低,Fibronectin、Twist1蛋白表达显著升高(P<0.05)。干预14天,模型组的创面愈合率显著低于其他三组(P<0.05);与模型组比较,空白组和实验组E-cadherin表达显著降低,Fibronectin表达显著升高,差异具有统计学意义(P<0.05);与模型组比较,其他三组Twist1、Clusterin蛋白和mRNA表达显著升高,空白组和实验组TGF-β1蛋白和mRNA表达显著升高,差异具有统计学意义(P<0.05)。结论糖尿病性溃疡的愈合与EMT过程有关,丹黄消炎液能够促进EMT过程,加快愈合速度,其途径可能是通过促进TGF-β1表达,介导Twist1调控Clusterin实现的。

Objective To explore the effect of Dan Huang Xiaoyan liquid on the epithelial-mesenchymal transition(EMT)during the healing process of diabetic ulcers and its possible regulatory mechanism.Methods A rat model of diabetic ulcers was duplicated and randomly divided into blank group(normal wound group),model group(diabetic wound group),control group(iodophor group)and experimental group(Dan Huang anti-inflammatory liquid group).The healing rate of each group was observed and calculated.Western blot and RT-qPCR were used to detect the protein and mRNA expressions of transforming growth factor β1(TGF-β1),human twist related protein 1(Twist1),Clusterin,E-cadherin and Fibronectin of wound tissue on the 7 th and 14 th day of treatment.Results On the 7 th day of treatment,the wound healing rate of blank group and experimental group was significantly higher than that of model group and control group(P<0.05).Compared with the model group,the expression of E-cadherin in the blank group and the experimental group was significantly decreased,the expression of Fibronectin was significantly increased,and the expression of TGF-β1 was significantly decreased.The expressions of Twist1,Clusterin protein and mRNA were significantly increased(P<0.05).Compared with the control group,the expression of E-cadherin in the experimental group was significantly decreased,while the expressions of Fibronectin and Twist1 protein were significantly increased(P<0.05).After 14 days of treatment,the wound healing rate of the model group was significantly lower than that of the other three groups(P<0.05).Compared with the model group,the expression of E-cadherin in the blank group and the experimental group was significantly decreased,while the expression of Fibronectin was significantly increased,with statistical significance(P<0.05).Compared with the model group,the protein and mRNA expressions of Twist1 and clusterin in the other three groups were significantly higher than those in the blank group and the experimental group.The protein and mRNA expressions of TGF-β1 were significantly increased(P<0.05).Conclusion The healing of diabetic ulcers is related to the EMT process.The Danhuang anti-inflammatory liquid can promote the EMT process and accelerate the healing speed,which may be promoted by TGF-β1 expression,which mediates Twist1 regulation of Clusterin.