基于Wnt/β-catenin通路探讨拔毒生肌散对糖尿病大鼠皮肤溃疡愈合的促进作用

Promoting Effect of Badu Shengji Powder(拔毒生肌散)on Skin Ulcer Healing in Diabetic Rats Based on Wnt/β-catenin Pathway

目的探讨拔毒生肌散通过调控Wnt/β-连环蛋白(β-catenin)通路对糖尿病大鼠皮肤溃疡愈合的促进作用。方法随机将60只Wistar雄性大鼠分为正常对照组和糖尿病组。采用腹腔注射链脲佐菌素结合喂食高脂高糖饲料方法制备糖尿病模型。28 d后,对比正常对照组和糖尿病组大鼠的体质量和血糖,然后制备背部皮肤溃疡模型。造模成功的糖尿病大鼠分为模型组、拔毒生肌散组、一效膏组、清创胶组。各组用药14 d。在第7、14天,采用Image-pro plus 6.0软件测量各组大鼠创面愈合率,采用苏木素-伊红(HE)染色法检测肉芽组织病理变化,酶联免疫吸附测定(ELISA)法检测肉芽组织晚期糖基化终末产物(advanced glycation end products,AGEs)、晚期糖基化终末产物受体(receptor for advanced glycation end-products,RAGE)、Wnt1、β-catenin、糖原合成酶激-3β(glycogen synthase exciter-3β,GSK-3β)蛋白含量,逆转录-聚合酶链反应(RT-PCR)法检测肉芽组织RAGE、Wnt、β-catenin、GSK-3βmRNA表达水平。结果与正常对照组比较,糖尿病组大鼠的体质量下降(P<0.01),血糖升高(P<0.01)。与正常对照组比较,模型组第7、14天创面愈合率均较低(P<0.01);肉芽组织中炎症细胞较多,成纤维细胞、胶原纤维和毛细血管数量较少;肉芽组织Wnt1、β-catenin mRNA表达水平较低(P<0.01),AGEs蛋白、RAGE、GSK-3β蛋白和mRNA含量较高(P<0.01)。与模型组比较,拔毒生肌散组第7、14天创面愈合率较高(P<0.01);肉芽组织AGEs蛋白、RAGE、GSK-3β蛋白和mRNA含量较低(P<0.05,P<0.01),Wnt1、β-catenin mRNA表达水平较高(P<0.01)。结论拔毒生肌散通过调节Wnt/β-catenin通路促进糖尿病大鼠皮肤溃疡的愈合。AGEs的沉积导致Wnt/β-catenin通路的抑制。

Objective To explore promoting effect of Badu Shengji Powder(拔毒生肌散)on skin ulcer healing in diabetic rats based on the regulation of Wnt/β-catenin pathway.Methods Sixty Wistar male rats were randomly divided into normal control group and diabetic group.The diabetes model was established by intraperitoneal injection of streptozotocin combined with high-fat and high-sugar feed.After 28 d,the body weight and blood glucose of rats in the normal control group and the diabetic group were compared,the back skin ulcer model was established.The successfully modeled diabetic rats were divided into model group,Badu Shengji Powder group,Yixiao Ointment(一效膏)group and debridement glue group.Each group was treated for 14 d.On the 7th and 14th day,the ulcer healing rate of rats in each group was measured by Image Pro Plus 6.0 software,the pathological changes of granulation tissue were detected by hematoxylin-eosin(HE)staining,the advanced glycation end products(AGEs),the contents of receptor for advanced glycation end products(RAGE),Wnt,β-catenin,glycogen synthase exciter-3β(GSK-3β)protein of granulation tissue were detected by enzyme-linked immunosorbent assay(ELISA),and the expression levels of RAGE,Wnt,β-catenin and GSK-3βmRNA were detected by Reverse Transcription-Polymerase Chain Reaction(RT-PCR).Results Compared with those of the normal control group,the weight of the rats in the diabetic group was decreased(P<0.01),and the blood sugar was increased(P<0.01).Compared with that of the normal control group,the ulcer healing rate of the model group on the 7th and 14th day was lower(P<0.01).There were more inflammatory cells and fewer fibroblasts,collagen fibers and capillaries in granulation tissue.The expression levels of Wnt1 andβ-catenin mRNA of granulation tissue were lower(P<0.01).The contents of AGEs protein,RAGE and GSK-3βprotein and mRNA were higher(P<0.01).Compared with those of the model group,the ulcer healing rate of the Badu Shengji Powder group on the 7th and 14th day was higher(P<0.01).The expression levels of AGEs protein,RAGE and GSK-3βprotein and mRNA of granulation tissue were lower(P<0.01)and the expression levels of Wnt1 andβ-catenin mRNA were higher(P<0.01).Conclusion Badu Shengji Powder pro⁃motes the healing of skin ulcers in diabetic rats by regulating the Wnt/β-catenin pathway.The deposition of AGEs lead to the inhibition of the Wnt/β-catenin pathway.