基于Wnt/β-catenin信号通路探讨环黄芪醇对糖尿病性皮肤溃疡模型大鼠创面愈合的作用

Effect of Cycloastragenol on Wound Healing of Diabetic Skin Ulcers in Rats Based on Wnt/β-catenin Pathway

目的:本研究旨在探讨环黄芪醇对糖尿病性皮肤溃疡模型大鼠创面愈合的影响及其作用机制。方法:受试动物设正常对照组、模型对照组、环黄芪醇20 mg/kg组、环黄芪醇20 mg/kg+IWR-1(Wnt通路抑制剂)8.2μg/kg组、环黄芪醇20 mg/kg+STF-31(GLUT1抑制剂)5.8 mg/kg组。除正常对照组外,其余大鼠经腹腔注射链脲佐菌素建立糖尿病模型,剪去大鼠背部标记区域的皮肤和皮下组织建立皮肤溃疡模型。大鼠按分组灌胃给药14 d,并在给药第5、10、14 d测定大鼠创面愈合率;检测血清中超氧化物歧化酶(SOD)活力、丙二醛(MDA)和还原性谷胱甘肽(GSH)的含量;HE染色检测创面组织的病理变化;免疫组化法检测创面组织中血管内皮生长因子A(VEGFA)和内皮型一氧化氮合酶(ENOS)的表达情况;细胞划痕法检测环黄芪醇对脐静脉内皮细胞(HUVEC)在LPS诱导的炎症状态下迁移率的影响;Western blot法检测给药第10 d的创面组织和LPS诱导后的HUVEC细胞中β-连环蛋白(β-catenin)、糖原合成酶激酶-3β(GSK-3β)、特异性顶部盘状底板反应蛋白-3(RSPO3)、G1/S-特异性周期蛋白-D1(Cyclin D1)、基质金属蛋白酶-2(MMP2)、基质金属蛋白酶-9(MMP9)蛋白的表达情况。结果:与正常对照组比较,模型对照组大鼠在给药第5、10 d时创面愈合率显著降低(P<0.01),创面组织中ENOS的表达和血清SOD活力明显降低(P<0.05或P<0.01),血清MDA含量明显升高(P<0.05或P<0.01);在给药第10 d时,β-catenin、Cyclin D1和MMP2蛋白的表达显著下调(P<0.01);与模型对照组比较,环黄芪醇20 mg/kg组大鼠在给药第10 d时创面愈合率显著升高(P<0.01),在给药第5、10 d时血清中SOD活力和创面组织中ENOS的蛋白表达显著升高(P<0.01),β-catenin、cyclin D1和MMP2蛋白的表达明显上调(P<0.05或P<0.01)。与空白对照组比较,模型对照组细胞的迁移率明显降低(P<0.05或P<0.01),Cyclin D1、MMP2和MMP9蛋白的表达显著下调(P<0.01);与模型对照组比较,环黄芪醇25μg/m L组细胞的迁移率明显升高(P<0.05或P<0.01),Cyclin D1和MMP9蛋白的表达显著上调(P<0.01)。结论:环黄芪醇能促进糖尿病性皮肤溃疡模型大鼠的创面愈合,其作用机制涉及Wnt/β-catenin信号通路的激活。

Objective:To investigate the effect of Cycloastragenol(CAG)on wound healing and its mechanism in a rat model of diabetic skin ulcer.Methods:The rats were divided into control group,model group,CAG 20 mg/kg group,CAG 20 mg/kg+IWR-1(Wnt pathway inhibitor)8.2μg/kg group,and CAG 20 mg/kg+STF-31(GLUT1 inhibitor)5.8 mg/kg group.Except for the control group,all other rats were injected intraperitoneally with streptozotocin to establish diabetes model,and the marked skin and subcutaneous tissue on rat back were removed to establish skin ulcer model.The rats were orally administered with corresponding drugs for 14 days,and wound healing rates were measured on the 5th,10th,and 14th days.Serum contents of superoxide dismutase(SOD),malondialdehyde(MDA),and reduced glutathione(GSH)were assessed.HE staining was used to detect pathological changes in the wound granulation tissue,and immunohistochemistry was used to examine the expressions of vascular endothelial growth factor A(VEGFA)and endothelial-type nitric oxide synthase(ENOS).The effect of CAG on human umbilical vein endothelial cell(HUVEC)migration during LPS-induced inflammation was measured by cell scratch assay.Western blot was used to analyze the protein expressions ofβ-catenin,glycogen synthase kinase-3β(GSK-3β),R-spondin3(RSPO3),G1/Sspecific Cyclin-D1,Matrix Metalloproteinase-2(MMP2),and Matrix Metalloproteinase-9(MMP9)in wound granulation tissues on 10th day of administration and LPS induced HUVEC cells.Results:Compared with control group,the wound healing rate was significantly reduced on the 5th and 10th day of administration in model group(P<0.01),the ENOS expression and serum content of SOD were significantly reduced(P<0.05 or P<0.01),while serum content of MDA was significantly increased(P<0.01).On the 10th day of administration,the protein expressions ofβ-catenin,cyclinD1 and MMP2 were significantly down regulated(P<0.01).Compared with model group,the wound healing rate in CAG 20mg/kg group was significantly increased on the 10th day(P<0.01),the serum content of SOD and ENOS expression in wound granulation tissue were significantly increased on the 5th and 10th day of administration(P<0.01),the protein expressions ofβ-catenin,cyclinD1and MMP2 were significantly up regulated(P<0.05 or P<0.01).Compared with control group,the cell migration rate was significantly reduced in model group(P<0.05 or P<0.01),the protein expressions of cyclinD1,MMP2 and MMP9 were down regulated(P<0.01).Compared with model group,the cell immigration rate in CAG 25μg/mL group was increased(P<0.05 or P<0.01),while the protein expressions of cyclinD1 and MMP9 were up regulated(P<0.01).Conclusion:CAG can promote wound healing in diabetic skin ulcer model rats,and its mechanism may be related to the activation of the Wnt/β-catenin signaling pathway.