四妙勇安汤调控神经生长因子/蛋白激酶B/糖原合成酶激酶3β通路对糖尿病足大鼠血管生成和微循环及创面愈合的影响

Regulation effects of Simiao Yong′an decoction on angiogenesis,microcirculation and wound healing in diabetic foot rats based on nerve growth factor/protein kinase B/glycogen synthase kinase 3βpathway

目的探讨四妙勇安汤调控神经生长因子(NGF)/蛋白激酶B(Akt)/糖原合成酶激酶3β(GSK3β)通路对糖尿病足大鼠血管生成和微循环及创面愈合的影响。方法选取40只无特定病原体级SD雄性大鼠,分为正常对照组(NO组)、模型组(MO组)、二甲双胍组(DM组)、四妙勇安汤组(SD组),每组10只。对MO组、DM组、SD组采用腹腔注射链脲佐菌素+足部造矩形切口建立糖尿病足模型,NO组不建立该模型,建模成功后,对DM组灌胃0.2 g/kg二甲双胍,对SD组灌胃50 g/kg四妙勇安汤,NO组、MO组同期灌胃等容积0.9%氯化钠溶液。观察并记录创面愈合情况,苏木精-伊红染色检测皮肤组织病理形态,激光多普勒仪检测微循环相关指标,蛋白质印迹法检测皮肤组织血管生成及NGF/Akt/GSK3β通路相关蛋白表达,体外细胞实验验证四妙勇安汤对NGF/Akt/GSK3β通路的作用。结果MO组给药后1、2、3、4周末创面愈合率、足部血流灌注量(PU)以及皮肤组织血管内皮生长因子(VEGF)、NGF、Akt蛋白表达均低于NO组,GSK3β蛋白表达高于NO组,DM组、SD组给药后1、2、3、4周末创面愈合率、PU以及皮肤组织VEGF、NGF、Akt蛋白表达均高于MO组,GSK3β蛋白表达低于MO组,且SD组比DM组变化更显著(均P<0.05)。NO组皮肤组织细胞结构正常,MO组皮肤组织出现明显病理改变,与MO组比较,DM组、SD组病理形态明显改善。体外实验结果显示,大鼠原代胰岛细胞加入50 g/kg四妙勇安汤后VEGF、NGF、Akt蛋白表达高于,而GSK3β蛋白表达低于加入等容积磷酸盐缓冲液[(2.35±0.23)比(1.48±0.16)、(2.18±0.22)比(1.39±0.15)、(2.21±0.24)比(1.43±0.16)、(1.19±0.13)比(2.22±0.24)](均P<0.001)。结论四妙勇安汤可显著促进糖尿病足大鼠血管生成及创面愈合,并有效改善微循环,其作用机制可能与调控NGF/Akt/GSK3β通路有关。

Objective To investigate the regulation effects of Simiao Yong′an decoction on angiogenesis,microcirculation and wound healing in diabetic foot rats based on nerve growth factor(NGF)/protein kinase B(Akt)/glycogen synthase kinase 3β(GSK3β)pathway.Methods Totally 40 SD male rats without specific pathogen level were selected,and they were divided into normal control group(NO group),model group(MO group),metformin group(DM group),Simiao Yong′an decoction group(SD group),with 10 rats in each group.For MO group,DM group and SD group,the model of diabetic foot was established by intraperitoneal injection of streptozotocin and rectangular incision of foot,and NO group would not establish the model.After successful modeling,0.2 g/kg metformin was given in DM group,50 g/kg Simiao Yong′an decoction was given in SD group,and an equal volume of 0.9%sodium chloride solution was simultaneously gavaged in MO group.Wound healing was observed and recorded.The pathological morphology of skin tissue was detected by hematoxylin eosin staining,the microcirculation related indicators were detected by laser Doppler instrument,the skin tissue angiogenesis and the NGF/Akt/GSK3βpathway related protein expressions were detected by Western blotting,and the effect of Simiao Yong′an decoction on NGF/Akt/GSK3βpathway was verified in vitro experiment.Results At the end of 1,2,3 and 4 weeks after drug administration,wound healing rate,foot blood flow perfusion(PU)value,vascular endothelial growth factor(VEGF),NGF and Akt protein expressions of skin tissue in MO group were lower than those in NO group,GSK3βprotein expression was higher than that in NO group,wound healing rate,PU value,VEGF,NGF and Akt protein expressions in skin tissue in DM group and SD group were higher than those in MO group,GSK3βprotein expression was lower than that in MO group,and the change in SD group was more significant than that in DM group(all P<0.05).The structure of skin histiocyte in NO group was normal,and the skin tissue in MO group showed obvious pathological changes.Compared with MO group,the pathological morphology in group DM and SD was significantly improved.In vitro experiment results showed that VEGF,NGF and Akt protein expressions in primary pancreatic islet cells of rats after added with 50 g/kg Simiao Yong′an decoction were higher and GSK3βprotein expression was lower than those after added with equal volume phosphate buffer[(2.35±0.23)vs(1.48±0.16),(2.18±0.22)vs(1.39±0.15),(2.21±0.24)vs(1.43±0.16),(1.19±0.13)vs(2.22±0.24)](all P<0.001).Conclusion Simiao Yong′an decoction can significantly promote angiogenesis and wound healing in diabetic foot rats,and effectively improve microcirculation.Its mechanism may be related to the regulation of NGF/Akt/GSK3βpathway.