白芷提取物促糖尿病溃疡新生血管成熟作用及机制研究

Effect and mechanism of Angelica dahurica extract on promoting neovascularization maturation of diabetic ulcer

目的研究白芷提取物对自发2型糖尿病db/db(C57BLKS/J)小鼠皮肤溃疡新生血管成熟的影响与机制。方法选取8周龄雄性db/db小鼠随机分为模型组及白芷提取物组,同窝雄性db/m小鼠作为对照组。背部打孔法制备糖尿病溃疡小鼠模型。白芷提取物组以白芷提取物1.8 g/kg ig给药,对照组和模型组ig等量生理盐水,连续给药21 d。记录创伤后2、4、7、11、14、21 d小鼠创面大致情况;对创伤后11 d各组小鼠创面进行HE、免疫组化、免疫荧光染色观察创面组织形态、新生血管数量与成熟情况以及创面叉头转录因子(FOXO1)、血管生成素-2(Ang-2)在创面组织中的定位表达;体外根据不同干预条件,将内皮细胞分为正常糖浓度缺氧组、高糖缺氧组、高渗缺氧组、高糖缺氧+白芷提取物组。并将不同干预条件下内皮细胞与周细胞共培养,观察白芷提取物干预高糖缺氧下内皮细胞对周细胞趋化作用及体外管状结构形成的影响。Western bloting法检测各组创面组织及内皮细胞中FOXO1、p-FOXO1、Ang-2蛋白水平及其上游蛋白激酶B(Akt)蛋白及磷酸化蛋白水平。结果创伤后4、7、11、14、21 d,白芷提取物组创面愈合速率、创面恢复大致情况、创面组织中血管形成数量及新生血管成熟情况明显优于模型组(P<0.05);白芷提取物组中FOXO1和Ang-2蛋白表达水平明显高于模型组(P<0.05),白芷提取物组中p-Akt、p-FOXO1蛋白表达均低于模型组(P<0.05);体外实验中白芷提取物干预高糖缺氧下内皮细胞后,细胞中FOXO1和Ang-2蛋白表达水平明显降低(P<0.05),p-Akt,p-FOXO1蛋白表达明显增加(P<0.05);此外,白芷提取物能提高高糖缺氧下内皮细胞对周细胞的趋化作用,促进高糖缺氧下内皮细胞与周细胞体外管状结构形成。结论白芷提取物能促进新生血管形成与成熟,从而提高db/db小鼠创面愈合速率和质量,其作用机制可能与下调FOXO1/Ang-2通路,实现内皮细胞对周细胞募集及管状结构形成有关。

Objective To study the effect and mechanism of Angelica dahurica extract(AD)on neovascularization maturation in db/db mice.Methods Forty-eight 8-week-old male db/db mice were randomly divided into model group and A.dahurica extract-treated group,and 24 littermate male db/m mice were set as control group.A mouse model of diabetic ulcer was prepared by punching the back.The A.dahuricae extract-treated group was administered with AD 1.8 g/kg ig,and control group and model group were ig administered with the same amount of normal saline for 21 d.The healing condition of mouse wounds was recorded at 2,4,7,11,14 and 21 d after trauma.The number and maturation of new blood vessels in wound tissues was observed by HE,immunohistochemistry and immunofluorescence staining at 11 d after trauma.Expression of fork head transcription factor(FOXO1)and angiopoietin-2(Ang-2)in wound tissues were detected by Western blotting and immunohistochemistry.According to different intervention conditions in vitro,endothelial cells were divided into normal glucose and hypoxia group,high glucose and hypoxia group,hypertonic and hypoxic group,high glucose and hypoxia+A.dahurica extract-treated group.Endothelial cells were eo-cultured with pericytes under different intervention conditions,and the effects of A.dahurica intervention on endothelial cells,chemotaxis and tubular structure formation in vitro under high glucose and hypoxia were observed.Western blotting was used to detect the levels of FOXO1,p-FOXO1 and Ang-2 proteins and their upstream protein kinase B(Akt)and phosphorylation protein changes in wound tissues and endothelial cells.Results The wound healing rate of the A.dahurica extract-treated group,the general recovery of the wound,the number of blood vessels and the maturation of new blood vessels in the wound tissue were significantly better than the model group(P<0.05).FOXO1 and Ang-2 protein expression levels in the A.dahurica extract-treated group were significantly higher than the model group(P<0.05);p-Akt and p-FOXOl protein expression in the A.dahurica extract-treated group were lower than model group(P<0.05).After A.dahurica intervention in endothelial cells under high glucose and hypoxia in vitro,the expression levels of FOXO1 and Ang-2 protein in the cells were significantly reduced(P<0.05),p-Akt and p-FOXO1 protein expression were significantly increased(P<0.05);In addition,A.dahurica extract could increase the chemotaxis of endothelial cells to pericytes under high glucose and hypoxia and promote the formation of tubular structures of endothelial cells and pericytes in vitro under high glucose and hypoxia.Conclusion A.dahurica extract can promote the formation and maturation of neovascularization,so as to improve the rate and quality of wound healing in db/db mice.The mechanism may be related to the down-regulation of FOXO1/Ang-2 pathway,the recruitment of endothelial cells to pericytes,and the promotion of the maturation of wound new blood vessels.