白芷通过缺氧诱导因子-1α介导血小板衍生的生长因子受体-β的表达促进糖尿病小鼠足创面愈合的实验研究

Angelica dahurica via hypoxia inducible factor-1αmediates platelet derived growth factor receptors-βsecretion to promote wound healing in type 2 diabetic mice

目的探讨白芷是否通过缺氧诱导因子-1α(HIF-1α)/血小板衍生的生长因子受体-β(PDGFR-β)信号通路促进2型糖尿病小鼠血管生成和加速创面愈合。方法将48只自发2型糖尿病db/db雄性小鼠按照随机数字表法分为糖尿病溃疡组(db/db组)和白芷组(db/db+Angelica dahurica组),每组24只,另选取24只同龄同源db/m雄性小鼠作为正常溃疡对照组(db/m组)。采用皮肤打孔器在各组小鼠背部打孔制备皮肤溃疡模型。白芷组给予白芷颗粒1.8 g/kg灌胃,正常溃疡对照组和糖尿病溃疡组给予等量生理盐水灌胃。每天给药一次,连续给药11 d,分别于创伤后第1、4、7、11天处死小鼠取材分析,每次每组6只小鼠。使用HE、Masson染色观察创面血管形成及胶原纤维形成等组织形态学改变;免疫组织化学染色、免疫荧光双染及Western blotting法从蛋白水平检测白芷干预后db/db小鼠创面组织中血管化及新生血管成熟相关因子[磷酸化磷脂酰肌醇-3激酶/磷脂酰肌醇-3激酶(p-PI3K/PI3K)、磷酸化蛋白激酶B/蛋白激酶B(p-AKT/AKT)、HIF-1α和PDGFR-β]的表达情况。将人脑微血管周细胞(HBVP)分为以下5组:对照组、高糖组、白芷干预组、高渗对照组和白芷+PI3K抑制剂(LY294002)干预组,应用Western blotting法检测各组细胞中HIF-1α、p-PI3K/AKT、PDGFR-β的表达,使用PI3K抑制剂(LY294002)以及使用HIF-1α-小干扰RNA(siRNA)静默HIF-1α或pReceiver-M11-HIF-1α过表达HIF-1α后检测HBVP募集因子PDGFR-β的表达情况。组间比较采用单因素方差分析。结果动物实验显示,糖尿病溃疡组小鼠各时间点创面愈合率均明显低于正常溃疡对照组(P<0.05),创伤后第4、7、11天,白芷组的创面修复、缩短创面愈合时间均优于糖尿病溃疡组(P<0.05);与正常溃疡对照组比较,糖尿病溃疡组创面的HIF-1α和PDGFR-β阳性表达明显减少(P<0.05);与糖尿病溃疡组比较,白芷干预组上述因子的阳性表达明显增加(P<0.05)。细胞实验显示,与低糖组HBVP相比,高糖缺氧条件下HIF-1α蛋白表达及核转位明显减少(P<0.05)。白芷干预组可明显促进HIF-1α的核转位和转录活性,还可增加HIF-1α的蛋白水平(P<0.05);而且,白芷可激活PI3K/AKT信号通路,应用PI3K抑制剂LY294002能够明显抑制细胞HIF-1α、PDGFR-β的蛋白表达(P<0.05);应用HIF-1α-siRNA转染细胞后发现PDGFR-β的蛋白表达水平显著下降,而过表达HIF-1α后,PDGFR-β的表达相应增加(P<0.05)。结论白芷促进糖尿病创面血管新生的作用机制与激活PI3K/AKT、HIF-1α、PDGFR-β信号通路有关。

Objective To determine whether the hypoxia inducible factor-1α(HIF-1α)/platelet-derived growth factor receptor-β(PDGFR-β)signaling pathway is the mechanism by which Angelica dahurica stimulates angiogenesis and promotes angiogenesis and accelerates wound healing in type 2 diabetic mice.Methods A total of 48 spontaneous type 2 diabetic db/db male mice were randomly assigned to two groups using the random number table method:diabetic ulcer group(db/db group)and Angelica dahurica group(db/db+Angelica dahurica),with 24 mice in each group,24 homologous db/m male mice of the same age were selected as the normal ulcer control group(db/m group).The skin ulcer model was prepared using a skin punch on the back of mice.The normal ulcer control group and the diabetes ulcer group received the same amount of normal saline by gavage,while the Angelica dahurica group received 1.8 g/kg of Angelica dahurica granules.The treatment was administered once daily for 11 consecutive days and the mice were euthanized for analysis at 1,4,7,and 11 days post-trauma.There were six mice in each group.HE and Masson staining were used to observe the histomorphologic changes of wound angiogenesis and collagen fiber formation.Western blotting,immunohistochemical staining,and immunofluorescence double staining were used to identify the expression of factors related to angiogenesis and neovascularization,such as phosphorylated phosphatidylinositol 3-kinase/phosphatidylinositol 3-kinase(p-PI3K/PI3K),phosphorylated protein kinase B/protein kinase B(p-AKT/AKT),HIF-1α,and PDGFR-β,in the wound tissues of db/db mice following Angelica dahurica intervention at the protein levels.Five groups were created from human brain microvascular pericytes(HBVP):control group,high glucose group,Angelica dahurica intervention group,high osmotic control group,and angelica dahurica+PI3K inhibitor(LY294002)intervention group.Before detecting the expression of the pericytes recruitment factor PDGFR-β,HIF-1αor pReceiver-M11-HIF-1αoverexpression was silenced using PI3K inhibitor(LY294002)and HIF-1α-small interfering RNA(siRNA).Western blotting was used to detect the expression of HIF-1α,p-PI3K/AKT,and PDGFR-β.One-way analysis of variance was used to compare the groups.Results According to the animal studies,the wound healing rate of the diabetic ulcer group was significantly lower than that of the normal ulcer control group at each time point(P<0.05).On the 4th,7th,and 11th day after wounding,the Angelica dahurica group demonstrated improved wound repair and a shorter wound healing time than the diabetic ulcer group(P<0.05).Compared with normal ulcer control group,the positive expression of HIF-1αand PDGFR-βin the diabetic ulcer group was significantly decreased(P<0.05),while compared to the normal ulcer control group,the positive expression of HIF-1αand PDGFR-βin Angelica dahurica intervention group was significantly increased(P<0.05).The weekly cell experiment revealed that under high glucose and hypoxia conditions,there was a significant reduction in the expression of HIF-1αprotein and nuclear translocation compared to the low glucose group(P<0.05).Angelica dahurica intervention group significantly increased nuclear translocation of HIF-1α,transcriptional activity,and HIF-1αprotein level.Additionally,Angelica dahurica could activate the PI3K/AKT signaling pathway,and the application of PI3K inhibitor LY294002 could significantly inhibit HIF-1αand PDGFR-βprotein expression in cells.Following the transfection of cells with HIF-1α-siRNA,it was found that PDGFR-βprotein expression level significantly decreased,while overexpression of HIF-1αled to a corresponding increase in PDGFR-βexpression(P<0.05).Conclusions Angelica dahurica promotes angiogenesis in diabetic wounds by activating PI3K/AKT,HIF-1α,and PDGFR-βsignaling pathways.