兰州大学机构库 >第二临床医学院
黄芪甲苷调控TGF-β1/Smad2/3 通路改善高糖损伤的皮肤修复细胞的功能
Alternative TitleAstragaloside IV attenuates high glucose-induced skin wound repair cells injury via TGF-β1/Smad2/3 signaling pathway
高琼
Subtype博士
Thesis Advisor张选奋
2023-08-31
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name医学博士
Degree Discipline外科学
Keyword黄芪甲苷 astragaloside IV 皮肤损伤修复 wound healing TGF-β/Smads TGF-β/Smads
Abstract

背景:

慢性创面是糖尿病最常见的并发症之一,治疗难度大、复发率高、截肢风险大。黄芪是中医学用于托毒排脓、敛疮生肌的药材,有促进创面愈合之功效[1],而黄芪甲苷(astragaloside Ⅳ,AS-Ⅳ)是黄芪药理活性的有效成分之一[2],多项研究证实AS-Ⅳ具有降血糖、免疫调节、抗炎、抗氧化、抗衰老、抗纤维化、抗肿瘤等作用[3-6]。然而,AS-Ⅳ对高糖损伤的皮肤修复细胞的作用及其机制尚未完全阐明。

目的:

初步探究AS-Ⅳ对高糖损伤角质形成细胞(HaCaT)、血管内皮细胞(HUVEC)和成纤维细胞(HSF)增殖活力、细胞凋亡和迁移运动等生物学行为的影响及其机制,为糖尿病慢性创面的防治提供新思路。

方法:

以HaCaT、HUVEC和HSF为研究对象,实验分为四个部分。第一部分,设置AS-IV处理组(HG+AS-Ⅳ组)、高糖损伤组(HG+DMSO组)、正常对照组(NG+DMSO组),采用CCK-8、Annexin V-FITC/PI双染法结合流式细胞技术和体外划痕分别观察并记录增殖活力、细胞凋亡和迁移运动的变化;第二部分,设置HG+AS-Ⅳ组、HG+DMSO组、NG+DMSO组,采用ROS荧光探针结合流式细胞技术和ELISA法检测氧化应激标志物(ROS、SOD、MDA)和炎症相关细胞因子(IL-6、IL-8、IL-10)的变化;第三部分,设置HG+AS-Ⅳ组、HG+DMSO组、NG+DMSO组,采用Western blot和qRT-PCR实验技术检测TGF-β1/Smad2/3通路关键分子(TGF-β1、TβRⅠ、TβRⅡ、Smad2、p-Smad2、Smad3、p-Smad3、Smad7)的蛋白及mRNA的表达;第四部分,根据第三部分的结果,设置TGF-β1/Smad2/3通路抑制剂组(HG+AS-Ⅳ+SB431542组)或激活剂组(HG+AS-Ⅳ+SRI011381组)、AS-IV处理组(HG+AS-Ⅳ组)和高糖损伤组(HG+DMSO组),采用CCK-8、Annexin V-FITC/PI双染法结合流式细胞技术、体外划痕、ROS荧光探针结合流式细胞技术、ELISA法分别检测细胞生物学行为(增殖、凋亡、迁移)、氧化应激(ROS、MDA、SOD)、炎症反应(IL-6、IL-8、IL-10)的变化,以探究TGF-β1/Smad2/3通路在AS-Ⅳ保护高糖损伤的HaCaT、HUVEC和HSF中的作用。

结果:

1.AS-Ⅳ影响高糖损伤HaCaT、HUVEC和HSF的生物学行为

HG+DMSO组的细胞增殖活力、划痕愈合率均低于NG+DMSO组(P<0.01),而细胞凋亡率高于NG+DMSO组(P<0.01);HG+AS-Ⅳ组的细胞增殖活力、划痕愈合率均高于HG+DMSO组(P<0.01),而细胞凋亡率低于HG+DMSO组(P<0.01)。

2.AS-Ⅳ影响高糖损伤HaCaT、HUVEC和HSF的氧化应激和炎症反应

HG+DMSO组SOD浓度低于正常对照组(P<0.01),而ROS荧光阳性细胞占比、MDA浓度均高于NG+DMSO组(P<0.01);HG+DMSO组IL-10浓度低于NG+DMSO组(P<0.01),而IL-6和IL-8浓度均高于NG+DMSO组(P<0.01);HG+AS-Ⅳ组SOD浓度高于HG+DMSO组(P<0.05),而ROS荧光阳性的细胞占比、MDA浓度均低于HG+DMSO组(P<0.05);AS-Ⅳ处理组IL-10浓度高于HG+DMSO组(P<0.01),而IL-6和IL-8浓度均低于HG+DMSO组(P<0.01)。

3.AS-Ⅳ影响高糖损伤HaCaT、HUVEC和HSF的TGF-β1/Smad2/3通路

(1)HaCaT部分:HG+DMSO组TGF-β1蛋白和mRNA的表达、p-Smad2/Smad2和p-Smad3/Smad3的比值均低于NG+DMSO组(P<0.05),而Smad7蛋白和mRNA的表达高于NG+DMSO组(P<0.05);HG+AS-Ⅳ组TGF-β1蛋白和mRNA的表达、p-Smad2/Smad2和p-Smad3/Smad3的比值均高于HG+DMSO组(P<0.05),而Smad7蛋白和mRNA的表达低于HG+DMSO组(P<0.01)。

(2)HUVEC部分:HG+DMSO组TGF-β1蛋白和mRNA的表达、p-Smad2/Smad2和p-Smad3/Smad3的比值均高于NG+DMSO组(P<0.01),而Smad7蛋白和mRNA的表达低于NG+DMSO组(P<0.05);HG+AS-Ⅳ组TGF-β1蛋白和mRNA的表达、p-Smad2/Smad2和p-Smad3/Smad3的比值均低于HG+DMSO组(P<0.05),而Smad7蛋白和mRNA的表达高于HG+DMSO组(P<0.05)。

(3)HSF部分:HG+DMSO组TGF-β1、TβR1、TβR2蛋白和mRNA的表达、p-Smad2/Smad2和p-Smad3/Smad3的比值均低于NG+DMSO组(P<0.05);HG+AS-Ⅳ组TGF-β1、TβR1、TβR2蛋白和mRNA的表达、p-Smad2/Smad2和p-Smad3/Smad3的比值均高于HG+DMSO组(P<0.05)。

4.TGF-β1/Smad2/3通路介导AS-Ⅳ对高糖损伤HaCaT、HUVEC和HSF的保护作用

(1)HaCaT部分:HG+AS-Ⅳ+SB组的细胞增殖活力、划痕24 h和48 h的愈合率均低于HG+AS-Ⅳ组(P<0.01),而细胞凋亡率高于HG+AS-Ⅳ组(P<0.01);HG+AS-Ⅳ+SB组SOD浓度低于HG+AS-Ⅳ组(P<0.05),而ROS荧光阳性的细胞占比和MDA浓度均高于HG+AS-Ⅳ组(P<0.05);HG+AS-Ⅳ+SB组IL-10浓度低于HG+AS-Ⅳ组(P<0.05),而IL-6和IL-8浓度均高于HG+AS-Ⅳ组(P<0.05)。

(2)HUVEC部分:HG+AS-Ⅳ+SRI组的细胞增殖活力、划痕24 h和48 h的愈合率均低于HG+AS-Ⅳ组(P<0.05),而细胞凋亡率高于HG+AS-Ⅳ组(P<0.01);HG+AS-Ⅳ+SRI组SOD浓度低于HG+AS-Ⅳ组(P<0.01),而ROS荧光阳性的细胞占比和MDA浓度均高于HG+AS-Ⅳ组(P<0.05);HG+AS-Ⅳ+SRI组IL-10浓度低于HG+AS-Ⅳ组(P<0.01),而IL-6和IL-8浓度均高于HG+AS-Ⅳ组(P<0.01)。

(3)HSF部分:HG+AS-Ⅳ+SB组的细胞增殖活力、划痕12 h和24 h的愈合率均低于HG+AS-Ⅳ组(P<0.01),而细胞凋亡率高于HG+AS-Ⅳ组(P<0.05);HG+AS-Ⅳ+SB组SOD浓度低于HG+AS-Ⅳ组(P<0.01),而ROS荧光阳性的细胞占比和MDA浓度均高于HG+AS-Ⅳ组(P<0.05);HG+AS-Ⅳ+SB组IL-10浓度低于HG+AS-Ⅳ组(P<0.01),而IL-6和IL-8浓度均高于HG+AS-Ⅳ组(P<0.01)。

结论:

AS-Ⅳ通过调控高糖损伤HaCaT、HUVEC和HSF中TGF-β1/Smad2/3通路,发挥抗氧化、抗炎、抗凋亡的作用、保护HUVEC和HSF的增殖、迁移,可能有助于改善糖尿病慢性创面的再上皮化和肉芽组织形成、促进创面愈合。

 

关键词:黄芪甲苷,皮肤损伤修复,TGF-β/Smads

Other Abstract

Background:

As one of the most common complications of diabetes, diabetic wound is characterized by low cure rate, high recurrence rate, and high amputation risk. Astragaloside IV (AS-IV) is the primary active constituent of Astragalus membranaceus (Huangqi)[2], which is widely used for tissue regeneration[1]. Studies have demonstrated the role of AS-IV in lowering blood glucose, immunomodulatory, anti-inflammatory, anti-oxidant, anti-aging, anti-fibrotic, anti-tumor, and so on[3-6]. Nevertheless, the effects and underlying mechanisms of AS-IV in high glucose-induced skin wound repair cells have not been fully elucidated.

Objective:

To investigate the effects and underlying mechanisms of AS-Ⅳ on the biological behavior of high glucose-induced keratinocytes (HaCaT), vascular endothelial cells (HUVEC), and fibroblasts (HSF), and provides a new idea for the prevention and control of chronic diabetic wound.

Methods:

This experiment was divided into four parts. In the first part, AS-Ⅳ group (HG+AS-Ⅳ group), high glucose group (HG+DMSO group), and normal control group (NG+DMSO group) were set up. The cell viability, apoptosis and migration were detected by CCK-8, flow cytometry, and scratch assay. In the second part, the level of ROS, MDA, SOD, IL-6, IL-8, and IL-10 were detected by fluorescent probes and ELISA. In the third part, the expression of TGF-β1, TβRⅠ, TβRⅡ, Smad2, p-Smad2, Smad3, p-Smad3, and Smad7 were detected by WB and qRT-PCR. In the fourth part, according to the results from part three, inhibitor group (HG+AS-Ⅳ+SB431542 group) or activator group (HG+AS-Ⅳ+SRI011381 group), AS-Ⅳ group (HG+AS-Ⅳ group), and high glucose group (HG+DMSO group) were set up. The cell viability, apoptosis, migration, the level of ROS, MDA, SOD, IL-6, IL-8, and IL-10 were detected by CCK-8, flow cytometry, scratch assay, fluorescent probes, and ELISA to verify the role of TGF-β1/Smad2/3 pathway in the regulation of high glucose-induced skin wound repair cells with AS-Ⅳ.

Results:

1. AS-Ⅳ affect the biological behavior of high glucose-induced HaCaT, HUVEC, and HSF

The cell viability and the scratch healing rate in the HG+DMSO group were lower than that in the NG+DMSO group (P<0.01), but the apoptosis rate of cells was higher (P<0.01). The cell viability and the scratch healing rate in the HG+AS-Ⅳ group were higher than that in the HG+DMSO group (P<0.01), but the apoptosis rate of cells was lower (P<0.01).

2. AS-Ⅳ affect the oxidative stress and inflammatory response of high glucose-induced HaCaT, HUVEC, and HSF

The concentration of SOD in the HG+DMSO group was lower than that in the NG+DMSO group (P<0.01), but the ROS-positive cells and the concentration of MDA were higher (P<0.01); the concentration of IL-10 in the HG+DMSO group was lower than that in the NG+DMSO group (P<0.01), but the concentration of IL-6 and IL-8 were higher (P<0.01). The concentration of SOD in the HG+AS-Ⅳ group was higher than that in the HG+DMSO group (P<0.05), but the ROS-positive cells and the concentration of MDA were lower (P<0.05); the concentration of IL-10 in the HG+AS-Ⅳ group was higher than that in the HG+DMSO group (P<0.01), but the concentration of IL-6 and IL-8 were lower (P<0.01).

3. AS-Ⅳ affect the TGF-β1/Smad2/3 pathway of high glucose-induced HaCaT, HUVEC, and HSF

(1) HaCaT part: The expression of TGF-β1 at both the protein and RNA level, and the phosphorylation of Smad2 and Smad3 in the HG+DMSO group were lower than that in the NG+DMSO group (P<0.05), but the expression of Smad7 at both the protein and RNA level was higher (P<0.05). The expression of TGF-β1 at both the protein and RNA level, and the phosphorylation of Smad2 and Smad3 in the HG+AS-IV group were higher than that in the HG+DMSO group (P<0.05), but the expression of Smad7 at both the protein and RNA level was lower (P<0.01).

(2) HUVEC part: The expression of TGF-β1 at both the protein and RNA level, and the phosphorylation of Smad2 and Smad3 in the HG+DMSO group were higher than that in the NG+DMSO group (P<0.01), but the expression of Smad7 at both the protein and RNA level was lower (P<0.05). The expression of TGF-β1 at both the protein and RNA level, and the phosphorylation of Smad2 and Smad3 in the HG+AS-IV group were lower than that in the HG+DMSO group (P<0.05), but the expression of Smad7 at both the protein and RNA level was higher (P<0.05).

(3) HSF part: The expression of TGF-β1, TβR1, and TβR2 at both the protein and RNA level, and the phosphorylation of Smad2 and Smad3 in the HG+DMSO group were lower than that in the NG+DMSO group (P<0.05). The expression of TGF-β1, TβR1, and TβR2 at both the protein and RNA level, and the phosphorylation of Smad2 and Smad3 in the HG+AS-IV group were higher than that in the HG+DMSO group (P<0.05).

4. AS-Ⅳ attenuates high glucose-induced HaCaT, HUVEC, and HSF injury via TGF-β1/Smad2/3 signaling pathway.

(1) HaCaT part: The cell viability and the scratch healing rate at 24 h and 48 h in the HG+AS-IV+SB group were lower than that in the HG+AS-IV group (P<0.01), but the apoptosis rate of cells was higher (P<0.01); the concentration of SOD in the HG+AS-IV+SB group was lower than that in the HG+AS-IV group (P<0.05), but the ROS-positive cells and the concentration of MDA were higher (P<0.05); the concentration of IL-10 in the HG+AS-IV+SB group was lower than that in the HG+AS-IV group (P<0.05), but the concentration of IL-6 and IL-8 were higher (P<0.05).

(2) HUVEC part: The cell viability and the scratch healing rate at 24 h and 48 h in the HG+AS-IV+SRI group were lower than that in the HG+AS-IV group (P<0.05), but the apoptosis rate of cells was higher (P<0.01); the concentration of SOD in the HG+AS-IV+SRI group was lower than that in the HG+AS-IV group (P<0.01), but the ROS-positive cells and the concentration of MDA were higher (P<0.05); the concentration of IL-10 in the HG+AS-IV+SRI group was lower than that in the HG+AS-IV group (P<0.01), but the concentration of IL-6 and IL-8 were higher (P<0.01).

(3) HSF part: The cell viability and the scratch healing rate at 12 h and 24 h in the HG+AS-IV+SB group were lower than that in the HG+AS-IV group (P<0.01), but the apoptosis rate of cells was higher (P<0.05); the concentration of SOD in the HG+AS-IV+SB group was lower than that in the HG+AS-IV group (P<0.01), but the ROS-positive cells and the concentration of MDA were higher (P<0.05); the concentration of IL-10 in the HG+AS-IV+SB group was lower than that in the HG+AS-IV group (P<0.01), but the concentration of IL-6 and IL-8 were higher (P<0.01).

Conclusion:

AS-Ⅳ exerts anti-oxidant, anti-inflammatory, and anti-apoptotic effects to protect the proliferation and migration of high glucose-induced HaCaT, HUVEC, and HSF by regulating the TGF-β1/Smad2/3 signaling pathway, which may benefit for re-epithelialization and granulation tissue formation of chronic diabetic wound.

 

Keywords: astragaloside IV, wound healing, TGF-β/Smads

MOST Discipline Catalogue医学 - 临床医学 - 外科学
URL查看原文
Language中文
Other Code262010_120180900910
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/538031
Collection第二临床医学院
Affiliation
兰州大学第二临床医学院
Recommended Citation
GB/T 7714
高琼. 黄芪甲苷调控TGF-β1/Smad2/3 通路改善高糖损伤的皮肤修复细胞的功能[D]. 兰州. 兰州大学,2023.
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