黄芪甲苷介导的内皮祖细胞外泌体通过 miR-21/PTEN 轴促进高糖损伤内皮细胞自噬并抑制细胞凋亡。
Astragaloside IV - mediated endothelial progenitor cell exosomes promote autophagy and inhibit apoptosis in hyperglycemic damaged endothelial cells via miR-21/PTEN axis.
机构信息
Department of Burns and Plastic Surgery, the First Affiliated Hospital of Hunan University of Chinese Medicine, Hunan, China.
College of Integrated Traditional Chinese and Western Medicine, Hunan University of Chinese Medicine, Hunan, China.
出版信息
Folia Histochem Cytobiol. 2022;60(4):323-334. doi: 10.5603/FHC.a2022.0030. Epub 2022 Dec 12.
INTRODUCTION
As one of the basic components of Astragalus, Astragaloside IV (AS-IV) has a protective effect on endothelial injury caused by diabetes. AS-IV stimulated endothelial progenitor cells (EPCs) to secrete exosomes loaded with miR-21. This study aimed to investigate the effects of AS-IV-mediated EPCs exosomal miR-21 (EPC-exos-miR-21) on high glucose (HG) damaged endothelial cells.
MATERIALS AND METHODS
After the isolation of EPCs derived from fetal umbilical cord blood, exosomes of EPCs were obtained by differential centrifugation. The morphology of exosomes was observed by electron microscopy. The particle size distribution of exosomes was detected by Nanoparticle Tracking Analysis. Human umbilical vein endothelial cells (HUVECs) were treated with 33 mM glucose to establish an HG injury model. Flow cytometry and TUNEL assay were used to characterize the surface markers of primary EPCs and the apoptosis of HUVECs. The gene and protein expression were detected by qPCR, immunofluorescence, and Western blotting. A dual luciferase assay was used to verify the targeting relationship of miR-21 with PTEN.
RESULTS
HG environment led to time- and dose-dependent inhibition and enhancement of autophagy and apoptosis in HUVECs. AS-IV stimulated EPCs to secrete exosomes loaded with miR-21. Exosomes secreted by EPCs pretreated with AS-IV [EPC-exo(ASIV)] promoted autophagy and inhibited apoptosis in HG-impaired HUVECs. PTEN is a target of miR-21. MiR-21 carried by EPC-exo(ASIV) repressed PTEN expression in HG-impaired HUVECs. In contrast, p-AKT, p-mTOR, p-PI3K, cleaved PARP and PARP levels were upregulated. Compared to the HG group, the expression of autophagy regulatory genes (ATG5, beclin1 and LC3) was enhanced in the EPC-exo(ASIV) group and EPC-exo(ASIV)-miR-21 mimic group. In contrast, apoptosis-positive regulatory genes (Bax, caspase-3 and caspase-9) were attenuated. Further overexpression of PTEN reversed the expression of these genes.
CONCLUSIONS
AS-IV-mediated EPC-exos-miR-21 could enhance autophagy and depress apoptosis in HG-damaged endothelial cells via the miR-21/PTEN axis.
简介
作为黄芪的基本成分之一，黄芪甲苷（AS-IV）对糖尿病引起的内皮损伤具有保护作用。AS-IV 可刺激内皮祖细胞（EPCs）分泌含有 miR-21 的外泌体。本研究旨在探讨 AS-IV 介导的 EPC 外泌体 miR-21（EPC-exos-miR-21）对高糖（HG）损伤内皮细胞的影响。
材料与方法
从胎牛脐带血中分离出 EPCs 后，通过差速离心获得 EPCs 的外泌体。通过电子显微镜观察外泌体的形态。通过纳米颗粒跟踪分析检测外泌体的粒径分布。用 33mM 葡萄糖处理人脐静脉内皮细胞（HUVECs）建立 HG 损伤模型。流式细胞术和 TUNEL 测定法用于鉴定原代 EPCs 的表面标志物和 HUVECs 的凋亡情况。通过 qPCR、免疫荧光和 Western blot 检测基因和蛋白表达。双荧光素酶测定用于验证 miR-21 与 PTEN 的靶向关系。
结果
HG 环境导致 HUVECs 的自噬和凋亡呈时间和剂量依赖性抑制和增强。AS-IV 刺激 EPCs 分泌载有 miR-21 的外泌体。用 AS-IV 预处理的 EPCs 分泌的外泌体[EPC-exo（ASIV）]促进 HG 损伤的 HUVECs 中的自噬并抑制凋亡。PTEN 是 miR-21 的靶标。EPC-exo（ASIV）携带的 miR-21 抑制 HG 损伤的 HUVECs 中 PTEN 的表达。相反，p-AKT、p-mTOR、p-PI3K、cleaved PARP 和 PARP 水平升高。与 HG 组相比，EPC-exo（ASIV）组和 EPC-exo（ASIV）-miR-21 模拟物组的自噬调节基因（ATG5、beclin1 和 LC3）表达增强。相反，凋亡阳性调节基因（Bax、caspase-3 和 caspase-9）减弱。PTEN 的进一步过表达逆转了这些基因的表达。
结论
AS-IV 介导的 EPC-exos-miR-21 可通过 miR-21/PTEN 轴增强 HG 损伤内皮细胞中的自噬并抑制凋亡。
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