电针通过缺氧诱导的人脐静脉内皮细胞中介导的HIF-1α/VEGF/Notch 1信号通路中的外泌体miR-210促进血管生成。
Electro-acupuncture Promotes Angiogenesis via Exosomal miR-210 in the Hypoxia-induced HUVECs Mediated HIF-1α/VEGF/Notch 1 Signal Pathway.
作者信息
Xu Shu-Ying, Ni Si-Ming, Zeng Chun-Li, Peng Yong-Jun
机构信息
Department of Acupuncture and Rehabilitation, Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing 210029, Jiangsu Province, China.
出版信息
Curr Neurovasc Res. 2022;19(4):406-417. doi: 10.2174/1567202620666221024143408.
BACKGROUND
Acupuncture has been wildly applied for cerebral ischemia treatment in China for thousands of years, while the specific mechanism remains uncertain. Recently, many studies have shown that acupuncture promotes angiogenesis after ischemia occurs. Here, we examined the effect of electro-acupuncture (EA) exosomes on angiogenesis in hypoxia-induced human umbilical vein endothelial cells (HUVECs).
OBJECTIVE
To investigate whether EA exosomal miR-210 promotes angiogenesis in the hypoxiainduced HUVECs via the HIF-1α/VEGF/Notch 1 signal pathway.
METHODS
The middle cerebral artery occlusion (MCAO) model was established and treated with EA therapy. Then, exosomes were identified and isolated from rats' plasma in the MCAO+EA group by transmission electron microscopy (TEM), surface markers expressions, and PKH26 reagent. MiR- 210 mimic, miR-210 inhibitor, and HIF-1α were transfected. Flow cytometry, CCK-8 assay, and Transwell assay were conducted to assess the migration, apoptosis, and proliferation of each group of cells. Western blot and quantitative PCR were performed to detect the CD34, HIF-1α, VEGF, Notch 1, and miR-210 expression levels in each group.
RESULTS
MiR-210 was significantly upregulated in exosomes of the MCAO plasma, and further enhanced by EA therapy. EA-EXOs and miR-210 mimic inhibited cell apoptosis, promoted cell proliferation and cell migration in hypoxia-induced HUVECs. However, the miR-210 inhibitor reversed the proliferation and migration number induced by EA-EXOs. Besides, EA-EXOs and miR- 210 mimic further enhanced those HIF-1α, VEGF, and Notch 1 levels compared to the hypoxia treatment only. Silencing HIF-1α or miR-210 reversed the high expressions of those three angiogenic factors induced by hypoxia and EA-EXO. qPCR showed similar trends with their relative mRNAs. To analyze these associations quantificationally, Spearman's rank correlation coefficient was calculated. As revealed by results, the expression of proteins and mRNA were highly correlative with each other.
CONCLUSION
These results indicated that EA-EXO miR-210 promotes angiogenesis in hypoxia conditions via HIF-1α/VEGF/Notch 1 signal pathway.
背景
数千年来，针灸在中国被广泛应用于脑缺血治疗，但其具体机制仍不明确。最近，许多研究表明，缺血发生后针灸可促进血管生成。在此，我们研究了电针（EA）外泌体对缺氧诱导的人脐静脉内皮细胞（HUVECs）血管生成的影响。
目的
探讨EA外泌体miR-210是否通过HIF-1α/VEGF/Notch 1信号通路促进缺氧诱导的HUVECs血管生成。
方法
建立大脑中动脉闭塞（MCAO）模型并给予EA治疗。然后，通过透射电子显微镜（TEM）、表面标志物表达和PKH26试剂，从MCAO+EA组大鼠血浆中鉴定并分离外泌体。转染miR-210模拟物、miR-210抑制剂和HIF-1α。采用流式细胞术、CCK-8法和Transwell法评估各组细胞的迁移、凋亡和增殖情况。进行蛋白质印迹和定量PCR检测各组中CD34、HIF-1α、VEGF、Notch 1和miR-210的表达水平。
结果
MCAO血浆外泌体中miR-210显著上调，并通过EA治疗进一步增强。EA-EXOs和miR-210模拟物抑制缺氧诱导的HUVECs细胞凋亡，促进细胞增殖和细胞迁移。然而，miR-210抑制剂逆转了EA-EXOs诱导的增殖和迁移数量。此外，与仅缺氧处理相比，EA-EXOs和miR-210模拟物进一步提高了HIF-1α、VEGF和Notch 1水平。沉默HIF-1α或miR-210可逆转缺氧和EA-EXO诱导的这三种血管生成因子的高表达。qPCR显示其相对mRNA有相似趋势。为定量分析这些关联，计算了Spearman等级相关系数。结果显示，蛋白质和mRNA的表达彼此高度相关。
结论
这些结果表明，EA-EXO miR-210通过HIF-1α/VEGF/Notch 1信号通路促进缺氧条件下血管生成。
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