Atherosclerosis is one common chronic inflammatory disease in which angiogenesis is involved. Here we established an in vitro cell model of angiogenesis made by human dermal microvascular endothelial cells (HMEC-1) and work to investigate the role of triptolide (TPL) in this model. To induce angiogenesis, HMEC-1 cells were cultured in Matrigel-conditioned medium. The ratio of tubes to nucleus was detected. To evaluate angiogenesis, Western blot assay was carried out to detect endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor receptor-2 (VEGFR2) and VEGF. Cell counting kit-8 was utilized to estimate the viability of HMEC-1 cells. microRNA (miR)-92a was analyzed by qRT-PCR. The targeting relationship between integrin subunit alpha 5 (ITGA5) and miR-92a was verified through luciferase activity assay. The effects of ITGA5 on signaling transducers (ERK, PI3K, and AKT) in a phosphorylated form were valued using Western blot method. After stimulated by TPL, LY294002 and PD98059, the alteration in phosphorylation of the signaling transducers was evaluated by Western blot assay. The ratio of tubes to nucleus and angiogenesis related factors were increased with the delaying of culture time. TPL decreased the expression of angiogenesis factors. Furthermore, miR-92a was upregulated by TPL and miR-92a silence upregulated angiogenesis factors. In addition, TPL decreased ITGA5 which was proved as a target of miR-92a. ITGA5 overexpression resulted in the abundance of angiogenesis factors while ITGA5 silence led to the opposite results. Meanwhile, ITGA5 overexpression increased phosphorylation of ERK, PI3K and AKT while ITGA5 silence reversed the trend. TPL (as an anti-angiogenesis agent) suppressed angiogenesis by upregulating miR-92a, and miR-92a-mediated down-regulation of ITGA5 blocked the signaling transduction of ERK and PI3K/AKT pathways.

中文翻译：

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雷公藤甲素通过调节 miR-92a 抑制微血管内皮细胞中的血管生成。

动脉粥样硬化是一种常见的慢性炎症性疾病，其中涉及血管生成。在这里，我们建立了由人真皮微血管内皮细胞 (HMEC-1) 制成的体外血管生成细胞模型，并努力研究雷公藤甲素 (TPL) 在该模型中的作用。为了诱导血管生成，HMEC-1 细胞在基质胶条件培养基中培养。检测管与核的比率。为了评估血管生成，进行蛋白质印迹测定以检测内皮一氧化氮合酶（eNOS）、血管内皮生长因子受体-2（VEGFR2）和VEGF。细胞计数试剂盒 8 用于估计 HMEC-1 细胞的活力。通过 qRT-PCR 分析 microRNA (miR)-92a。整合素亚基α5（ITGA5）与miR-92a之间的靶向关系通过荧光素酶活性测定得到验证。使用蛋白质印迹法评估了 ITGA5 对磷酸化形式的信号转导子（ERK、PI3K 和 AKT）的影响。经 TPL、LY294002 和 PD98059 刺激后，通过蛋白质印迹法评估信号转导子磷酸化的变化。随着培养时间的延长，管核比和血管生成相关因子增加。TPL降低了血管生成因子的表达。此外，miR-92a 被 TPL 上调，而 miR-92a 沉默上调的血管生成因子。此外，TPL 降低了 ITGA5，这被证明是 miR-92a 的靶标。ITGA5 过表达导致血管生成因子丰富，而 ITGA5 沉默导致相反的结果。同时，ITGA5 过表达增加了 ERK、PI3K 和 AKT 的磷酸化，而 ITGA5 沉默逆转了这一趋势。