Gannan Medical University, Ganzhou, Jiangxi, China.
Department of Orthopedic Surgery, The First Affiliated Hospital of Gannan Medical University, Ganzhou, Jiangxi, China.
PLoS One. 2024 Feb 23;19(2):e0299382. doi: 10.1371/journal.pone.0299382. eCollection 2024.
Endothelial cell injury is one of the important pathogenic mechanisms in thrombotic diseases, and also neutrophils are involved. MicroRNAs (miRNAs) have been demonstrated to act as essential players in endothelial cell injury, but the potential molecular processes are unknown. In this study, we used cellular tests to ascertain the protective effect of miR-328-3p on human umbilical vein endothelial cells (HUVECs) treated with oxygen-glucose deprivation (OGD).
In our study, an OGD-induced HUVECs model was established, and we constructed lentiviral vectors to establish stable HUVECs cell lines. miR-328-3p and Toll-like receptor 2 (TLR2) interacted, as demonstrated by the dual luciferase reporter assay. We used the CCK8, LDH release, and EdU assays to evaluate the proliferative capacity of each group of cells. To investigate the expression of TLR2, p-P65 NF-κB, P65 NF-κB, NLRP3, IL-1β, and IL-18, we employed Western blot and ELISA. Following OGD, each group's cell supernatants were gathered and co-cultured with neutrophils. An immunofluorescence assay and Transwell assay have been performed to determine whether miR-328-3p/TLR2 interferes with neutrophil migration and neutrophil extracellular traps (NETs) formation.
In OGD-treated HUVECs, the expression of miR-328-3p is downregulated. miR-328-3p directly targets TLR2, inhibits the NF-κB signaling pathway, and reverses the proliferative capacity of OGD-treated HUVECs, while inhibiting neutrophil migration and neutrophil extracellular trap formation.
miR-328-3p inhibits the NF-κB signaling pathway in OGD-treated HUVECs while inhibiting neutrophil migration and NETs formation, and ameliorating endothelial cell injury, which provides new ideas for the pathogenesis of thrombotic diseases.
内皮细胞损伤是血栓性疾病的重要发病机制之一，其中也涉及中性粒细胞。现已证实 microRNAs（miRNAs）在内皮细胞损伤中发挥重要作用，但潜在的分子过程尚不清楚。本研究通过细胞实验，确定 miR-328-3p 对氧葡萄糖剥夺（OGD）处理的人脐静脉内皮细胞（HUVEC）的保护作用。
本研究建立了 OGD 诱导的 HUVEC 模型，并构建了慢病毒载体，建立了稳定的 HUVEC 细胞系。双荧光素酶报告基因实验证实 miR-328-3p 与 Toll 样受体 2（TLR2）相互作用。我们使用 CCK8、LDH 释放和 EdU 测定评估每组细胞的增殖能力。通过 Western blot 和 ELISA 检测 TLR2、p-P65 NF-κB、P65 NF-κB、NLRP3、IL-1β 和 IL-18 的表达。OGD 后，收集各组细胞上清液与中性粒细胞共培养。通过免疫荧光和 Transwell 实验确定 miR-328-3p/TLR2 是否干扰中性粒细胞迁移和中性粒细胞胞外诱捕网（NETs）形成。
在 OGD 处理的 HUVEC 中，miR-328-3p 的表达下调。miR-328-3p 可直接靶向 TLR2，抑制 NF-κB 信号通路，逆转 OGD 处理的 HUVEC 的增殖能力，同时抑制中性粒细胞迁移和中性粒细胞胞外诱捕网形成。
miR-328-3p 在 OGD 处理的 HUVEC 中抑制 NF-κB 信号通路，同时抑制中性粒细胞迁移和 NETs 形成，改善内皮细胞损伤，为血栓性疾病的发病机制提供了新的思路。
