Study on visfatin-induced inflammation and necroptosis via LOX-1 in human umbilical vein endothelial cells

本研究旨在探索脂肪细胞因子内脏脂肪素（Visfatin）能否诱导血管内皮细胞（VEC）发生炎症及坏死性凋亡（Necroptosis），并初步探讨其机制。体外分离培养人脐静脉内皮细胞（HUVECs），单独使用 Visfatin 刺激或在阻断氧化低密度脂蛋白受体 1（LOX-1）后给予 Visfatin 刺激，采用 Western blot、RT-PCR、免疫细胞化学法、酶联免疫吸附法（ELISA）、MTT、流式细胞化学法等观察细胞炎症及 Necroptosis 的发生情况。结果显示，100 ng/mL 的 Visfatin 作用 24 h 可诱导 HUVECs 中单核细胞趋化蛋白-1（MCP-1）和 LOX-1 的 mRNA 及蛋白水平表达显著上调；而 LOX-1 抑制剂聚肌苷酸预处理可明显降低 Visfatin 诱导的 MCP-1 表达。另外，100 ng/mL 的 Visfatin 作用 24 h 可明显诱导 HUVECs 中出现坏死样特征，且 Necroptosis 特异性基因 BMF 的 mRNA 表达显著增加，细胞增殖率降低；而聚肌苷酸预处理后，细胞增殖率升高，BMF 的基因表达下调。实验结果提示，Visfatin 通过 LOX-1 介导，引起了血管内皮细胞炎症反应及 Necroptosis，在动脉粥样硬化的发生与发展中起着重要的作用。.; The aim of the study is to identify the effects and underlying mechanisms of visfatin on inflammation and necroptosis in vascular endothelial cells. Human umbilical vein endothelial cells (HUVECs) were stimulated with visfatin or pretreated with Polyinosinic acid (LOX-1 inhibitor). By using the Western blot, RT-PCR, immunocytochemistry, enzyme-linked immunosorbent assay (ELISA), MTT and flow cytometry technique, the occurrence of inflammation and necroptosis in HUVECs were evaluated. Our results showed that 100 ng/mL visfatin significantly increased the mRNA and protein expression of monocyte chemotactic protein 1 (MCP-1) and LOX-1 after 24 hours' treatment in HUVECs. However, pretreatment with Polyinosinic acid could significantly reduce the expression of MCP-1 compared with visfatin group. Additionally, 100 ng/mL visfatin could induce the production of necrotic features and increase the mRNA expression of BMF (one of the markers of necroptosis), while pretreating with Polyinosinic acid markedly downregulated the mRNA expression of BMF gene and promoted the cell proliferation. These results indicate that visfatin might induce inflammation and necroptosis via LOX-1 in HUVECs, suggesting that visfatin plays a central role in the development of atherosclerosis.