PDK1在ox-LDL诱导的血管内皮细胞损伤及其功能障碍中的作用研究

Research on the role of PDK1 in ox-LDL-induced vascular endothelial cell injury and dysfunction

摘要:

目的]探讨丙酮酸脱氢酶激酶同工酶1(PDK1)对内皮细胞损伤及其功能障碍的影响。 [方法]采用siRNA-PDK1敲低PDK1的表达,并使用磷酸盐缓冲液(PBS)或氧化型低密度脂蛋白(ox-LDL)处理人脐静脉内皮细胞(HUVEC),以探究PDK1在ox-LDL诱导的血管内皮细胞损伤及其生物学功能障碍中的作用。使用活性氧(ROS)及线粒体活性染色试剂盒检测HUVEC中ROS的产生及线粒体损伤状况。使用流式细胞凋亡试剂盒检测HUVEC的凋亡水平,并通过结晶紫染色和Dil染色检测HUVEC的损伤及其对THP-1细胞的募集和黏附能力的改变。通过Western blot和RT-qPCR检测CD86、CD206、NOD样受体蛋白3(NLRP3)、白细胞介素1β(IL-1β)、单核细胞趋化蛋白1(MCP-1)等的mRNA和蛋白表达。通过免疫荧光染色评估CD86和CD206的表达水平。使用试剂盒测定超氧化物歧化酶(SOD)的活性和丙二醛(MDA)的含量,采用ELISA试剂盒检测MCP-1、细胞间黏附分子1(ICAM-1)、血管细胞黏附分子1(VCAM-1)等细胞因子的分泌情况。 [结果]与ox-LDL处理的HUVEC相比,敲低PDK1表达后,ROS的产生减少,线粒体损伤减轻,SOD活性恢复,MDA含量减少,细胞凋亡水平降低,核因子κB(NF-κB)和NLRP3炎症小体的激活受到抑制,炎症因子(IL-1β、IL-18)和趋化黏附因子(MCP-1、ICAM-1、VCAM-1)的释放减少,黏附的THP-1细胞数量减少,THP-1细胞中CD206、精氨酸1(Arg-1)的表达增加,THP-1细胞中CD86、诱导型一氧化氮合酶(iNOS)的表达受到抑制(均P＜0.05)。 [结论]敲低血管内皮细胞中PDK1的表达可以减少ox-LDL处理引起的内皮细胞氧化应激和炎症反应,进而改善内皮细胞生物学功能。

Abstract:

Aim To explore the effects of pyruvate dehydrogenase kinase isoenzyme 1 (PDK1) on endothelial cell injury and dysfunction. Methods siRNA-PDK1 was used to knock down PDK1 expression, and human umbilical vein endothelial cells (HUVEC) were treated with phosphate-buffered saline (PBS) or oxidized low density lipoprotein (ox-LDL) to investigate the role of PDK1 in ox-LDL-induced vascular endothelial cell injury and its biological dysfunction.Reactive oxygen species (ROS) production and mitochondrial damage in HUVEC were detected using ROS and mitochondrial activity staining kits. The apoptosis levels of HUVEC were detected via flow cytometry apoptosis assay kit, and the damage of HUVEC and its changes in recruitment and adhesion ability to THP-1 cells were detected through crystal violet staining and Dil staining. The mRNA and protein expression of CD86, CD206, NOD-like receptor protein 3 (NLRP3), interleukin-1β (IL-1β) and monocyte chemoattractant protein-1 (MCP-1) were detected by Western blot and RT-qPCR.The expression levels of CD86 and CD206 were evaluated by immunofluorescence staining. Superoxide dismutase (SOD) and malondialdehyde (MDA) concentrations were measured with corresponding kits, while the secretions of cytokines including MCP-1, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were detected with ELISA kits. Results Compared with HUVEC treated with ox-LDL, knocking down PDK1 expression resulted in reduced ROS production, reduced mitochondrial damage, restored SOD activity, decreased MDA concentrations, decreased apoptosis levels, inhibited activation of nuclear factor-κB (NF-κB) and NLRP3 inflammasomes, decreased release of inflammatory factors (IL-1β and IL-18) and chemokines (MCP-1, ICAM-1 and VCAM-1), decreased number of THP-1 cells, increased expression of CD206 and arginine-1 (Arg-1) in THP-1 cells, and inhibited expression of CD86 and inducible nitric oxide synthase (iNOS) in THP-1 cells (all P＜0.05). Conclusion Knocking down the expression of PDK1 in endothelial cells can reduce oxidative stress and inflammatory response caused by ox-LDL, thereby improving the biological function of endothelial cells.