PCSK3通过调控血管平滑肌细胞炎症反应及细胞表型转化标志物的表达抑制细胞迁移

PCSK3 inhibits cell migration by regulating the inflammatory response of vascular smooth muscle cells and the expression of cell phenotype transformation markers

目的:探讨前蛋白转化酶枯草溶菌素3(PCSK3)调控小鼠血管平滑肌细胞增殖和迁移的作用。方法:首先采用0、25、50、100 mg/L的氧化型低密度脂蛋白(ox-LDL)刺激小鼠血管平滑肌细胞(VSMCs),通过RT-PCR检测PCSK3 mRNA表达情况。将100 mg/L的ox-LDL刺激小鼠血管平滑肌细胞作为模型组,将VSMCs分为对照组(NC组,0 mg/L ox-LDL刺激的VSMCs+scramble siRNA,SCR),ox-LDL组(模型组+SCR),ox-LDL+si-PCSK31#组(模型组+si-PCSK31#质粒)以及ox-LDL+si-PCSK32#组(模型组+si-PCSK32#质粒)。EdU法检测各组VSMCs增殖活性,Transwell小室实验观察细胞迁移情况,ELISA试剂盒检测IL-1β,TNF-α,IL-6,细胞间黏附分子-1(ICAM-1)含量,RT-PCR检测各组SMCs细胞的Ⅰ型及Ⅲ型胶原,平滑肌肌动蛋白(α-SMA)的mRNA表达水平的变化。结果:100 mg/L ox-LDL处理浓度下的VSMCs中PCSK3明显增高,能够诱导VSMCs的增殖。干扰PCSK3内源性表达抑制ox-LDL刺激下的VSMCs的迁移能力与增殖能力,减少IL-1β,TNF-α,IL-6和ICAM-1水平分泌。与ox-LDL组相比,si-PCSK3处理的细胞组中Ⅰ型胶原和OPN mRNA表达明显下调,α-SMA mRNA表达明显上调。结论:下调PCSK3表达可减弱小鼠血管平滑肌细胞增殖和迁移,可能与PCSK3对细胞内的炎症反应及细胞表型转化的调控作用有关。

Objective To investigate the effect of Recombinant Proprotein Convertase Subtilisin/Kexin Type 3(PCSK3)on the proliferation and migration of vascular smooth muscle cells.Methods First,the cell model was established by stimulating vascular smooth muscle cells(VSMCs)of mouse with 0,25,50,100 mg/L ox-LDL,and the expression of PCSK3 mRNA was detected by RT-PCR.The atherosclerotic model cells stimulated by 100 mg/L ox-LDL were used as the model group,VSMCs were divided into NC group(0 mg/L ox-LDL stimulated VSMCs+scramble siRNA,SCR),ox-LDL group(model group+SCR),ox LDL+si-PCSK31#group(model group+si-PCSK31#plasmid)and ox-LDL+si-PCSK32#group(model group+si-PCSK32#plasmid).The proliferation activity of VSMCs in each group was detected by EdU method,and cell migration was observed by Transwell,ELISA kit for detecting IL-1β,TNFα,IL-6,intercellular adhesion molecule-1(ICAM-1)content,RT-PCR was used to detect the changes of mRNA expression of type I andⅲcollagen and smooth muscle actin(α-SMA)in SMC cells of each group.Results Under the concentration of 100 mg/L ox-LDL treatment,PCSK3 in VSMCs significantly increased,which can induce the proliferation of VSMCs.Interference with the endogenous expression of PCSK3 can inhibit the migration and proliferation ability of VSMCs stimulated by ox-LDL,reduce the secretion of IL-1β,TNFα,IL-6,and ICAM-1 levels.Compared with the ox-LDL group,the expression of type I collagen and OPN mRNA was significantly downregulated in the si-PCSK3 treated cell group,while the expression ofα-SMA mRNA was significantly upregulated.Conclusion Down-regulation of PCSK3 can attenuate the proliferation and migration of vascular smooth muscle cells,which may be related to the regulation of PCSK3 on intracellular inflammatory response and cell phenotype transformation.