ERK1/2和PI3K/PKB信号通路在调节大鼠肺动脉平滑肌细胞外基质基因表达中的作用

摘要

目的：探讨细胞外调节蛋白激酶（ERK1/2）信号通路抑制剂PD98059和三磷酸肌醇激酶（PI3K/PKB）信号通路抑制剂LY294002在结缔组织生长因子（CTGF）调控大鼠肺动脉平滑肌细胞（PASMC）外基质异常沉积中的作用机制。方法体外培养SD大鼠PASMC，并分为空白组、CTGF组、CP组（CTGF+PD98059）、CL组（CTGF+LY294002）、CPL组（CTGF+PD98059+ LY294002）。实时荧光定量RT-PCR检测CTGF、PD98059和LY294002对PASMC Collagen Ⅲ和Fibronectin mRNA表达的影响，免疫组化和Western-blot检测其对CollagenⅢ蛋白表达的影响。结果与空白组比较，CTGF （50 ng/ml）刺激PASMC 48 h后CollagenⅢ和Fibronectin mRNA表达明显增强，72 h后PASMC CollagenⅢ蛋白表达减少，差异均有统计学意义（P<0.05）。与CTGF组比较，经PD98059（20μmol/L）和(或)LY294002（10μmol/L）干预48 h后CollagenⅢ和Fibronectin mRNA表达明显减弱，72 h后PASMC CollagenⅢ蛋白表达增加，差异均有统计学意义（P<0.05）；且PD98059和LY294002联合干预PASMC CollagenⅢ和Fibronectin mRNA表达下调更显著。结论 CTGF可刺激PASMC细胞外基质CollagenⅢ和Fibronectin mRNA表达，可能是促进肺动脉高压肺血管重构中基质沉积的重要因素之一。PD98059和LY294002可分别通过抑制ERK1/2和PI3K/PKB信号通路干预CTGF的生物学效应。%Objective To explore the effects of the extracellular regulated protein kinase’s (ERK1/2) inhibitor PD98059 and ino-sitol triphosphate kinase (PI3K/PKB) signaling pathway’s inhibitor LY294002 on extracellular matrix (ECM) deposition in pulmonary arterial smooth muscle cells (PASMCs) stimulated by connective tissue growth factor (CTGF). Methods PASMCs of SD rat were cul-tured in vitro. The PASMCs were divided into control group, CTGF group, CP (CTGF+PD98059) group, CL (CTGF+LY294002) group and CPL (CTGF+PD98059+LY294002) group. Real-time lfuorescent quantitative RT-PCR was used to detect the expression of colla-gen III and ifbronectin mRNA of PASMCs, and the expression of collagenШprotein of PASMCs was detected by immunohistochem-istry and western-blot. Results The expressions of collagenШand ifbronectin mRNA of PASMCs stimulated with CTGF (50 ng/ml) for 48 h were signiifcantly higher than those in control group, and the collagen proteinШof PASMCs was decreased signiifcantly after stimulation with CTGF (50 ng/ml) for 72 h (P<0.05). The expressions of collagenШand ifbronectin mRNA in PASMCs cultured with PD98059 (20μmol/L) and/or LY294002 (10μmol/L) for 48 h was signiifcantly lower than those in CTGF group (P<0.05). The collagen proteinШin PASMCs cultured with PD98059 (20μmol/L) and/or LY294002 (10μmol/L) for 72 h was increased (P<0.05). The expres-sions of collagenШand ifbronectin mRNA of PASMCs stimulated with both PD98059 and LY294002 were more signiifcant. Conclu-sions CTGF may increase the expression of collagenШand ifbronectin mRNA in PASMCs, which may contribute to the deposition of ECM in PASMCs during pulmonary vascular remodeling. PD98059 and LY294002 may repress ERK1/2 and PI3K/PKB signaling pathways and interfere with the biological effect of CTGF.