PD98059抑制Peroxynitrite诱导人脑血管平滑肌细胞DDR_2的表达

PD98059 inhibits Peroxynitrite-induced discoidin domain receptor-2 expression in human brain vascular smooth muscle cells

目的探讨过氧亚硝酸盐(ONOO-)诱导人脑血管平滑肌细胞(HBVSMCs)盘状结构域受体2(DDR2)表达的机制。方法体外培养HBVSMCs,用倒置相差显微镜、吖啶橙染色法和MTT比色法对PD98059预处理的细胞进行细胞形态学及细胞生存率检测,采用Western blot及Real-time PCR技术,检测ONOO-作用下HBVSMCs中DDR2表达及施加ERK1/2抑制剂PD98059后DDR2/MMP-9表达的变化。结果 10μmol/L ONOO-诱导DDR2在蛋白及mRNA水平上呈现高表达(P<0.05),随着ONOO-浓度的增加,DDR2逐渐呈现低表达(P<0.05)。PD98059预处理后的HBVSMCs,无明显形态学变化,对其进行生存率检测亦无显著影响(P>0.05)。PD98059显著抑制ONOO-诱导的DDR2/MMP-9表达(P<0.05)。结论 ERK1/2信号转导途径抑制剂PD98059可以抑制ONOO-诱导的DDR2/MMP-9表达,ONOO-诱导HBVSMCs DDR2的表达机制与ERK1/2信号转导途径有关。

Objective To explore the possible mechanism underlying peroxynitrite（ONOO-）-induced discoidin domain receptor-2（DDR2） expression in human brain vascular smooth muscle cells（HBVSMCs）.Methods HBVSMCs were cultured in vitro in different concentrations of ONOO-,and PD98059,a specific inhibitor of ERK1/2.Morphology and viability of HBVSMCs pretreated with PD98059 were detected by an inverted phase contrast microscope,acridine orange staining and MTT assay.Western blot and Real-time PCR were employed to determine expressions of DDR2/ MMP-9.Results 10 μmol/L of ONOO-significantly induced expression of DDR2 at both protein and mRNA levels（P0.05）.With increase in concentrations of ONOO-,expression of DDR2 was markedly decreased（P0.05）.After pretreatment on HBVSMCs with PD98059,cellular morphology and viability had no significant change by an inverted phase contrast microscope,acridine orange staining and MTT assay（P0.05）.Pretreatment on HBVSMCs with PD98059 significantly inhibited expressions of DDR2/ MMP-9 in HBVSMCs induced by 10 μmol/L of ONOO-（P0.05）.Conclusion PD98059 inhibits ONOO——induced DDR2 expression.The ERK1/2 signal transduction pathway might involve in ONOO——induced DDR2 expression in HBVSMCs.