C-型钠尿肽对大鼠心肌成纤维细胞分泌单核细胞趋化蛋白-1及纤溶酶原激活物抑制剂-1的抑制作用

Role of C-type natriuretic peptide in inhibiting monocyte chemoattractant protein-1 and plasminogen activator inhibitor-1 of cardiac fibroblasts in rats

目的探讨C-型钠尿肽(C-type natriuretic peptide,CNP)对心肌成纤维细胞分泌单核细胞趋化蛋白-1(monocyte chemoattractant protein-1,MCP-1)以及纤溶酶原激活物抑制剂-1(plasminogen activator inhibitor-1,PAI-1)的作用,并检验CNP对心肌成纤维细胞的下游信号通路的影响。方法将大鼠心肌成纤维细胞分别置于不含CNP的培养基(对照组)和含10-9 mol/L CNP(CNP1组)、10-8 mol/L CNP(CNP2组)、10-7 mol/L CNP(CNP3组)培养基中培养24h;采用反转录定量PCR检测各组心肌成纤维细胞中MCP-1和PAI-1 mRNA的表达;ELISA法测定各组心肌成纤维细胞MCP-1和PAI-1蛋白水平。将心肌成纤维细胞分别应用10-9 mol/L的CNP(实验1组)、ERK1/2抑制剂U0126(10-4 mol/L,实验2组)、联合应用CNP及U0126(联合实验组)预处理30 min,空白对照组不进行预处理,实验分析CNP对细胞外信号调节激酶(extracellular signal-regulated kinase,ERK)1/2的激活作用。结果 CNP1组、CNP2组及CNP3组细胞中MCP-1 mRNA(0.716±0.016、0.671±0.104、0.635±0.108)、PAI-1 mRNA表达(0.802±0.072、0.780±0.021、0.654±0.017)及MCP-1蛋白[(0.814±0.211)、(0.781±0.280)、(0.752±0.171)g/L]、PAI-1蛋白水平[(0.728±0.217)、(0.696±0.274)、(0.643±0.301)g/L)]均低于对照组[1.192±0.037、1.107±0.156,(1.021±0.263)、(1.047±0.207)g/L](P<0.05),CNP1组、CNP2组和CNP3组组间MCP-1、PAI-1 mRNA表达及MCP-1、PAI-1蛋白水平比较差异均无统计学意义(P>0.05);实验1组、实验2组和联合实验组P-ERK1/2蛋白[(0.672±0.301)、(0.569±0.298)、(0.536±0.197)g/L]及MCP-1蛋白[(0.814±0.211)、(0.801±0.188)、(0.764±0.261)g/L)]、PAI-1蛋白[(0.728±0.217)、(0.682±0.287)、(0.543±0.301)g/L)]均低于空白对照组[(1.028±0.129)g/L、(1.021±0.263)g/L、(1.047±0.207)g/L](P<0.05),实验1组、实验2组及联合实验组组间以上指标比较差异均无统计学意义(P>0.05)。结论 CNP可抑制心肌成纤维细胞分泌MCP-1、PAI-1,可能是CNP抗纤维化的新机制。

To investigate the role of C-type natriuretic peptide （CNP） in inhibiting monocyte chemoattractant protein-1 （MCP-1） and plasminogen activator inhibitors-1 （PAl-l） of cardiac fibroblasts in rats as well as to assess the influence of CNP on downstream signal pathway of cardiac fibroblasts. Methods The cardiac fibroblasts of rats were cultured in the medium containing no CNP （control group）, containing 10-9 mol/L CNP （CNP1 group）, 10-8 mol/L CNP （CNP2 group）, and 10-7 mol/L CNP （CNP3 group） for 24 h. The mRNA expressions of MCP-1 and PAI-1 in cardiac fibroblasts were detected by reverse transcription quantitative PCR. The MCP-1 and PAI-1 proteins were detected by ELISA. The cardiac {ibroblasts were pretreated with 10-9 mol/L CNP （experiment 1 group）, 10 4 mol/L ERK1/2 inhibitor U0126 （experiment 2 group） and combination of CNP and U0126 （combined experiment group） for 30 min, while blank control group was not pretreated. The effect of CNP on activation of extracellular signal-regulated kinase （ERK）1/2 was analyzed. Results The levels of MCP-1 mRNA （0. 716±0. 016, 0. 671±0. 104, 0. 635±0. 108）, PAbl mRNA （0. 802±0. 072, 0.780±0. 021, 0.654±0.017）, MCP-1 protein （（0.814±0.211）, （0.781±0.280）, （0.752±0.171）g/L）, andPAI-1 protein （（0.728±0.217）, （0.696±0.274）, （0.643±0.301）g/L） in CNP1, CNP2 and CNP3 groups were significantly lower than those in control group （1. 192±0. 037, 1. 107±0. 156, （1. 021±0. 263）g/L, （1. 047±0. 207）g/L） （P〈O. 05）, and there were no significant differences between each two of CNP1, CNP2 and CNP3 groups （P〉0.05）. The levels of P-ERK1/2 protein （（0. 672±0. 301）, （0. 569±0. 298）, （0. 536±0. 197）g/L）, MCP-1 protein （（0. 814±0. 211）, （0. 801±0. 188）, （0. 764±0. 261）g/L）, and PAl 1 protein （（0. 728±0. 217）, （0. 682±0. 287）, （0. 543±0. 301）g/L） in experiment 1 group, experiment 2 group and combined experiment group were significantly lower than those in control group （（1.028±0.129）, （1. 021±0. 263）, （1. 047±0.207）g/L） （P〈0.05）, and there were no significant differences between each two of experiment groups （P〉0.05）. Conclusion CNP can inhibit the secretions of MCP-1 and PAI-1 by cardiac fibroblasts, which may be a new mechanism of anti-fibrosis in CNP.