咯利普兰通过MAPK/ERK信号通路抑制中性粒细胞的黏附

摘要

[目的]研究磷酸二酯酶4 (PDE4)特异性抑制剂咯利普兰抑制中性粒细胞黏附的有关分子机制,明确其抗炎的主要信号通路,为PDE4选择性中药抑制剂的药理研究提供参考.[方法]猪中性粒细胞的提取采用密度梯度离心法,中性粒细胞与猪内皮细胞的黏附采用虎红比色法,中性粒细胞黏附分LFA-1和Mac-1表达量的检测采用流式细胞技术,中性粒细胞p38MAPK、ERK等磷酸化活性检测采用western blot法.[结果]咯利普兰可显著抑制fMLP刺激的中性粒细胞和内皮细胞的黏附(P＜0.05),极显著抑制fMLP刺激的中性粒细胞LFA-1和Mac-1的表达(P＜0.01),阻断fMLP刺激的中性粒细胞ERK磷酸化活性(P＜0.01),而对p38MAPK磷酸化活性无显著影响.[结论]咯利普兰可抑制中性粒细胞和内皮细胞黏附,其机制与阻断中性粒细胞ERK磷酸化进而抑制LFA-1和Mac-1表达有关.%[Objective]To study the molecular mechanism of rolipram,a specific inhibitor of phosphodiesterase type 4 (PDE4),on the inhibition of neutrophils adhesion,elucidate the main pathway of anti-inflammation,and provide a reference for the pharmacological study of PDE4 selective herbal inhibitors.[Methods]Density gradient centrifugation was used to extract porcine neutrophils,Tiger red colorimetric method was used to detect the adhesion of neutrophils to porcine endothelial cells,the adhesion molecules,LFA-1 and Mac-1,of neutrophils were detected by flow cytometry,and the phosphorylation of p38MAPK and ERK in neutrophils was detected by Western blot.[Results]Rolipram could significantly inhibit fMLP-stimulated adhesion of neutrophils and endothelial cells (P ＜ 0.05),significantly inhibit fMLP-stimulated expression of LFA-1 and Mac-1 on neutrophils (P ＜ 0.01),block fMLP-stimulated ERK phosphorylation activity in neutrophils (P ＜ 0.01),but could not show any significant effect on p38MAPK phosphorylation.[Conclusion]Rolipram can inhibit the adhesion of neutrophils and endothelial cells,and its mechanism maybe relate to blocking ERK phosphorylation and inhibiting LFA-1 and Mac-1 expression of neutrophils.