咯利普兰对猪中性粒细胞表达Mac-1的作用及有关信号机制  

Effect of Rolipram on expression of Mac-1 in porcine neutrophils and its signaling mechanism

作　　者：张旭日 张溪园 王恩慈 郭雨楠 李姣 黄安琦 姜代勋 ZHANGXuri;ZHANG Xiyuan;WANG Enci;GUO Yunan;LI Jiao;HUANG Anqi;JIANG Daixun(Beijing Key Laboratory of Traditional Chinese Veterinary Medicine,College of Animal Science and Technology,Beijing University of Agriculture,Beijing 102206,China)

机构地区：[1]北京农学院动物科学技术学院/兽医学(中医药)北京市重点实验室,北京102206

出　　处：《北京农学院学报》2024年第1期52-57,共6页Journal of Beijing University of Agriculture

基　　金：北京市基金-市教委联合项目(KZ202010020030)。

摘　　要：【目的】阐明咯利普兰为代表的磷酸二酯酶4抑制剂的抗炎新机制。【方法】采用密度梯度离心法分离猪中性粒细胞,流式细胞术检测猪中性粒细胞表达巨噬细胞表面分子抗原-1(Macrophage surface molecular antigen-1,Mac-1);Real-time qPCR和Western blot技术检测猪中性粒细胞p38丝裂原活化蛋白激酶(p38 MAPK)、细胞外调节蛋白激酶(ERK)、p65核转录因子(p65 NF-κB)mRNA表达和磷酸化活性。【结果】佛波酯(phorbol myristate acetate, PMA)可显著上调中性粒细胞表达Mac-1(P<0.01),咯利普兰对PMA上调的中性粒细胞表达Mac-1有一定抑制作用,其中,5μmol/L咯利普兰可极显著抑制中性粒细胞表达Mac-1(P<0.01);PMA可极显著上调中性粒细胞p38 MAPK、ERK和p65 NF-κB mRNA表达和磷酸化活性(P<0.01),5μmol/L咯利普兰对PMA升高的中性粒细胞p38 MAPK、p65 NF-κB mRNA表达有极显著抑制作用(P<0.01),对ERK mRNA表达有显著抑制作用(P<0.05),对PMA升高的中性粒细胞p38 MAPK、ERK和p65 NF-κB磷酸化活性有极显著抑制作用(P<0.01)。【结论】咯利普兰可显著抑制中性粒细胞表达Mac-1,其机制与阻遏p38 MAPK、ERK、p65 NF-κB基因表达和磷酸化活性有关。[Objective]The inhibitory effect of rolipram on neutrophil adhesion is associated with the expression of Macrophage surface molecular antigen-1(Mac-1).In this study,we investigated the role of rolipram in regulating Mac-1 and uncovered a novel anti-inflammatory mechanism of phosphodiesterase 4 inhibitors using phorbol myristate acetate(PMA).[Methods]Porcine peripheral blood neutrophils were isolated and purified using density gradient centrifugation.The expression of Mac-l on neutrophils was determined using Flow Cytometry.The gene expression and phosphorylation activity of signaling proteins,including p38 MAPK,ERK and p65 NF-κB,were detected by Real-Time fluorescence quantitative PCR and Western Blot,respectively.[Results]PMA significantly increased the expression of Mac-1 on neutrophils(P<0.01).However,rolipram was able to inhibit the PMA-induced expression of Mac-1 on neutrophils.Specifically,at a concentration of 5μmol/L,rolipram significantly inhibited the expression of Mac-1(P<0.01).Furthermore,PMA greatly increased the gene expression and phosphorylation activity of p38 MAPK,ERK,and p65 NF-κB in neutrophils(P<0.01).In contrast,rolipram at 5μmol/L effectively suppressed the PMA-induced gene expression of p38 MAPK and p65 NF-κB in neutrophils(P<0.01).It also inhibited the PMA-induced gene expression of ERK(P<0.05).Additionally,rolipram significantly inhibited the PMA-induced phosphorylation activity of p38 MAPK,ERK,and p65 NF-κB in neutrophils(P<0.01).[Conclusion]Rolipram has been found to effectively suppress the expression of Mac-1 on neutrophils.This inhibitory effect is attributed to its ability to inhibit gene expression and phosphorylation activity of p38 MAPK,ERK,and p65 NF-κB.

关 键 词：巨噬细胞表面分子抗原-1 p38丝裂原活化蛋白激酶 细胞外调节蛋白激酶 p65核转录因子 咯利普兰 中性粒细胞 猪 

分 类 号：S859.1[农业科学—临床兽医学]