【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。更多还原

【Abstract】 [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 5 μmol/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 5 μmol/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.更多还原