Effect of Rolipram on expression of Mac-1 in porcine neutrophils and its signaling mechanism
[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]Por-cine 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,in-cluding p38 MAPK,ERK and p65 NF-κB,were detected by Real-Time fluorescence quantitative PCR and Western Blot,re-spectively.[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 sig-nificantly inhibited the expression of Mac-1(P＜0.01).Furthermore,PMA greatly increased the gene expression and phos-phorylation activity of p38 MAPK,ERK,and p65 NF-κB in neutrophils(P＜0.01).In contrast,rolipram at 5 μmol/L effec-tively 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 phos-phorylation 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 ex-pression and phosphorylation activity of p38 MAPK,ERK,and p65 NF-κB.
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