Activation of RAW264.7 macrophages by active fraction of Albizia julibrissin saponin via Ca2+-ERK1/2-CREB-lncRNA pathways.

The saponin active fraction from the stem bark of Albizia julibrissin (AJSAF) is an ideal vaccine adjuvant, but its mechanism of action remains unclear. The recent evidences indicate that long noncoding RNAs (lncRNAs) play essential roles in regulating the activation and function of macrophages. The current experiments were designed to investigate the effects of AJSAF on the activation of RAW264.7 macrophages and to explore its intracellular molecular mechanisms using a global gene expression microarray. AJSAF could significantly enhance phagocytic activity, induce reactive oxygen species (ROS), promote surface molecule expression, and up-regulate the mRNA and protein expression of cytokines and chemokines in RAW264.7 cells. AJSAF induced the differential expression of 223 mRNAs and 103 lncRNAs in RAW264.7 cells. Bioinformatics were used to predict the potential target mRNAs and function of up-regulated lncRNA A_30_P01018532 in RAW264.7 cells induced by AJSAF. The total 99 co-expressed mRNAs were classified as putative target genes of A_30_P01018532. A_30_P01018532 was associated with the inflammatory and immune response. AJSAF significantly increased the intracellular free Ca²⁺ levels and induced the phosphorylation of ERK1/2 and CREB in RAW264.7 cells. Moreover, Ca²⁺ chelator BAPTA-AM, ERK1/2 inhibitor PD98059 and CREB inhibitor KG-501 significantly inhibited the up-regulation of TNF-α, CCL2, CXCL2, CCL22, and A_30_P01018532 in RAW264.7 cells induced by AJSAF. These results suggested that AJSAF could activate RAW264.7 cells via Ca²⁺-ERK1/2-CREB pathways and that A_30_P01018532 might be an important regulator of mRNA expression in AJSAF-activated macrophage. This study may provide insights into the molecular mechanisms of action of AJSAF.