Immunomodulatory effect of APS and PSP is mediated by Ca2-cAMP and TLR4/NF-κB signaling pathway in macrophage.

OBJECTIVE

This study is to investigate the role of second messengers and TLR4/NF-κB signaling pathway in the immunomodulatory activities of Astragalus polysaccharide (APS) and Polysaccharopeptide (PSP) in macrophages.

METHODS

RAW 264.7 macrophage cells were treated with APS, PSP, lipopolysaccharide (LPS), or NiCl. Power-spectral method was used to detect protein kinase C (PKC) and Griess reaction to detect nitric oxide (NO). ELISA was conducted to detect cyclic adenosine monophosphate (cAMP), diglycerides (DAG), inositol 1, 4, 5-triphosphate (IP3), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Confocal laser scanning microscopy was performed to detect calcium level. qRT-PCR and Western blot was used to detect mRNA and protein expression of NF-κB.

RESULTS

APS and PSP significantly increased the concentrations of intracellular second messengers (NO, cAMP, DAG, IP3, Ca) and the activity of PKC in macrophages (p<0.05).The intracellular NF-κB mRNA and protein levels were significantly increased in macrophages treated by APS and PSP (p<0.05), whereas those were significantly decreased after NiCl incubation (p<0.05). Similarly, the secretion of TNF-α and IL-6 were significantly decreased by the treatment of NiCl.

CONCLUSION

Our findings strongly suggest that Ca-cAMP and TLR4/NF-κB signaling pathways are, at least partly, involved in APS and PSP mediated immunomodulatory activities in macrophages.