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-kappa B signaling pathway 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 NiCl2. 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-alpha (TNF-alpha) 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-kappa B. Results: APS and PSP significantly increased the concentrations of intracellular second messengers (NO, cAMP, DAG, IP3, Ca2+) and the activity of PKC in macrophages (p <0.05).The intracellular NF-kappa B mRNA and protein levels were significantly increased in macrophages treated by APS and PSP (p < 0.05), whereas those were significantly decreased after NiCl2 incubation (p <0.05). Similarly, the secretion of TNF-alpha and IL-6 were significantly decreased by the treatment of NiCl2. Conclusion: Our findings strongly suggest that Ca2+-cAMP and TLR4/NF-kappa B signaling pathways are, at least partly, involved in APS and PSP mediated immunomodulatory activities in macrophages. (C) 2016 Elsevier B.V. All rights reserved.