title: Immunomodulatory effect of APS and PSP is mediated by Ca2+-cAMP and TLR4/NF-κB signaling pathway in macrophage.
abstract: 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 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-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, Ca2+) 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 NiCl2incubation (p<0.05). Similarly, the secretion of TNF-α and IL-6 were significantly decreased by the treatment of NiCl2.
  
  
  CONCLUSIONS
  Our findings strongly suggest that Ca2+-cAMP and TLR4/NF-κB signaling pathways are, at least partly, involved in APS and PSP mediated immunomodulatory activities in macrophages.
authors.affiliation: Department of Clinical Laboratory, the Second Affiliated Hospital of Chongqing Medical University, Chongqing 400010, China.
authors.forename: Zhixue
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Department of Clinical Medicine, Xinjiang Medical University, Urumqi 830054, China.
authors.forename: Zijing
authors.initials: Z
authors.lastname: Liu
authors.affiliation: Department of Clinical Laboratory, the Second Affiliated Hospital of Chongqing Medical University, Chongqing 400010, China.
authors.forename: Lijng
authors.initials: L
authors.lastname: Zhou
authors.affiliation: Department of Clinical Laboratory, the Second Affiliated Hospital of Chongqing Medical University, Chongqing 400010, China.
authors.forename: Tingting
authors.initials: T
authors.lastname: Long
authors.affiliation: Department of Clinical Laboratory, the Second Affiliated Hospital of Chongqing Medical University, Chongqing 400010, China.
authors.forename: Xing
authors.initials: X
authors.lastname: Zhou
authors.affiliation: Department of Clinical Laboratory, the Second Affiliated Hospital of Chongqing Medical University, Chongqing 400010, China. Electronic address: yixibao@163.com.
authors.forename: Yixi
authors.initials: Y
authors.lastname: Bao
mesh_terms: D000818:Animals; D027883:Astragalus Plant / Q000737:chemistry; D020013:Calcium Signaling / Q000187:drug effects*; D000242:Cyclic AMP / Q000378:metabolism; D004353:Drug Evaluation, Preclinical; D007155:Immunologic Factors / Q000494:pharmacology*; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D010936:Plant Extracts / Q000494:pharmacology*; D011134:Polysaccharides / Q000494:pharmacology; D000067996:RAW 264.7 Cells; D051197:Toll-Like Receptor 4 / Q000378:metabolism*
chemical_list: D007155:Immunologic Factors; D016328:NF-kappa B; D010936:Plant Extracts; D011134:Polysaccharides; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4; D009569:Nitric Oxide; D000242:Cyclic AMP
keywords: APS; PSP; Second messenger