title: TLR-4 may mediate signaling pathways of Astragalus polysaccharide RAP induced cytokine expression of RAW264.7 cells.
abstract: BACKGROUND
  Polysaccharides of Radix Astragali (Astragalus membranaceus (Fisch) Bge.; Huangqi) are able to induce cytokine production of macrophages and are considered the main active ingredient for the immune-enhancing effect of this commonly used medicinal herb.
  
  
  OBJECTIVE
  To investigate the molecular mechanism of immunomodulating activities of a reported Astragalus polysaccharide, RAP, which is a hyperbranched heteroglycan with average molecular weight of 1334kDa.
  
  
  METHODS
  The cytokine production of RAW264.7 cells were analyzed by using ELISA assays while cell viability was assessed by MTT method. Western blot analysis was used for determining protein contents of mitogen-activated protein kinases (MAPKs). In addition, the level of IL-6, iNOS, and TNF-α mRNA was determined by RT-PCR.
  
  
  RESULTS
  It has been found that RAP itself did not have any cytotoxic effect on mouse mammary carcinoma 4T1 cells, but it significantly enhanced cytotoxicity of the supernatant of RAW264.7cells on 4T1 cells. Furthermore, RAP enhanced the production of NO and cytokines in RAW264.7 cells, and significantly up-regulated gene expressions of TNF-α, IL-6, iNOS. All these bioactivities were blocked by the inhibitor of TLR4 (Toll-like receptor 4), suggesting that TLR4 is a receptor of RAP and mediates its immunomodulating activity. Further analyses demonstrated that RAP rapidly activated TLR4-related MAPKs, including phosphorylated ERK, phosphorylated JNK, and phosphorylated p38, and induced translocation of NF-κB as well as degradation of IκB-α. These results are helpful to better understand the immunomodulating effects of Radix Astragali.
  
  
  CONCLUSIONS
  RAP may induce cytokine production of RAW264.7 cells through TLR4-mediated activation of MAPKs and NF-κB.
authors.affiliation: School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Wei
authors.affiliation: School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong, China.
authors.forename: Hai-Tao
authors.initials: HT
authors.lastname: Xiao
authors.affiliation: School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong, China.
authors.forename: Wan-Rong
authors.initials: WR
authors.lastname: Bao
authors.affiliation: Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong, China.
authors.forename: Dik-Lung
authors.initials: DL
authors.lastname: Ma
authors.affiliation: State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao, China.
authors.forename: Chung-Hang
authors.initials: CH
authors.lastname: Leung
authors.affiliation: State Key Laboratory of Phytochemistry and Plant Resources in West China, Institute of Chinese Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China.
authors.forename: Xiao-Qiang
authors.initials: XQ
authors.lastname: Han
authors.affiliation: State Key Laboratory of Phytochemistry and Plant Resources in West China, Institute of Chinese Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China.
authors.forename: Chun-Hay
authors.initials: CH
authors.lastname: Ko
authors.affiliation: State Key Laboratory of Phytochemistry and Plant Resources in West China, Institute of Chinese Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China.
authors.forename: Clara Bik-San
authors.initials: CB
authors.lastname: Lau
authors.affiliation: State Key Laboratory of Phytochemistry and Plant Resources in West China, Institute of Chinese Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China.
authors.forename: Chun-Kwok
authors.initials: CK
authors.lastname: Wong
authors.affiliation: State Key Laboratory of Phytochemistry and Plant Resources in West China, Institute of Chinese Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China.
authors.forename: Kwok-Pui
authors.initials: KP
authors.lastname: Fung
authors.affiliation: State Key Laboratory of Phytochemistry and Plant Resources in West China, Institute of Chinese Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China.
authors.forename: Ping-Chung
authors.initials: PC
authors.lastname: Leung
authors.affiliation: School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong, China. Electronic address: bzxiang@hkbu.edu.hk.
authors.forename: Zhao-Xiang
authors.initials: ZX
authors.lastname: Bian
authors.affiliation: School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong, China. Electronic address: simonhan@hkbu.edu.hk.
authors.forename: Quan-Bin
authors.initials: QB
authors.lastname: Han
mesh_terms: D000818:Animals; D027883:Astragalus Plant; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D016207:Cytokines / Q000378:metabolism*; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011134:Polysaccharides / Q000302:isolation & purification / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects*; D051197:Toll-Like Receptor 4 / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D016207:Cytokines; D011134:Polysaccharides; D051197:Toll-Like Receptor 4; D009569:Nitric Oxide; D020928:Mitogen-Activated Protein Kinases
keywords: Astragalus polysaccharides; MAPKs; NF-κB; RAW264.7 macrophages