Activation of RAW264.7 macrophages by the polysaccharide from the roots of Actinidia eriantha and its molecular mechanisms
Introduction
Polysaccharides obtained from natural sources represent a structurally diverse class of macromolecules, and are known to affect a variety of biological responses, especially the immune response. Most polysaccharides derived from higher plants are relatively nontoxic and do not cause significant side effects, which is a major problem associated with bacterial polysaccharides and synthetic compounds. The plant polysaccharides are recognized as an effective biological response modifier with low toxicity (Petrovsky & Cooper, 2011). Recently, many polysaccharides have been shown to possess adjuvant potential on specific cellular and humoral immune responses against antigen, and be excellent adjuvant candidates for many vaccines (Licciardi & Underwood, 2011). Thus, the plant polysaccharides have enormous potential for use as an adjuvants in vaccines against both pathogens and cancer (Granell, Fernández-del-Carmen, & Orzáez, 2010).
The roots of Actinidia eriantha Benth (Actinidiaceae) have been used for gastric carcinoma, nasopharyngeal carcinoma, breast carcinoma, and hepatitis in traditional Chinese medicine. The modern pharmacological experiments proved that the water extracts of this drug possessed antitumor and immunopotentiating activities. In our previous works, the polysaccharide from the roots of A. eriantha (AEPS) has been shown to be the main active principles responsible for the antitumor and immunomodulatory effect of this drug (Xu, Yao, Sun, & Wu, 2009). AEPS was also proved to possess the immunological adjuvant activity on specific cellular and humoral immune responses to ovalbumin (OVA) in mice, and elicit a Th1 and Th2 immune responses (Sun, Wang, Xu, & Ni, 2009). Moreover, its low toxicity, no side effects and availability all make it as a safe and efficacious adjuvant candidate suitable for a wide spectrum of prophylactic and therapeutic vaccines. However, the underlying mechanisms of AEPS in the regulation of immune response need to be investigated.
Macrophages occupy a unique niche in the immune system, in that they can not only initiate innate immune responses, but also be effector cells that contribute to fight infection and inflammation. Macrophages can kill pathogens directly by phagocytosis and indirectly via the secretion of pro-inflammatory factors. Macrophages also exert an important role as an interface between innate and adaptive immunity. They are responsible for processes such as antigen processing and presentation to antigen-specific T cells. Following activation, macrophages can induce expression of accessory and costimulatory molecules that promote sustained stimulatory interactions with T cells and the generation of adaptive immunity. Indeed, the basic mechanisms of the immunostimulatory, anti-tumor, bactericidal and other therapeutic effects of plant polysaccharides are thought to occur via activation of immune cells resulting in the induction of immune responses (Beutler, 2004). Macrophages were thought to be the important target cells of some antitumor and immunomodulatory drug (Cheng, Wan, Wang, Jin, & Xu, 2008).
RAW264.7 cells are commonly accepted as a tool to investigate the molecular mechanisms of macrophages involved in regulating immunity (Hartley et al., 2008). The current experiments were designed to investigate the immunomodulatory effects of AEPS on RAW264.7 macrophages by determining the effect on the production and expression of nitric oxide, cytokines, chemokines, accessory and costimulatory molecules, and explore its molecular mechanisms using a high-throughput mouse Toll-like receptor (TLR) signaling pathway PCR array.
Section snippets
Materials
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), lipopolysaccharide (LPS), and polymyxin B (PMB) were purchased from Sigma Chemical Co., Saint Louis, MO, USA; DMEM medium was from Gibco, NY, USA. Griess reagent was purchased from Sigma-Aldrich (NSW, Australia). Mouse TNF-α, IL-1β, IL-6 and IL-10 detecting ELISA kits were from Wuhan Boster Biological Technology Co. Ltd., Hubei, China. Trizol was purchased from Invitrogen, USA; revert Aid™ M-MuLV reverse transcriptase was from
Effects of AEPS on RAW264.7 cell viability
To investigate the effects of AEPS on the growth of RAW264.7 cells, after treated with AEPS (0–100 μg/m1) for 24 h, cells were detected for the viability using MTT assay. As shown in Fig. 1A, AEPS was not cytotoxic to RAW264.7 cells up to the concentration of 200 μg/ml, even promote the cell proliferation at the concentration of 25–100 μg/ml (P < 0.01).
AEPS increased the pinocytic and phagocytic activity of RAW264.7 cells
The effect of AEPS on the pinocytic activity of RAW264.7 cells was examined by the uptake of neutral red. As shown in Fig. 1B, AEPS significantly
Discussion
AEPS has previously been proved to possess the immunological adjuvant effect and antitumor activity through improving immune response. However, its molecular mechanism responsible for regulating immune response is not fully understood. In the present study the activation of AEPS on the macrophages was investigated and its subsequent intracellular signaling pathways were explored using RAW264.7 macrophage as a cellular model.
One of the most distinguished features of macrophage activation would
Acknowledgments
This work was supported by Grant-in-Aid from the National Natural Science Foundation of China (no. 31272597), the Zhejiang Provincial Natural Science Foundation of China (no. R3080027), the National Key Project of Scientific and Technical Supporting Programs Funded by Ministry of Science & Technology of China (no. 2008BADB4B06-2), the Key Scientific and Technological Innovation Team of Zhejiang Province (no. 2010R50031-13), Chinese Universities Scientific Fund (no. 2010KLEP007), and the
References (37)
- et al.
Up-regulation of TLR9 gene expression by LPS in mouse macrophages via activation of NF-(B, ERK and p38 MAPK signal pathways
Immunology Letters
(2002) Innate immunity: An overview
Molecular Immunology
(2004)- et al.
Structure and expression pattern of teleost caspase recruitment domain (CARD) containing proteins that are potentially involved in NF-kB signalling
Development and Comparative Immunology
(2010) - et al.
Stephanthraniline A inhibits the proliferation and activation of T cells in vitro and in vivo
European Journal of Pharmacology
(2012) - et al.
Macrophage immunomodulatory activity of polysaccharides isolated from Glycyrrhiza uralensis fish
International Immunopharmacology
(2008) - et al.
Plant-derived medicines: A novel class of immunological adjuvants
International Immunopharmacology
(2011) - et al.
Expression of the nitric oxide synthase gene in mouse macrophages activated for tumor cell killing. Molecular basis for the synergy between interferon-gamma and lipopolysaccharide
Journal of Biological Chemistry
(1993) - et al.
Macrophage polarization: Tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes
Trends in Immunology
(2002) - et al.
The chemokine system in diverse forms of macrophage activation and polarization
Trends in Immunology
(2004) - et al.
Molecular cloning and characterization of the human doublestranded RNA-activated protein kinase induced by interferon
Cell
(1990)
Toll-like receptors and acquired immunity
Seminars in Immunology
SHIP represses the generation of alternatively activated macrophages
Immunity
Panax notoginseng attenuates LPS-induced pro-inflammatory mediators in RAW264.7 cells
Journal of Ethnopharmacology
Novel polysaccharide adjuvant from the roots of Actinidia eriantha with dual Th1 and Th2 potentiating activity
Vaccine
The ubiquitin-editing enzyme A20 (TNFAIP3) is a central regulator of immunopathology
Trends in Immunology
Role of transcription factor NF-κB/Rel in induction of nitric oxide synthase
Journal of Biological Chemistry
Chemical composition and antitumor activity of different polysaccharides from the roots of Actinidia eriantha
Carbohydrate Polymers
Cutting edge: Cell surface expression and lipopolysaccharide signalling via the Toll-like receptor 4-MD-2 complex on mouse peritoneal macrophages
The Journal of Immunology
Cited by (198)
Structural characterization and immunomodulatory activity of a polysaccharide from Dioscotea opposita
2024, International Journal of Biological MacromoleculesThe isolation, characterization and biological activities of the non-glucan polysaccharides from the high-starch-content plant Pueraria mirifica
2024, International Journal of Biological MacromoleculesComprehensive studies of the anti-inflammatory effect of tetraprenyltoluquinone, a quinone from Garcinia cowa Roxb
2024, Journal of EthnopharmacologyCnidium officinale polysaccharide enhanced RAW 264.7 cells activation and NK-92 cells cytotoxicity against colon cancer via NF-κB and MAPKs signaling pathways
2023, International Journal of Biological Macromolecules