Immunoregulation Of The Polysaccharides From The Roots Of Actinidia Eriantha On RAW264.7 Cell And Its Mechanisms

Actinidia eriantha Benth (Actinidiaceae) is a liana plant that commonly grows in temperate climate zones.Its roots have been used for gastric carcinoma, nasopharyngeal carcinoma, breast carcinoma, and hepatitis in traditional Chinese medicine. AEPS(Actinidia eriantha polysaccharides) was the water-soluble polysaccharide isolated from the roots of Actinidia eriantha, and the main active principles responsible for the antitumor and immunomodulatory effect of this drug. AEPS has been proved to not only significantly inhibit the growth of mouse transplantable tumor, but also remarkably promote splenocytes proliferation, NK cell and CTL activity, IL-2 and IFN-γproduction from splenocytes, and serum antigen-specific antibody levels in tumor-bearing mice. It has also been reported that AEPS had strong potential to increase both cellular and humoral immune responses and elicit a balanced Th1/Th2 response to ovalbumin (OVA) in mice.Moreover, its low toxicity, no side effects and availability all make it as a safe and efficacious immunostimulants candidate suitable for a wide spectrum of prophylactic and therapeutic vaccines and antitumor agent with immunomodulatory activity.Macrophages represent a family of mononuclear leukocytes that are widely distributed throughout the body. As the first defense effector of host body, macrophages can recognize the invading microorganisms and tumor cells, as well as eliminate the invaders. Also, the innate immune function of macrophage contributes to the activation of adaptive humoral and cellular immunity. In addition, macrophage is one of three kinds of professional antigen-presenting cells including dendritic cells, macrophages and B cells.Macrophages can produce many kinds of cytokines and inflammatory mediators, such as TNF-α, IL-1β, IL-6, IL-10 and NO which are involved in the defense functions and inflammation. Therefore, the primary aim of this study is to investigate the effects of AEPS on the macrophage functions and its related mechanisms.We selected the murine macrophage cell line RAW264.7 as cellular model to observe the effect of AEPS on the macrophage functions. By using MTT method to detect cell proliferation, we found that AEPS was not cytotoxic to RAW264.7 cells up to the concentration of 200μg/ml. We determined the effects of AEPS on the phagocytosis, NO release and cytokine production of RAW264.7 cells by neutral red method, Griess reagent and ELISA, respectively. AEPS could significantly increased phagocytosis, the secretion of NO and the production of cytokines TNF-a, IL-10 and IL-6 production in RAW264.7 in the concentration-dependent manner. The mRNA expression of inflammatory factor, chemokines, TLR receptor and its signaling moleculer in RAW264.7 cell were also analyzed by reverse transcription polymerase chain rcaction (RT-PCR) AEPS remarkedly up-regulate the mRNA expression levels of inflammatory factor, chemokines, TLR2, TLR9, TRIF and NF-κB. We further examined the nuclear transcription factor NF-κB activation in RAW264.7 cells using western-blot analysis, and found that AEPS inhibited the expression of IKK-a and NF-κB p65 in cytoplasm of RAW264.7 cells.In conclusion, these results demonstrated that AEPS promote macrophage function through up-regulation of TLRs and activation of NF-κB.