PAMK has the function of promoting the lymphocytes active and proliferation, maintaining the stable cell morphology, increasing the secretion of cytokines such as TNF-α, IL-4, IL-6, IL-10, IL-17, IL-17A, IFN-γ, alleviating the immunosuppression caused by CTX, and increasing the index of immune organs, which was considered one of the characteristic of the immune level enhance15; 16. We all know that cytokines play a very important role in body immunity15; 17; 18 and macrophages play an important role in defending the body against invading pathogens15; 17; 18. Increased level of IL-4, IL-2, TNF-α and IFN-γ could enhance the macrophage activity and improve specific immunity. In our research, we found that PAMK could upregulated the mRNA and protein expression of IL-2, IL-4, TNF-α and IFN-γ caused by siTLR4. IL-2 can enhance the secretion of TNF-α, IL-1, NO and other cytokines from macrophages, enhance the cytotoxic effect of macrophages and increase the killing activity of macrophages. IL-4 can increase the tumor killing ability of bone marrow Mφ and the expression of Fc(II) receptor, and promote the transformation of macrophages from M1 to M2 type. From our research, the results indicated that the relative mRNA expression level andcellular supernatant level of IL-2, IL-4 were significant decreased by siTLR4, but it significant increased or had increase trend after PAMK treated. Thus, the proliferative activity of macrophages is also increased, and the secretion levels of TNF-α and IFN-γ also increased when treated with PAMK, just as our research showed. According, we suggested that PAMK could enhanced macrophage activity and enhanced the release of TNF-α and IFN-γ to increased macrophage immunomodulation. S, Li, Shi etc found polysaccharide from Auricularia auricula, fungal polysaccharide could promoted the activation of macrophages and induced the secretion of cytokines such as IL-1β, IL-6, NO and TNF-α to enhance the phagocytic activity of macrophages and increase macrophage immunomodulation19; 21; 22. However, Zeng, Ma.G, Wang etc indicated that Lonicerae Japonicae Flos and Lonicerae Flos, polysaccharide from Pleurotus eryngii and Procyanidin A2 could inhibit of NO secretion in RAW264.7, down-regulate of IL-1β, IL-6 and TNF-α release, down-regulate of iNOS protein and COX2, NF-κB (p-IKKα/β, p-IκBα and p-p65) and MAPK (p-p38, p-JNK and p-ERK) protein levels caused by LPS23–25. These results were similar to our research. Consequently, we suggested that PAMK could maintain stable immune levels of macrophages and maintain the body's immune balance when the body is stimulated by either LPS or siTLR4, etc.
TLR4, an important member of the TLR family, was the first upstream gene for NF-kappaB (NF-κB) and has been shown to be involved in immune responses26. Some research suggested that when TLR4 is activated, there are two conduction signal pathway: dependent myeloiddifferentiationfactor88 (MyD88) signaling pathway and non-dependent MyD88 signaling pathway. The activated TLR domain of TLR4 binds to PAMK in a complex that can bind to MyD88. Interleukin-1 receptor associated kinase 1 (IRAK1), IRAK4 and TNF receptor associated factor 6 (TRAF6) recruit to the TLR-MyD88 complex, resulting in phosphorylation of IRAK1 and TRAF6. The signal is propagated by the phosphorylated linker molecular complex to downstream MAP kinases (JNK, p38 MAPK), Apetala 1 (AP1) and the nuclear factor kappa-B kinase (IKK) complex NF-κB. Activation of inhibitor of IKKα/β phosphorylates inhibitor of NF-κB (IκB), which is shed from NF-κB and ubiquitinated, allowing NF-κB to be activated by the inhibitory state leading to transcription factor activation12; 27; 28. In our research, we indicated that PAMK could increase the relative mRNA expression level and protein expression level of TLR4, MyD88, IKKα/β, NF-κB in siTLR4 + PAMK group when it compared with siTLR4 group, and we found that the tend of mRNA and protein level was the same. Thus, We suggested that PAMK activate IKK through a MyD88-dependent pathway, which phosphorylates IKB and make it off NF-κB and allow NF-κB to enter the nucleus to exert immunoregulatory effects. When NF-κB signal pathway was activated, cells would secrete cytokines to maintain the immunity balance while the organism is stimulated, such as IL-2, IL-4, TNF-α and IFN-γ,just as our research showed. Li indicated that PAMK could increase the serum level of IL-2, IL-4, TNF-α and IFN-γ, enhance splenocytes immunomodulatoryactivity by TLR4/MyD88/NF-κB signal pathway9. Some researchers showed that Polysaccharides from Cordyceps gunnii mycelia could increase the spleen thymus index, up-regulate the serum level of IL-2, IL-12, IFN-γ, IgG and other cytokines secretion levels, protect immunosuppressed mice through TLR4/TRAF6/NF-κB signaling pathway29. These findings were consistent with previous research. However, Zheng, He and Jun suggested that Diethyl Blechnic Exhibits, an inulin-type fructan from Codonopsis pilosula, a novel polysaccharide from Acorus tatarinowii could inhibit release of pro-inflammatory factors caused by LPS such as prostaglandin E2 (PGE2) and relative mRNA expression of TNF-α, IL-1β, IL-6 and myeloperoxidase (MPO) through TLR4/MyD88/NF-κB in RAW264.730–33. That also indicated that polysaccharide regulate RAW264.7 immune function by TLR4/MyD88/NF-κB signal pathway.
In conclusion, our research indicated that PAMK could increase the relative mRNA expression level and serum cytokines level of IL-2, IL-4, TNF-α and IFN-γ, enhance the immunomodulatory activity of RAW264.7 by TLR4/MyD88/NF-κB signal pathway