Study Of The Immunomodulatory And Anti-tumor Effects Of Flammulina Velutipes Polysaccharides And The Underlying Mechanisms

Numerous studies have demonstrated that Flammulina velutipes polysaccharides have anti-tumor,immunomodulatory,anti-virus,anti-inflammation,anti-fatigue and anti-hypoxia effects.Flammulina velutipes have many biological active components and people have paid much attention to Flammulina velutipes polysaccharides(FVP), whose main functions are immunomodulatory action and anti-tumor effect.The research on immunomodulation has testified that FVP can promote spleen cells transformation in normal and S₁₈₀ tumor-bearing mice,enhance activities of NK cells and the production of interleukin 2,strengthen phagocytosis of peritoneal macrophages in normal mice,and increase the amount of serum hemolysin in normal mice.The study of anti-tumor effect have proven that FVP can inhibit S₁₈₀ sarcoma and H₂₂ ascitic type hepatic tumor,and FVP have favorable effects on ovarian cancer and breast cancer.But the mechanism of immunomodulatory and anti-tumor effects of FVP on cytokines is still unclear.It is,therefore,necessary for the mechanism to be further studied.Objective:1.To study the effect of Flammulina velutipes polysaccharides(FVP) on the production of tumor necrosis factorα(TNF-α),interferonγ(INF-γ) and interleukin 2 (IL-2) by murine spleen cells,and the effect on spleen cells metabolic activity with methylthiazolyl tetrazolium(MTT) colorimetry assay.2.To investigate the effect of Flammulina velutipes polysaccharides(FVP) on the production of tumor necrosis factorα(TNF-α) by murine peritoneal exudate cells and the effect on peritoneal exudate cells metabolic activity with methylthiazolyl tetrazolium(MTT) colorimetry assay.3.To study the effect of Flammulina velutipes polysaccharides(FVP) on the serum amount of tumor necrosis factorα(TNF-α) and interferonγ(INF-γ) in S₁₈₀ tumor-bearing mice,and the tumor growth inhibition rate.4.To explore the mechanisms of immunomodulatory and anti-tumor actions of Flammulina velutipes polysaccharides(FVP).Methods:1.Splenocytes from normal mouse were incubated at 37℃in the presence of Flammulina velutipes polysaccharides(FVP) for 48h.ELISA was employed to determine the amount of TNF-α,IFN-γ,and IL-2 in the culture supernatants.And MTT colorimetry was used for the assay of cell metabolic activity.2.Peritoneal exudate cells from normal mouse were incubated at 37℃in the presence of Flammulina velutipes polysaccharides(FVP) for 10h.ELISA was employed to determine the amount of TNF-αin the culture supernatants.And MTT colorimetry was used for the assay of cell metabolic activity.3.Mouse models bearing S₁₈₀ solid tumor were established.The control group was then administered intraperitoneally with sterile physiological saline solution at 0.1ml/10g body weight.The administered volume of other groups was the same as that of control.The positive control group was administered with Ganoderma lucidum polysaccharides(GLP),at 100 mg/kg intraperitoneally and the low,median and high dose groups of Flammulina velutipes polysaccharides were administered at 25,50,and 100mg/kg intraperitoneally.After 7 consecutive days,the serum samples were collected.Then TNF-αand IFN-γlevels in the samples were measured by using enzyme-linked immunosorbent assay(ELISA).Then the implanted tumors and spleens were removed and weighed,and the tumor growth inhibition rate and spleen index were calculated.Results:1.FVP(50,100 and 200μg/ml) could promote the metabolic activity of murine splenocytes and increase the amount of TNF-α,INF-γand IL-2 in the supernatants of splenocyte cultures.The effect increased along with the increment of the concentration of FVP was in a concentration-dependent manner in a certain range of concentrations.Compared with control group,the promotion effect on TNF-αlevel appeared to be strongest among the cytokines examined.2.FVP(50,100,and 200μg/ml) could promote the metabolic activity of murine peritoneal exudate cells and obviously increase the amount of TNF-αin peritoneal exudate cells cultures(P=0.000,0.000,and 0.000) in a concentration-dependent manner.3.FVP(25,50,and 100mg/kg) could inhibite the growth of S₁₈₀ solid tumor (P=0.000,0.000,and 0.000),and increase the index of spleen in S₁₈₀ solid tumor mice (P=0.000,0.000,and 0.000).The median and high doses of FVP could raise the serum levels of TNF-α(P=0.005 and 0.000) and INF-γ(P=0.047 and 0.009) in the S₁₈₀ tumor-bearing mice in a dose-dependent manner.4.FVP(200μg/ml) promoted the production of TNF-αby murine peritoneal exudate cells.Anti-TLR4 antibody(1 and 2μg/ml) showed marked,although not complete,suppression on this effect(P=0.034 and 0.010).However,specific p38 MAPK blocker SB203580(20μM) and NF-κB blocker helenalin(10μM) could completely abolish the stimulatory effect of FVP on the production of TNF-αby murine peritoneal exudate cells(P=0.006 and 0.003).Conclusion:1.FVP can promote the metabolic activity of murine splenocytes and increase the amount of TNF-α,INF-γand IL-2 in the supernatants of splenocytes cultures. 2.FVP can promote the metabolic activity of murine peritoneal exudate cells and obviously increase the amount of TNF-αin peritoneal exudate cells cultures.3.FVP can inhibite the growth of S₁₈₀ solid tumor,and increase the serum levels of TNF-αand INF-γin S₁₈₀ solid tumor mice.4.The signaling mechanism by which FVP promote TNF-αproduction may be as follows,the polysaccharides act on the TLR4 receptors on murine peritoneal exudate cells,signal through p38 MAPK pathway,and then activate NF-κB.The activated NF-κB in turn initiates the gene transcription of TNF-α.5.The mechanism of immunomodulatory and anti-tumor effects of FVP may be related with their potentiation of the production of TNF-αin mice.