| Objective:To study the effects of Anoectochilus formosanus polysaccharide(ARP)on immunological activities of both murine splenic lymphocytes and macrophage RAW254.7 cells in vitro.Methods:The safe concentration of ARP on murine splenic lymphocytes and RAW264.7 cells,the effects of ARP synergistical stimulation with ConA and LPS on murine splenic lymphocytes proliferationwere determined by MTT method in vitro.Flow cytometry were used to observe the influences of ARP on cell cycle of murine splenic lymphocytes and apoptosis of RAW264.7 cells.The phagocytic activity of RAW264.7 cells were performed by Neutralred method.The NO production of murine splenic lymphocytes and RAW264.7 cells were performed by Griess method.Enzyme-linked immunosorbent assay(ELISA)was used to detect the secretion of murine splenic lymphocytes(IL-2,IL-4,IL-6,IFN-y)induced by ConA and RAW-264.7 cells(TNF-?,IL-1?,IL-10)in the supernatant;The mRNA expression of IL-2,IL-4,IL-6,IFN-y,TNF-a,T-bet,GATA3 in murine splenic lymphocytes and TNF-a,IL-6,iNOS in RAW264.7 cells were deceted by RT-PCR.The expression of protein I?B-p65 in RAW264.7 cells was established by western blot.Results:1.Effects of ARP on the immune activities of murine splenic lymphocytes in vitro:The results of MTT method were showed that OD values could significantly increase with the dose-dependent(P<0.05),comparing to the control group,and it reached highest when the dose of ARP was 400?g/mL.Compared to ARP alone,ARP supplemented with ConA or LPS promoted the OD value significantly(P<0.05).Results of Flow cytometry compared with control group,in ARP group,the proportion of spleen lymphocytes in G0/G1 phase tend to be decreased,while increa-sed in the S phase and G2/M phase(P<0.01).As compared to the control group,ARP could promote the secretion of NO in splenic lymphocytes and the secretion levels of IL-2,IL-4,IL-6,IFN-y were higher stimulated,the mRNA levels of IL-2,IL-4,IL-6,IFN-?,TNF-?,T-bet,GATA3 quantified by RT-PCR were significantly increased by ARP(P<0.01).2.Effects of ARP on the immune activities of RAW264.7 cells in vitro:The results of MTT method to RAW264.7 cells were showed that ARP could increase the OD value and the value was significantly increased when the concentration of ARP reached to 25?g/mL,compared with control group.The apoptosis rate of RAW264.7cells tested by Flow cytometry was declined by ARP.The phagocytosis function of RAW264.7 cells could significantly improved by ARP,and the OD value was significantly increased when the concentration of 50?g/mL is reached.As compared to the control group,the secretion of NO in RAW264.7cells detected by Griess method was accelerated when the ARP concentration reached to 25?g/mL.Results of ELISA indicated that ARP also could promote the TNF-?,IL-1?and IL-10 secretion in RAW264.7cells(P<0.01).The mRNA expression of TNF-a,IL-6 and iNOS determined by RT-PCR were promoted by ARP in RAW264.7cells.However,the result of Western blot showed that the relative protein expression of I?B p65 in RAW264.7 cells cytoplasm was declined by ARP.Conclusions 1.ARP possesses strong immunoenhancement activity on murine splenic lymphocytes in vitro,the mechanism may be related to the regulation of cell cycle and prompt lymphocytes proliferation,or associated with the secretion and mRNA expression of IL-2,IL-4,IL-6,IFN-?,TNF-?,T-bet,GATA3.2.Cellular immune response in vitro of RAW264.7 cells was stimulated by ARP,which may due to the reduction the apoptosis rate of RAW264.7cells.and improvement its phagocytosis function,NO production,the secretion of TNF-?,IL-1?,IL-10 and mRNA expression of TNF-a,IL-6,iNOS,but down-regulated the protein expression of I?B p65. |
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