Immune Enhancement Effect And Its Mechanism Of Three Components Of Mongolian Milkvetch Root In Macrophage RAW264.7 Of Mice

Objective The effects of ASIV,APS and FAC on the proliferation,the contents and gene expression of cytokines,secretion of surface stimulating factors,cell cycle,the expression level of the NF-?B/MAPK signal pathway in RAW264.7 cells.Methods RAW264.7 cells,which were in the logarithmic growth phase,were chosen and treated with 0,25,50,100 ?g/mL ASIV,0,25,50,100 ?g/mL APS and 0,0.1,1,10 ?g/mL FAC with or without the inhibitor of NF kappa B PDTC,respectively.The most suitable concentration of the drug and the cell activity were determined by CCK-8.The contents of IL-6,IL-1?,TNF-?,NO and the mRNA expression levels of IL-6,IL-1?,TNF-?,iNOS were determined by ELISA and RT-PCR,respectively.The secretion of CD40,CD80 and CD86 and cell cycle were determined by flow cytometry.The protein expression levels of p50,p65,p-p65,p38,p-p38,ERK,p-ERK,JNK,p-JNK protein in ASIV treatment groups and the protein expression levels of p50,p65,p-p65 in APS treatment groups were determined by western blotting,the protein expression of p65 in the nucleus was determined by EMSA.Results:1.Effect of ASIV on the immune function of RAW264.7 cellsCompared with control group,the contents of IL-6,IL-1?,TNF-?,NO and the mRNA expression levels of IL-6,IL-1?,TNF-?,iNOS in 100 ?g/mL ASIV treatment significantly increased(p<0.01).The contents of IL-6 in 50 ?g /mL ASIV treatment group significantly increased(p<0.01)and mRNA expression levels of IL-1?,TNF-? and iNOS increased or significantly increased(p<0.05 or p<0.01).The contents of IL-6,IL-1?,TNF-?,NO and the mRNA expression levels of IL-6,IL-1?,TNF-?,iNOS in 100 ?g/mL ASIV+ PDTC treatment group significantly decreased than that in 100 ?g/mL ASIV group(p<0.05 or p<0.01).ASIV promoted the secretion of CD40 and CD86,increased the number of cells in G2/M phase to promote cell proliferation.Compared with control group,the ratio of p-p65/p65 in 25 ?g/mL ASIV treatment group,the ratios of p50/?-actin,p-p65/p65,p-p38/p38 protein in 50 ?g/mL ASIV treatment group significantly increased(p<0.01),the ratios of p-ERK/ERK and p-JNK/JNK significantly increased(p<0.01)in 100 ?g/mL ASIV treatment group,while PDTC could significantly reduce the increasing effect of ASIV.2.Effect of APS on the immune function of RAW264.7 cellsCompared with control group,the contents of IL-6,IL-1?,TNF-?,NO and the mRNA expression levels of IL-6,IL-1?,TNF-?,iNOS in 100 ?g/mL APS treatment significantly increased(p<0.01).The contents and mRNA expression levels of IL-6 and the mRNA expression levels of IL-1? in 50 ?g/mL APS treatment group significantly increased(p<0.01).The mRNA expression levels of TNF-? and iNOS decreased or significantly decreased in PDTC treatment group(p<0.05 or p<0.01).The contents of IL-6,NO and the mRNA expression levels of IL-6,IL-1?,TNF-?,iNOS in 100 ?g/mL APS+PDTC treatment group decreased or significantly decreased than that in 100 ?g/mL APS treatmeng group(p<0.05 or p<0.01).With the increase of APS concentration,the secretion of CD40 and CD86 increased,while the PDTC decreased the secretion.The number of cells in the G2/M phase increased in 100 ?g/mL APS treatmeng group.Compared with control group,the expression of p65 in the nucleus in 25 ?g/mL APS treatment group increased(p<0.05),the expression of p65 in the nuclues in 50 ?g/mL and 100 ?g/mL APS treatment group significantly increased(p<0.01).Compared with 100 ?g/mL APS treatment group,the expression of p65 in the nuclues significantly decreased in 100 ?g/mL APS+PDTC treatment group(p<0.01).3.Effect of FAC on the immune function of RAW264.7 cellsCompared with control group,the contents of IL-6,IL-1?,TNF-?,NO and the mRNA expression levels of IL-6,IL-1?,TNF-?,iNOS in 1 ?g/mL and 10 ?g/mL FAC treatment groups significantly increased(p<0.01).The contents of NO and the mRNA expression levels of TNF-?,iNOS in PDTC treatment group decreased or significantly decreased(p<0.05 or p<0.01).The contents of IL-6,IL-1?,TNF-?,NO and the mRNA expression levels of IL-6,IL-1?,TNF-?,iNOS in 10 ?g/mL FAC+ PDTC-treatment group significantly decreased than that in 10 ?g/mL FAC treatment group(p<0.01).FAC promoted the secretion of CD40 and CD86,whereas PDTC inhibited the promoting effect.FAC had a slight increase effect in the number of cells in the G2/M phase.Compared with the control group,with the increase of FAC concentration,the expression of p50 significantly increased in 0.1 ?g/mL FAC treatment group(p<0.01)and the expression of p50 in 10 ?g/mL FAC treatment group significantly decreased(p<0.01),while PDTC could further promote this reducing effect.Compared with control group,the p-p65/p65 ratio in 0.1 ?g/mL FAC treatment group significantly increased(p<0.01).Compared with 10 ?g/mL FAC treatment group,the p-p65/p65 ratio in PDTC+10 ?g/mL FAC treatment group significantly decreased(p<0.01).Conclusion ASIV by activating the NF-?B /MAPK signal pathway,APS and FAC by activating the NF-?B signal pathway improved immune function in RAW264.7 cells.