Preliminary Study On The Mechanism Of Fomes Officinals Polysaccharide In RAW264.7 Cell

Objective: To investigate the effect of crude Fomes Officinals polysaccharide(FOPS)and its components(FOPS-a)on Toll-like receptor 4(TLR4)mediated Nuclear factor-?Bp65(NF-?Bp65)and Mitogen-activated Protein Kinases(MAPK)signaling pathway in macrophage RAW264.7,and to explore FOPS and FOPS-a immunomodulatory mechanism effects in macrophage RAW264.7.Methods: 1)Use the method of Real-Time quantitative PCR to detect the effect of gene mRNA expression on TLR4?NF-?Bp65?iNOS?TNF-??IL-1??IL-6?and MAPK signaling pathways(P38?ERK and JNK)by different concentrations(50,100,200?g/mL)of FOPS and FOPS-a.2)By Western Blot,to inspect the effect of signal transduction protein expression and phosphorylation levels on the amount of TLR4?MyD88?TRAF6?NF-?B(NF-?B p65?I?b?)?MAPK(P38?ERK and JNK)by FOPS and FOPS-a.3)After pretreatment of cells with NF-?B inhibitor BAY11-7082 and MAPK inhibitor PD98059?SP600105?SB203580,the effects of TNF-?,IL-1? and IL-6 secretion in supernatant were detected by ELISA.Results: 1)The results of qRT-PCR showed that compared with the blank control group,FOPS and FOPS-a could significantly up-regulated the relative mRNA expression of TLR4?p65?ERK and P38 genes,and increase the gene content of iNOS?TNF-??IL-1? and IL-6.2)Western Blot results showed that compared with the blank control group,FOPS and FOPS-a could significantly up-regulated the protein expression levels and phosphorylation level of TLR4?MyD88?TRAF6?p-NF-?B p65?p-I?b??p-P38?p-ERK.3)Blocking NF-?B signaling pathway could significantly inhibit the secretion of FOPS and FOPS-a on TNF-? ? IL-1? and IL-6;Blocking MAPK signaling pathway can significantly inhibit the activation of ERK ? P38 and JNK pathway by FOPS and FOPS-a,and can significantly decrease the secretion of TNF-? by macrophage RAW264.7 induced by FOPS and FOPS-a.Conclusion: One of the mechanism by which FOPS and FOPS-a immune reaction on RAW264.7 macrophage is activating the TLR4_MyD88_TRAF6_NF-?B/MAPK signaling pathway.The process of FOPS and FOPS-a induced macrophage release of cytokines such as TNF-??IL-1? and IL-6 is related to NF-?B and MAPK signaling pathway.