The Impact Of Hericium Erinaceus Polysaccharide On The TLR3-TRIF-TRAF6 Signaling Pathway In MDRV-infected RAW264.7 Cells

Muscovy duck reovirus?MDRV?is a virus,which is widely infectious and immunosuppressive,and has caused large damage to the Muscovy duck industry in recent years.Hericium erinaceus polysaccharide?HEP?,extracted from hericium erinaceus,is an important active polysaccharide.HEP can antagonise MDRV-induced immunosuppression in Muscovy ducks,while the detailed mechanism remains unknown.Toll-like receptors?TLRs?are a class of highly conserved proteins and involved in innate immunity,since TLR3 is a member of the TLRs and can bind to dsRNA viruses,TLR3-associated signaling pathway probably plays an important role in enhanced host resistance to live virus.This experiment was aimed to explore the impact of HEP on the TLR3-TRIF-TRAF6 signaling pathway in MDRV-infected RAW264.7 cells and further provide references for elaborating the mechanism of how to HEP regulate cells resistance to MDRV-induced immunosuppression.Methods:in the present study,safe concentrations of HEP were firstly determined on mouse monocyte-macrophage leukemia cells?RAW264.7 cells?by MTT assay,and then tissue culture infective dose(TCID₅₀)of MDRV-YB strain was also detected in RAW264.7 cells.Cells were divided into 5 groups as follows:HEP group,HEP prevention group,HEP treatment group,MDRV-YB₄ infection group as well as blank control.All the samples were collected at 12 and 24 hour post-infection?hpi?,respectively,real-time PCR was performed to examine transcriptional level of TLR3 gene,and Western blot was used to determine the expression of TLR3,TRIF and TRAF6 protein,and ELISA was utilized to detect the contents of TNF-a,IL-10,IL-6,IL-1? and IFN-? in each groups?24 hpi?.Results:?1?HEP can contribute the growth of RAW264.7 cells in vitro when the concentration of HEP was in the range of 46.88ug/mL to 187.5ug/mL;?2?MDRV-YB₄,whose TCID₅₀ was 10-3.46/0.1 mL,can infect and replicate in the RAW264.7 cells.?3?Real-time PCR results showed that MDRV-infected RAW264.7 cells displayed an extremely significant?p<0.01?increase in relative expression of TLR3 mRNA in comparison with blank control,when compared with MDRV-YB₄ infection group,TLR3 mRNA levels of HEP prevention group and HEP treatment group were pulled down at an extremely significant degree?p<0.01?.?4?As expected,Western blot analysis showed that TLR3 as well as TRIF,TRAF6 proteins were increased extremely significant?p<0.01?in comparison with blank control,likewise,prevention or treatment with HEP groups could reverse the increasing trend at an extremely significant degree?p<0.01?.?5?Downstream product of TLR3-associated signaling pathway,TNF-a,IL-10,IL-6,IL-1? and IFN-? were detected with ELISA,results showed that all above indexes were jaggedly promoted at 24 hpi when compared with blank control,expectably,all above indexes in HEP prevention or treatment group decreased at different degrees in comparison with blank control,with one exception for further increase of IFN-?.Conclusions:considering all obtained results,we concluded that HEP could suppress the excessive activation of TLR3 expression at mRNA and protein levels during early phase of MDRV-YB₄ infection,the similar regulation of HEP was observed on TRIF and TRAF6.Additionally,HEP could antagonize the overexpression of downstream product of TLR3-associated signaling pathway?TNF-a,IL-10,IL-6,IL-1??and up-regulated IFN-? expression,thus playing useful part in antiviral activity.