Activation Effect of Human TLR4 Signaling Pathway by Polysaccharide from Phellinus Igniarius

OBJECTIVE To observe the effect of polysaccharide from Phellinus igniarius (PPI) on human Toll like receptor 4 (TLR4) pathway.METHODS The activation of TLR4 pathway was evaluated rapidly by using HEK-Blue^TM hTLR4 cells transfected with human TLR4 gene and secreted embryonic alkaline phosphatase reporter gene, and the PMA stimulated- THP-1 cells were used to observe the effects of drugs on the TLR4 pathway. The cell viability was detected by MTT assay. The cytokine secretion was evaluated by ELISA. mRNA expression level of the cells was analysed by Real-time PCR. RESULTS The treatment of PPI for 24 h did not affect cell viability but dose-dependently increased the expression of secreted embryonic alkaline phosphatase in HEK-Blue^TM hTLR4 cells. In addition, the treatment of PPI leaded to increased secretion of TLR4 pathway-associated cytokines such as TNF-α and IP-10 in THP-1 cells. The TLR4 receptor blocker TAK-242 significantly reduced PPI-induced cytokine secretion (P<0.01). The results of Real-time PCR showed that PPI not only increased the mRNA expression of cytokines such as TNF-α, IL-6, IL-12, IL-1β and COX-2 in MyD88 pathway, but also increased the mRNA expression of cytokines such as IP-10 and IFN-β in TRIF pathway. CONCLUSION PPI is an agonist of the human TLR4, it can activate both MyD88 and TRIF branches on the downstream of TLR4 and show no selective activation of the two TLR4 branching pathways.