Effects Of FKN On ERK Activation And TNF-α,MMP-2 Expression In Human PBMC And The Role Of Syk

Background: Recent research has shown that inflammation plays a key role in atherogenesis. CX3CL1 (fractalkine), the only member of the subclass of chemokines, is a known chemotactic factor for monocytes /macrophages as well as NK cells and T lymphocytes. In several pathologies, excessive production of CX3CL1 at specific sites leads primarily to monocyte/macrophage recruitment, which causes tissue and vascular damage. TNF-αand MMPS are prototypic inflammatory mediators produced by various cell types, including mast cells, macrophages, lymphocytes, and fibroblasts. Elevated serum levels of TNF-αand MMPS are associated with the pathophysiology of atherogenesis.Despite their clinical relevance, the mechanisms underlying monocyte/macrophage chemotaxis to CX3CL1 remain poorly documented. The present report addresses this issue and identifies cell signaling crucial for this process. Using the MonoMac6 cell line in response to soluble CX3CL1, Cambien et al. have identified PI3K and members of the MAPK family, ERK1/2, p38MAPK, and JNK1, as signaling components required for cell adhesion to fibronectin. Activation of Src and phosphorylation of Syk tyrosine kinases downstream of CX3CR1 have also been reported, although a putative functional role for these enzymes in any CX3CL1-induced cell response has not been investigated.Objective:The aims of the study are as follows: (1) To research the effects of FKN on ERK activation and TNF-α,MMP-2 expression in PBMC. (2) To research the function of the spleen tyrosine kinase in the signal transduction mechanism of FKN/CX3CR1 affecting atherosclerosis. (3) To research the function of ERK in the the signal conductive mechanism of FKN/CX3CR1 affecting atherosclerosis. (4) To research the relations among of the FKN,TNF-α,MMP-2,Syk and ERK.Methods(:1)Peripheral blood monocytes were isolated from fresh blood of healthy volunteers by Ficoll-Paque gradient centrifugation.(2)Divided the extractive Peripheral blood monocytes into five groups:control group,FKN group,FKN+PD98059(ERK inhibitor) group, FKN+Piceatannol (Syk inhibitor) group, Syk inhibitor Piceatannol group.(3)Measure the phospho-ERK actived by monocytes from the first four groups of five by western blot method.(4)Collected the supernatant of monocytes from each group ,measured the expression of MMP-2 in monocytes from each group by SDS-page-Zymograph method. ( 5 ) Collected the supernatant of monocytes from each group, determined the expression of TNF-αby euzyme-linked immunosorbent assay(ELISA).Result: (1)The expressions of ERKand TNF-αof FKN group was increased compared with the control group . (2)The expression of MMP-2 of FKN group was decreased compared with the control group.(3)The expressions of ERK,MMP-2 and TNF-αof FKN+PD98059(ERK inhibitor) group were decreased compared with the FKN group.(4)The expression of ERK of FKN+Piceatannol(Syk inhibitor) group was decreased compared with the FKN group.(5)The expressions of MMP-2 and TNF-αof FKN+Piceatannol(Syk inhibitor) group were increased compared with the FKN group. (6)The expression of TNF-αof Syk inhibitor Piceatannol group was increased compared with the control group. (7)The expression of TNF-αof FKN+Piceatannol(Syk inhibitor) group was decreased compared with the Syk inhibitor Piceatannol group.Conclusion: (1)FKN/CX3CR1 increases the expression of TNF-αin Peripheral blood monocytes as one of the mechanism of contributing to the progression of atherosclerosis. (2)FKN/CX3CR1 decreases the expression of MMP-2 in Peripheral blood monocytes.(3)Extracellular signal-regulated kinase (ERK) plays an important role during the expression of TNF-αin FKN-induced. FKN/CX3CR1 probably initiates intracellular signal transduction mechanism through the process that FKN/CX3CR1 activates the ERK and then improves the expression of TNF-α. (4)Spleen tyrosine kinase (Syk) plays an important role during the process that Peripheral blood monocytes synthesize ERK induced by FKN/CX3CR1. FKN/CX3CR1 perhaps initiates intracellular signal conductive mechanism through the process that FKN/CX3CR1 activates the ERK by Syk and then improves the expression of TNF-α.