| Background:Since Russel Ross developed his popular "response to injury" hypothesis of atherogenesis, the view that atherosclerosis is indeed a chronic inflammatory disease initiated by monocyte/lymphocyte adhesion to activated endothelial cells (EC) is now widely accepted and substantiated by experimental and clinical observations. Inflammatory is implicated in the formation of early fatty streaks, when the endothelium is activated and expresses chemokines, including monocyte chemotactic protein MCP-1 and interleukin IL-8, and adhesion molecules, including intercellular adhesion molecule ICAM-1, vascular adhesion molecule VCAM-1, leading to monocyte/ lymphocyte recruitment and infiltration into the subendothelium . FKN, a new discovered chemokine CX3CL1(FKN) which is over expressed in atherosclerosis contributes to platelet activation and increased thrombogenesis in vascular diseases. Combination with its receptor CX3CR1 mostly expressed on monocytes, lymphocytes, and natural killer (NK) cells the FKN can inhance the NK cell-mediated endothelium damage, which may result in vascular injury. At the same time, FKN can also mediate capture , firm adhesion and migration of monocytes and lymphocytes to the vascular endothelial cells under physiologic flow conditions. Up to now,studies at foreign country merely demonstrated that FKN stimulated angiogenesis by activating the MAPK system and PI3K signal pathways via the G protein-coupled receptor CX3CR1, but the underlying mechanism has not been exclu-dated. The signal transduction pathways of FKN/CX3CR1 in atherosclerosis is PKC/NF-ΚB/TNF-αand PI3K/NF-ΚB/TNF-αsignal pathways at domestic.It was studied by my supervisor professor.This experiment to be adopted the ELISA and Western blot and Zymographic method of molecular biology detection means,through research the role of PKC, ERK, TNF-αand MMP-2 in the FKN-CX3CR1 affect atherosclerosis First exploration that the possible existence of a cell signal transduction mechanisms of FKN-CX3CR1 and the relationship between FKN and MMP-2, and explore the possible mechanism of .FKN in atherosclerosis.Objective:(1) To investigate the sigal transductive mechanism of FKN/CX3CR1 affecting atherosclerosis;(2)To research the function of ERK,TNF-αand MMP-2 in the signal transduction mechanism of FKN /CX3CR1 affecting atherosclerosis and mutual relationship.Methods:(1)Peripheral blood monocytes(PBMC) were isolated from fresh blood of healthy volunteers by Ficoll-Paque gradient centrifugation;(2) The extractive PBMC were div-ided into four groups :control group,FKN group,FKN+Ro31-8220group,FKN+PD98059 dithiocarbamate (PDTC) group and FKN+captopril group.Each group was divided into two shares. We meas-ured phospho-ERK in one share using western blot analysis after 45min of incubation with FKN, collected culture liquid of the other share and evaluated TNF-αexpression by enzyme-linked immuno-sorbent assay(ELISA) and MMP-2 expression by gelatinase zymography after 24 h of incubation with FKN.We also detected FKN-mediated phospho-ERK and TNF-α,MMP-2 level in PBMC pretreated with PKC inhibitor Ro31-8220,ERK inhibitor PD98059;(3) The expressiong of ERK in monocytes of each group were detected by western blotting;(4)Culture solution of each group were collected, and detect the concentration of TNF-αby euzyme-linkedimmunosorbent assay (ELISA) and MMP-2 expression by gelatinase zymography.Result:1.Effect of fractalkine on ERK activation expression in human PBMC : In this experiment, observed that the FKN induced stimulation, mononuclear cells, the expression of phosphorylated ERK increased significantly increased over the control group, there were significant differences (P <0.05 ). Therefore, FKN within target cells can be induced ERK activation, FKN involved in atherosclerosis mechanisms may be related to the activation of ERK.2.Effect of fractalkine on TNF-α,MMP-2 expression in human PBMC:In this experiments,FKN stimulation of the cell culture medium and TNF-αconcentrations than control group increased significantly, there was significant difference (P<0.05);The group FKN stimulate cell culture medium of MMP-2 concentration than the control group decreased significantly, the difference was significant (P <0.05).Therefore, FKN and CX3CR1 promote mononuclear cells TNF-αsynthesis, and inhibit the expression of MMP-2, Fractakline possible through the promotion of TNF-α,involved in atherosclerosis in the inflammatory response.3.Effect of ERK on TNF-αand MMP-2 expression in human PBMC induced by FKN: The experimental use of the specificity of ERK inhibitor PD98059 and the role of mononuclear cells after induction by adding FKN. The results, PD98059 group of TNF-α,MMP-2 concentration than FKN stimulate group decreased significantly (P <0.05). Description after blocking ERK activation can reduce TNF-α,MMP-2 Synthesis, FKN possible through the pathway of ERK induced the synthesis of TNF-α,MMP-2 to participate in the inflammatory response.4. Effect of PKC on ERK expression in human PBMC induced by FKN: In this experiment, a specific inhibitor of PKC activity Ro31-8220 and the role of monocyte blocking PKC signaling pathway, and then to join the FKN induced ERK.Western Blot detection expression. The results, Ro31-8220 group ERK expression was significantly less than FKN stimulation, the difference was significant (P <0.05). Note FKN-induced ERK activation in part dependent on PKC signaling pathways, PKC may be upstream of ERK activation signal.5. Effect of PKC on TNF-αand MMP-2 expression in human PBMC induced by FKN: Collection medium, respectively gelatin enzyme assay and ELISA supernatant the concentration of TNF-αand MMP-2. The results, TNF-αconcentration of PKC inhibitor than FKN stimulation reduced, the difference was significant (P<0.05). PKC inhibitor group MMP-2 concentration than FKN stimulation significantly increased, the difference was significant (P <0.05). Therefore, PKC blocker can reduce TNF-αsynthesis, and promote MMP-2 synthesis. FKN may be through PKC ways to promote the synthesis of TNF-α, and inhibit MMP-2 synthesis.Conclusion: (1) FKN /CX3CR1 may increase the expression of ERK and the synthesis of TNF-αin PBMC as signal tranductive mechanisms whichcontribute to the progression of atherosclerosis; (2) Protein PKC play an important role during the process that PBMC synthesize ERK,TNF-αinduced by FKN /CX3CR1;(3)ERK may promote the expression of TNF-αand MMP-2 induced by FKN /CX3CR1;(4) FKN-CX3CR1 might PKC pathway on mononuclear cells in the expression of MMP-2 inhibited,; At the same time, FKN-CX3CR1 possible through ERK ways to promote mononuclear cells in the expression of MMP-2,but,overall, FKN-CX3CR1 of mononuclear cells the expression of MMP-2 inhibited.;(5) FKN/CX3CR1 affect atherosclerosis signal transduction mechanisms in the mononuclear cells may be realized through the following cascade: FKN/CX3CR1→→PKC→→ERK→→TNF-α→→AS the formation and development.
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