Effects Of FKN On The Expression Of P38 And NF-κB In Human PBMC And The Role Of PI3K

Coronary atherosclerotic heart disease(CAD)is one of the diseases with the highest morbidity and mortality all over the world. Atherosclerosis(AS) has already been known as the initial pathological change of CAD. The Pathogenesis of AS which is still under research would have far-reaching significance in the treatment and prevention of CAD. In recent years, a growing number of researches have focused on the inflammatory mechanisms of AS, and It has been proved by great amount of basic and clinical research that inflammation plays a vital role in the process of AS formation, development and rupture.Irregular chemokine (Fractalkine, FKN) is a newly discovered chemokine. FKN has the functions of adhesion and chemotaxis, without the participation of other adhesion molecule and selectin, which is different from other chemokines. FKN can promote the occurrence and development of AS by mediating leukocyte adhesion and chemotaxis, migration of vascular smooth muscle cell, promoting platelet activation, enhancing natural killer cell cytotoxicity and by increasing the expression of a variety of inflammatory factors. Thus, to improve the mechanisms of inflammation in AS, it will has great significance to study the mechanisms of signal transductions of FKN.Phosphatidylinositol-3 kinase (PI3K) is a kind of kinases which can specifically catalyze phosphatidylinositol 3-hydroxy phosphorylation, produce the second messenger—inositol lipid,so that it can transduct extracellular stimulate signal into the cell through the cell membrane. PI3K signaling pathway activated by many cytokines, growth factors and some physical and chemical factors can regulate a variety of cell functions, such as proliferation, apoptosis, metabolism, growth, transformation, vesicle transport, secretion, chemokine and so on. PI3K signaling pathway has been considered to be correlated to tumor occurrence and development, In recent years, it is indicated that atherosclerosis is closely related to PI3K.Mitogen-activated protein kinase (MAPK) is a cascade of protein kinase that can activate intracellular serine/threonine, which is important intracellular signal transduction pathway by transferring extracellular signal to intracellular signal in the eukaryotic. p38, as a member of MAPK family, can be activated by a variety of extracellular stressor, such as ultraviolet light, radiation, inflammatory cytokines, growth factors, osmotic pressure, heat shock, LPS and so on. Then, p38 can activate a variety of cytokines, inflammatory factors, transcription factors through the highly conserved 3 kinase "cascade" form of signal transduction.In recent years, it is showed that activation of p38 MAPK signaling pathway is one of the molecular biology mechanisms in the formation and progress of atherosclerosis.Nuclear factor- kappa B (NF-κB) is a transcription factor with functions of multi-gene transcription regulation,playing an important intermediary role in the inflammatory response. It mediates cell differentiation, proliferation, apoptosis and cytokine IL-6, IL-8, IL-10, TNF-αexpression,which are closely related to the formation of atherosclerosis. It is revealed that the expression of NF-κB is found in endothelial cells, mononuclear cells, vascular smooth muscle cells in the development of atherosclerosis.Objective: To investigate effects of FKN on the expression of p38 and NF-κB in human peripheral blood mononuclear cells and the role of PI3K in the p38 and NF-κB activation, in order to further explore the molecular mechanism by which FKN participate inflammation and signal transduction in the formation and progress of atherosclerosis.Methods: (1)Peripheral blood monocytes(PBMC) were isolated from fresh blood of healthy volunteers by Ficoll-Paque gradient centrifugation under sterile conditions; (2) The extractive PBMC were divided into four groups: control group, FKN group, FKN+PI3K inhibitor (LY294002) group, FKN+p38 inhibitor (SB203580). We prepare three samples for each group and detect phosphorylation p38 and NF-κB in mononuclear cells by western blot in 30min; (3) Scanning film: optical density of protein was anlalyzed by Quantity One gel imaging and analysis system and relative optical density ratio of phosphorylation p38 and NF-κB was calculated accordingly.(4) Statistical methods.Result:1. Effect of fractalkine on activation of p38 in human PBMC: In this experiment, observed that the FKN induced stimulation, mononuclear cells, the expression of phosphorylated p38 increased significantly over the control group, there were significant differences (P<0.01). Therefore, FKN within target cells can be induced p38 activation, FKN involved in atherosclerosis mechanisms may be related to the activation of p38.2. Effect of fractalkine on activation of NF-κB in human PBMC: In this experiment, observed that the FKN induced stimulation, mononuclear cells, the expression of NF-κB increased significantly over the control group, there were significant differences (P<0.01). Therefore, FKN within target cells can be induced NF-κB activation, FKN involved in atherosclerosis mechanisms may be related to the activation of NF-κB.3. Effect of p38 on activation of NF-κB in human PBMC induced by FKN: The experimental use of the specificity of p38 inhibitor SB203580 and the role of mononuclear cells after induction by adding FKN. The results, SB203580 group of NF-κB than FKN stimulate group decreased significantly (P<0.01). Description after blocking p38 can inhibit activation of NF-κB. FKN induced possibly activation of NF-κB through the pathway of p38 to participate in the inflammatory response.4. Effect of PI3K on activation of p38 in human PBMC induced by FKN: In this experiment, a specific inhibitor of PI3K activity LY294002 and the role of monocyte blocking PI3K signaling pathway, and then to join the FKN induced NF-κB.Western Blot detection expression. The results, LY294002 group phosphorylated p38 was significantly less than FKN stimulation, the difference was significant (P<0.01). Therefore, FKN induced p38 activation in part dependent on PI3K signaling pathways.5. Effect of PI3K on activation of NF-κB in human PBMC induced by FKN: In this experiment, a specific inhibitor of PI3K activity LY294002 and the role of monocyte blocking PI3K signaling pathway, and then to join the FKN induced NF-κB.Western Blot detection expression. The results, LY294002 group NF-κB was significantly less than FKN stimulation, the difference was significant (P<0.01). Therefore, FKN induced NF-κB activation in part dependent on PI3K signaling pathways.Conclusion:1. FKN may induce activation of p38 and activation of NF-κB in PBMC as signal tranductive mechanisms which contribute to the progression of atherosclerosis;2. p38 may promote activation of NF-κB induced by FKN in PBMC;3. PI3K may promote activation of p38 and activation of NF-κB induced by FKN in PBMC;4. FKN affects atherosclerosis signal transduction mechanisms in the mononuclear cells may be realized through the following cascade:...