Effect Of Fractalkine On Akt Activation And TNF-α Expression In Human PBMC And The Intervention Of Captopril

Recent research has shown that inflammation plays a key role in a therogenesis.Fractalkine(FKN),a recently discovered chemokine o- verexpressed in atherosclerotic plaque,binding to CX3CR1 on tar- get cells,promotes leukocyte adhesion and recruitment,vascular sm- ooth muscle cell(VSMC) migration, inflammatory factor expressi- on, as well as platelet activation,so FKN is thought to take part in inducing inflammation of atherosclerosis(AS). Protein kinase B (PKB,Akt),signal molecule tightly concerned with inflammation and chemotaxis of leucocytes,exerts effects in atherogenesis by mediating leucocyte adhesion and chemotaxis,VSMC migration a- nd activation of platelet.Tumor necrosis factor-α(TNF-α),one of in- flammatory mediators,damaging vascular endothelial cells, promot- ing endothelial cells to express adhesive and chemotactic factors, s- timulating VSMC proliferation,destructing coagulation-anticoagul- ation balance,producing pro-inflammatory and pro-coagulatory eff- ect,plays an important role in formation and progress of AS.Recen- tly,Angiotensin converting enzyme inhibitor(ACEI) have antiather- osclerotic effects,including antiinflammatory antithrombosis, and a-ntioxidative effect except basic antihypertensive, myocardial and v- ascular hypertrophy reversing effect.Research have suggested that abundant foam cells derived from monocytes and activated T cells exist in atherosclerotic plaque, monocyte recruitment into intima is the key step of AS,foam cells and lymphocytes take part in partial inflammation in plaque, and peripheral blood monouclear cell (PB- MC) are made up of monocyte and lymphocyte both expressing F- KN receptor CX3CR1. We associated FKN,Akt,TNF-αas well as PBMC,studied relationship of them and anti-inflammatory mecha- nism of captopril.ObjectiveThe aim of the study is to investigate the inflammatory and signal transduction mechanisms of FKN in atherogenesis and the anti-in- flammatory mechanism of captopril.(1)to examine the effect of F- KN on Akt activation and TNF-αexpression in PBMC.(2) to inv- estigate the influence of PI3K and NF-κB on Akt activation and TNF-αexpression in human PBMC. (3)to research the the interv- ention of captopril on Akt activation and TNF-αproduction in hu- man PBMC mediated by FKN.MethodsPBMC were isolated from fresh blood of healthy volunteers by F- icoll-Paque gradient centrifugation,the extractive PBMC were div- ided into five groups :control group,FKN group,FKN+LY294002 group,FKN+pyrrolidine dithiocarbamate (PDTC) group and FKN+ captopril group.Each group was divided into two shares. We meas- ured phospho-Akt in one share using western blot analysis after 45 min of incubation with FKN, collected culture liquid of the other share and evaluated TNF-αexpression by enzyme-linked immuno- sorbent assay(ELISA) after 24 h of incubation with FKN.We also detected FKN-mediated phospho-Akt and TNF-αlevel in PBMC pretreated with PI3K inhibitor LY294002,NF-κB inhibitor PDTC, as well as captopril .ResultsPhospho-Akt and TNF-αlevel of FKN group enhanced compared with control group(0.583±0.131 vs 0.082±0.020,P<0.05; 3053.78±427.25 pg/mL vs 357.52±137.64 pg/mL,P<0.05).TNF-αlevel of FKN+LY294002 group reduced compared with FKN group(1026. 56±234.73 pg/mL vs 3053.78±427.25 pg/mL,P<0.05). Phospho-Akt and TNF-αlevel of FKN+PDTC group decreased compared w- ith FKN group(0.307±0.064 vs 0.583±0.131,P<0.05 ;721.33±145. 22 pg/mL vs 3053.78±427.25 pg/mL,P<0.05).Phospho-Akt and T- NF-αlevel of FKN+captopril group reduced compared with FKN group(0.296±0.005 vs 0.583±0.131,P<0.05 ; 1658.11±221. 70 vs 3053.78±427.25 pg/mL,P<0.05).Difference of Phospho-Akt level between FKN+LY294002 group and FKN group were not signific- ant in statistics(0.693±0.059 vs 0.583±0.131,P>0.05). Phospho-Akt and TNF-αexpression correlated tightly with each other(r=0.552, p<0.05).Conclusion(1)FKN participates inflammation in atherogenesis by activating Akt and upreguleting TNF-αin human PBMC.(2) PI3K is not the upstream signal of Akt activation promoted by FKN in human P- BMC.(3)NF-κB is involved in Akt activation of human PBMC i- nduced by FKN.(4)PI3K and NF-κB mediates the expression of TNF-αin human PBMC stimulated by FKN.(5)Akt activation is closely related with TNF-αexpression of human PBMC induced by FKN.(6)Captopril may exert antiatherosclerotic effects by preventi-ng FKN-induced Akt activation and TNF-αexpression in human P- BMC.