Mechanism And Effect Of IL-1 On The ACAT-1 Expression In The Mononuclear Cells Differentiation To Foam Cells

Atherosclerosis(AS)is artery disease of chronic inflammation, related to multiple risk factors including high blood pressure,diabetes, smoking and lipoprotein abnormalities,etc.The important early pathological change of atherosclerosis is recruitment of blood mononuclear cells(monocyte,MC)to the vascular endothelial cell dysfunction.Then MCs differentiate into macrophages(macrophage cell, MP),and uptake lipid,contributes to foam cell formation.FC is the main reason participating in formation of the fatty streak forming in the vascular wall in the early stage of atherosclerosis.Cholesterol esters(CHE)are synthesized in an intracellular reaction catalyzed by acyl coenzyme A(CoA):cholesterol acyltransferase(ACAT) enzymes.Cholesterol ester,the main storage form of cholesterol(CH),is the major lipid in FC lipid droplets.In cells,it is that ACAT catalyzes the formation of cholesteryl esters from cholesterol.When CH in cells reaches a certain amount,ACAT,a intracellular enzyme,catalyzes the formation of cholesteryl esters from cholesterol,so as to avoid the toxic effect of high concentration of CH on cells.But in pathological conditions,the abnormal increase of ACAT activity,which precede the MP excessive lipid uptaking,promote a large number of CHE accumulation in MPs.The abnormal increase of ACAT activity plays a key role in the early FC formation.Therefore,Researchers attaches great importance to the role of ACAT in atherosclerosis.And ACAT has become one of the hot areas of atherosclerosis mechanism.Since the ACAT1 gene was first identified in 1993,two ACAT genes have been identified in mammals:ACAT1 and ACAT2.There are a lot of progress in the ACAT research,particularly in the gene structure and recombinant expression.ACAT1 exists in almost all human tissues,while ACAT2 only distributes in the liver and intestine.Recent research shows that ACAT1 activity,related to the formation of atherosclerosis,is markedly up-regulated in the mononuclear cells differentiation to foam cells.But so far,up-regulation mechanism of ACAT activity is not very clear up to now.We need further studying in the future.In the development of Atherosclerosis,there are various pro-inflammatory cytokine,such as IL-1,IL-6,IL-8(interleukin-1,6,8), tumor necrosis factor-a(TNF-a),interferon-γ(INF-γ),and so on, secreted from macrophages,vascular smooth muscle cells(VSMC)and the endothelial cells.These cytokine promote blood vessel inflammation; result in vascular smooth muscle proliferation and migration,lipid accumulating and endothelial dysfunction.It has been reported that IL-1, TNF-a can increase intracellular ACAT respectively.It is speculated that IL-1 may be an important trigger of ACAT up-regulation in varying stages of lipid overloading.However,this speculation has yet to be confirmed.IL-1 is the classic regulator,switching on inflammation. Macrophages cells can synthesize IL-1,which up-regulate ACAT mRNA expression at the transcriptional level,promote cholesterol esterification and deposition,VSMC transformation into FC,and participate in the formation and development of atherosclerosis.But the mechanism is not clear.It has been confirmed interleukin induce inflammatory response through mitogen-activated protein kinase(MAPK)signaling pathway; Raf-1,MAPK upstream oncogene product,can be phosphorylated by protein kinase C(PKC)directly,thereby activates ras-mediated MAPK activation.This paper intends to investigate the course of MC/MP into FC, using cell culture and molecular biology techniques.Aim to address the following issues:①IL-1 is the initiation factor switching on ACAT up-regulation in MP lipid overloading;②Measuring cells ACAT mRNA levels,protein content and enzyme activity in the mononuclear cells differentiation to foam cells,to clear up the role of ACAT link;③IL-1 up-regulates the intracellular signal transduction pathway of ACAT-1 through PKC/MAPK signaling pathway. Part 1 Effects of IL—1 on the ACAT-1 mRNA expression in the mononuclear cells differentiation to foam cellsObjectiveTo explore the effects of ACAT-1 mRNA expression in the differentiation from the mononuclear cells to the foam cells.Methods1.The mononuclear cells line THP-1 was induced resusci- tation and cultured;2.The mononuclear cells line THP-1 were cocultured with phorbol myristate acetate and induced into differentiation to macropahges.The reverse microscope was applied to detected cell morphology.Flow cytometry was employed to examine CD14 expression and to ensure mononuclear cells differentiation to macrophages.3.The macropahges were cultured with oxidized low density lipoprotein and induced into differentiation to the foam cells.The reverse microscope was applied to detected cell morphology,and oil red O staining was employed to examine cytoplasm lipid deposition and to ensure macrophages differentiation to the foam cells.4.RT-PCR assay were employed to examine the expression of ACAT-1 mRNA in different groups(the mononuclear cells group,the macrophages group,the foam cells group,the foam cells IL-1 group,the foam cells IL-1/IL-1 monoclonal antibody group)and to explore the influence of IL-1/IL-1 mAb on ACAT-1 mRNA expression.Results1.The mononuclear cell line THP-1 presented a suspension growth pattern after resuscitation,and showed a significant changes after 24 hours induction by PMA including the adhensive growth pattern, ovalisation shap,obvious enlarged volume,puffing of the cytolymph, increased cellular organelle and the pseudopod formation at the cellular membrane.The mononuclear cells line THP-1 expressed the CD14 with a 90%positive rate after induced by PMA.2.The macrophages presented an enlarged volume,adhensive growth pattern and the puffed cytolymph.Some red stained substance could be found in the cytolymph,indicating that the the macrophages have differentiated into foam calls.3.The expressions of ACAT-1 mRNA in the foam cells group and the foam cells IL-1 group are higher than in the mononuclear cells group and the macrophages group,especially in the foam cells IL-1 group. Compared the foam cells IL-1 group with the foam cells IL-1/IL-1 monoclonal antibody group,ACAT-1 mRNA expression of the latter were significantly decreased.Conclusion1.The mononuclear cells line THP-1 could differentiated to macrophage and foam cells,during which ACAT-1 mRNA expression increased,after induction by PMA and ox-LDL.2.IL-1 can up-regulate the expression of ACAT-1 mRNA during the mononuclear cells differentiation into the foam cells,and IL-1 mAb may retard the effect.3.The up-regulation effects of IL-1 on the ACAT-1 mRNA expression,which accelerated the formation of foam cell,presented in the experiment.IL-1 may be the initiation factor switching on ACAT up-regulation in MP lipid overloading. Part 2 Effects of IL—1 on the ACAT-1 protein expression and activity in the mononuclear cells differen tiation to foam cellsObjectiveTo explore the Effects of IL—1 on the ACAT-1 protein expression and activity in the mononuclear cells differentiation to foam cellsMethods1.The mononuclear cells line THP-1 was induced resuscitation and cultured;2.The mononuclear cells line THP-1 were co-cultured with phorbol myristate acetate and induced into differentiation to macrophages.The reverse microscope was applied to detected cell morphology.Flow cytometry was employed to examine CD14 expression and to ensure mononuclear cells differentiation to macrophages.3.The macrophages were cultured with oxidized low density lipoprotein and induced into differentiation to the foam cells.The reverse microscope was applied to detected cell morphology,and oil red O staining was employed to examine cytoplasm lipid deposition and to ensure macrophages differentiation to the foam cells.4.Western blotting assay and liquid phase scintillation counting were employed to examine the expression of ACAT-1 protein and ACAT-1 activity in different groups(the mononuclear cells group,the macrophages group,the foam cells group,the foam cells IL-1 group,the foam cells IL-1/IL-1 monoclonal antibody group).Results1.IL-1 can up-regulate the expression of ACAT-1 protein during the mononuclear cells differentiation into the foam cells.2.The expressions of ACAT-1 protein in the macrophages group,the foam cells group and the foam cells IL-1 group are higher than in the mononuclear cells group,especially in the foam cells IL-1 group.3.The up-regulation effects of IL-1 on the ACAT-1 protein expression and ACAT-1 activity will be remarkably decreased after IL-1 mAb added.Conclusion1.Without inflammatory cytokine,the expression of ACAT-1 protein increased during the mononuclear cells differentiation into the foam cells. with IL-1,the expression of ACAT-1 protein further increased,and IL-1 mAb may resist the effect.2.The effects of IL-1 on the ACAT-1 activity were accomplished by means of up-regulation of the expression of ACAT-1 mRNA transcription and ACAT-1 protein during the mononuclear cells differentiation into the foam cells. Part 3 Mechanism of signaling pathway of IL—1 on the expression of ACAT-1 in the mononuclear cells differentiation into the foam cellsObjectiveThe paper aims to discuss the mechanism of IL-1 on the expression of ACAT-1 in the mononclear cells differentiation into the foam cells and to explore the signaling pathway—whether by PKC/MAPK or not.Method1.The mononuclear cells line THP-1 was induced resuscitation and cultured,and PKC and MAPK activity was detected;2.The mononuclear cells line THP-1 were cocultured with phorbol myristate acetate.The reverse microscope was applied to detected cell morphology,and Flow cytometry was employed to examine CD14 expression,and PKC and MAPK activity was detected.3.The macropahges were cultured with oxidized low density lipoprotein for 24 hours.The reverse microscope was applied to detected cell morphology,and oil red O staining was employed to examine cytoplasm lipid deposition,and PKC and MAPK activities were detected.4.PKC and MAPK activity was detected in two different groups(the foam cells IL-1 group,the foam cells IL-1/IL-1 monoclonal antibody group). 5.PKC and MAPK inhibitors were separately added to the different groups(the mononuclear cells group,the macrophages group,the foam cells group,the foam cells IL-1 group,the foam cells IL-1/IL-1 monoclonal antibody group).RT-PCR assay were employed to examine the expression of ACAT-1 mRNA,and Western blotting assay and liquid phase scintillation counting were employed to examine the expression of ACAT-1 protein and ACAT-1 activity in different groups.Result1.The PKC and MAPK activities in the macrophages group,the foam cells group and the foam cells IL-1 group are higher than in the mononuclear cells group.2.After added PKC and MAPK inhibitors,the effects of IL-1 on the expression of ACAT-1 mRNA were remarkably decreased.3.After added PKC and MAPK inhibitors,the effects of IL-1 on the expression of ACAT-1 protein and the activity of ACAT-1 were markedly reduced.ConclusionPKC/MAPK signaling pathway could be involved in the IL-1 up-regulation effects on the ACAT-1 mRNA expression,the ACAT-1 protein expression and the ACAT-1 activity in the mononuclear cells differentiation into the foam cells.