| Atherosclerosis,the main pathological basis of coronary artery disease,is extremely complicated.At present most scholars agree with endothelial injury hypothesis that,endothelial cells are damaged by many risk factors,leading to an increase in vascular permeability and subsequent lipid depositions in tunica intima;infiltrating monocyte-derived macrophages swallow the lipid and form foam cells.As the foam cells die,cholesterol released from foam cells forms the lipid-rich core,macrophages in plaques secrete matrix metalloproteinases which degrade extracellular matrix and weaken the thin fibrous cap,gradually causing vulnerable plaque formation.Thus,macrophages play a vital role in the process of atherosclerosis.CD147 molecule,belonging to the immunoglobulin superfamily,is one member of transmembrane glycoprotein widely expressed on various cells in human body and can induce expression of matrix metalloproteinases(MMP).MMP can degrade extracellular matrix which is the main component of the atherosclerotic plaque,thereby promoting instability of the plaque.Homocysteine(Hcy)was identified as a new independent risk factor for atherosclerosis in the 1990s.Many international research findings have demonstrated that Hey can enhanced expression of MMP in many cells,but its mechanism has not yet been fully elucidated,and there are few reports about whether Hey can upregulate MMP expression through CD147-mediated pathway involved in the development of atherosclerosis.Statins not only have a strong lipid-lowering effect,and can stabilize and reverse artery atheromatous plaques.It is documented that statins can inhibit the expression of CD 147 and MMP in atherosclerotic plaques,Hcy is also a risk factor for atherosclerotic plaque progression,however,it remains rather unclear whether statins inhibit CD 147 expression induced by Hcy.Objectives1.Investigate the influence of Hey in different concentration on CD147 expression in human U-937 macrophages.2.Examine the inhibitory effect of rosuvastatin on CD 147 expression induced by Hcy.3.Determinate toxic effect of Hcy or rosuvastatin on macrophages viability using MTT assay to rule out the effect of drug toxicity on experiment result.Methods1.Macrophages formationHuman U-937 cells was maintained in RPMI 1640 medium supplemented with 10%fetal bovine serum at 37? in an atmosphere of 5%CO2.U-937 cells in cell plates(5×105/L)was stimulated with 10nmol/L PMA for 48 hours,then the adherent cells were identified as macrophages by morphology assessment under Light microscope.2.Hcy and rosuvastatin treatment?After U937 cells were differentiated into macrophage,plates were washed twice with 1 mL PBS,then cells were cultured in serum-free culture for 24h.Cell treatment measures are as follows:1)macrophages were stimulated with 0,50,100 and 500 umol/L of Hey for 48 hours respectively;2)macrophages was incubated with 500 umol/LHcy for 0,12,24,48 hours respectively;3)rosuvastatin in different concentration was added when U-937 macrophages were treated with 500umol/L homocysteine for 48 hours respectively.CD147expression was then analyzed using Western blot and RT-PCR.Cellular immumofluorescence method was used to detect the expression of membrane-bound CD 147.3.MTT assayPMA-induced U937 macrophages were seeded at 2 × 104cells per well with 150ul cell medium,After treating with Hey or rosuvastatin,cells were cultured with 10 ul MTT(5g/L)at 37? for 4 hours.100ulDMSO was added to each well after the medium is carefylly removed.The optical density was measured at ?= 490nm by Thermo Scientific Microplate Reader until farmazan was completely solubilized.4.Statistical analysisAll data were presented as meansąSD,the statistics were analyzed using SPSS 21.0,multiple means' comparison adopted method of One-way ANOVA.In cases those equal variances assumed,LSD test was used to compare the samples between the multiple samples.A value of P<0.05 was considered statistically significant.Result1.Macrophage cell identificationU937 cell were round or oval in shape and suspended in the culture medium.When treated with 10nmol/L PMA for 48h,cells became attaching to the dish and appeared as shuttle or stellate or irregular in shape.These changes suggested U937 cells were differentiated into macrophages.2.RT-PCRHomocysteine(100-500umol/L)significantly increased the expression of CD147mRNA in a dose-dependent manner(P<0.05).when macrophages incubated with 500umol/L homocysteine for different time points(24,48hours),CD147mRNA expression were upregulated in a time-dependent manner(P<0.05).macrophages were treated with rosuvastatin in different concentration and 500umol/L homocysteine for 48 hour,rosuvastatin(10-7-10-5mol/L)significantly reverse the expression of homcysteine-induced CD147mRNA in U-937 macrophages in a dose-dependent manner.3.Western blotIntervention of U937 cell with Hey for 48 h increased the protein expression of CD 147 gradually in a concentration-dependent manner,and Hcy(100-500umol/L)significantly increased the CD147 protein expression(P<0.05),Intervention with 500unol/L homocysteine for different time points(24,48hours)significantly increased CD 147 protein expression in a time-dependent manner(P<0.05).After treatment with different concentrations of rosuvastatin and 500umol/LHcy for 48h,rosuvastatin(10-7-10-5 mol/L)significantly inhibited homocystein-induced CD 147 protein in a dose-dependent manner(P<0.05).4.Cellular immumofluorescence.U937 macrophages was stimulated with Oumol/LHcy,500umol/LHcy,500umol/L Hcy+10-5 mol/L rosuvastatin for 48h.The fluorescence of membrane-bound CD147 from untreated macrophage is weak,A increase in cell fluorescence was observed after the macrophages were treated with 500umol/L Hey and actually decreased when the macrophages were treated 10-5mol/L rosuvastatin and 500umol/L Hey.5.MTTThere was no siginficant difference in OD value in control group,Hey groups,rosuvastastin groups(P>0.05).MTT results demonstrated that Hey or rosuvastatin and Hcy together exerted no significant cytotoxicity on U937 macrophage cells.ConclusionsHey can increase the expression of CD147 in PMA-induced U-937 macrophages.Additional extracellular rosuvastatin can decrease the expression of CD 147,in PMA-induced U-937 macrophages induced by homocysteine.Hcy(0-500umol/L)and rosuvastatin(10'8-10-5 mol/L)exerted no significant cytotoxicity on U937 macrophage cells. |
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