The Effect Of Advanced Glycation End Product Modified Low Density Lipoprotein On The Activity Of Mast Cells And Undlying Mechanism

Background:Diabetes is a major risk factor for coronary heart disease. Accumulation of advanced glycation end product modified low density lipoprotein (AGE-LDL) was shown to promote the formation and development of atherosclerosis(AS). It has been vertified that the activity of mast cells was associated with with AS and the stability of atherosclerotic plaques.Objective:AGE-LDL was used to stimulate mouse bone marrow derived mast cells (mBMMCs), and effects of AGE-LDL and rosuvastatin on the activity of mast cells and underlying signal pathways were investigated.Method:First,bone marrow derived cells were isolated from femur,tibia and fibula of male C57BL/6mouse (6-8weeks old), then induced to differentiate into mature mast cells by adding recombinant murine interleukin-3(rmIL-3) and recombinant murine stem cell factor(rmSCF). Then the phenotype of such cells was identified by staining with toluidin blue and fluorescence activate sorting5weeks later.Then,we investigated the effect of AGE-LDL on the activity of mast cells from two aspects as follow:(1)The effect of AGE-LDL on the releasing of histamine from mBMMCs:mBMMCs were stimulated with different doses of AGE-LDL,then measured the the levels of histamine released from the cells by the fluorescence;(2)The effect of AGE-LDL on the secretion of inflammatory factor from mast cells:We used different doses of AGE-LDL to stimulate mBMMCs for6h,then assayed the tumor necrotic factor-a(TNF-α), interleukin-6(IL-6) and interferon-y (IFN-y) mRNA expression of cells by real-time PCR,meanwhile we used Enzyme linked immunosorbent assay (ELISA) to measure the levels of these factors in supernatant of mBMMCs stimulated by AGE-LDL for24h.Finally,we investigated the potential signal pathways:(1)mBMMCs were stimulated with AGE-LDL,then observed the expression of phosphorylated NF-kB,phosphorylated p38MAPK,phosphorylated Erkl/2and phosphorylated JNK by western blot at different times (0,0.25h,0.5h,1h) 2h).(2)mBMMCs were preincubated with the PDTC,SB203580and PD98059,which was respectively the inhibitors of NF-κB,p38MAPK and Erkl/2,rosuvastatin,then stimulated with AGE-LDL, measured the levels of histamine released by cells and levels of inflammatory factors secreted by cells.(3)mBMMCs were preincubated with PDTC,SB203580and PD98059, then interfered with AGE-LDL,observed the expression of phosphorylated NF-κB by western blot.Results:(1) Mouse bone marrow derived mast cells were induced by rmIL-3and rmSCF.And5weeks later,more than95%of cells can be stained blue by toludin, the percentage of CD117/FCεRIa double positive cells was95.6%, indicating such cells were matured mast cells.(2)The effect of AGE-LDL on the activity of mBMMCs:AGE-LDL could promote mBMMCs to release histamine in a dose dependent manner (p<0.05), and the peak effect was at10minute, the peak concentration was50ug/ml.The mRNA expression protein concentration of TNF-α,IL-6,IFN-y of mBMMCs were increased in a dose depen-dent manner by incubation cells with AGE-LDL, and the effect was maximal at the concentration of50ug/ml.(3)Undelying signal pathways:The phosphorylation of NF-κB,p38MAPK and Erkl/2of mBMMCs significantly increased after stimulation with AGE-LDL, which could be partly inhibited by rosuvastatin.The phospho-rylation of NF-κB and p38MAPK was most obvious respectively after mBMMCs interfered with AGE-LDL for15minutes,and the peak value of phosphorylation of Erkl/2was seen after mBMMCs interfered with AGE-LDL for30minutes.AGE-LDL had no effect on the phosphorylation of JNK.The inhibitor of p38MAPK and Erkl/2could downregulated the expression of phospholated NF-κB of mBMMCs stimulated by AGE-LDL PDTC,the inhibitor of NF-κB and rosovastatin could inhibite the effect of AGE-LDL on the histamine releasing by mBMMCs. The inhibitor of NF-κB, Erkl/2and resovastatin could inhibit the effect of AGE-LDL on the synthesis and secretion of TNF-α,IL-6,IFN-γ from mBMMCs.Conclusion:AGE-LDL could active mast cells,promote such cells to release histamine and secrete inflammatory cytokinesTNF-α,IL-6,IFN-γ,which might be attributed to activation of NF-κB and Erkl/2. Rosuvastatin could inhibit the pathological effects of AGE-LDL. The effect of AGE-LDL on the activity of mast cells might explain why patients with diabetes have higher incidence of atherosclerosis and acute cardiovascular events.