Effects Of TNF-α And IL-10 On Cholesterol Content And ABCA1 Expression In THP-1 Macrophage-derived Foam Cells

PartⅠEffects of TNF-αand IL-10 on Cholesterol Content in THP-1 Macrophage-derived Foam CellsBackground A typical pathological hallmark of atherosclerosis is the excessive accumulation of cholesterol and cholesteryl ester in macrophages, leading to their conversion to foam cells.Objective To investigate the effects of tumor necrosis factor-α(TNF-α) and interleukin-10(IL-10) on cholesterol content in THP-1(human acute monocytic leukemia cells) macrophage-derived foam cells.Methods Cultured THP-1 monocyte cell was induced into macrophages by 160nmol/l phorbol myristate acetate(PMA)for 72 h and then incubated with 50mg/l oxidized low density lipoprotein(ox-LDL)for 48h,macrophages were induced to foam cells. Foam cells were randomly allocated into 4 groups: control group, culture medium had no other substances; TNF-αgroup,10μg/l TNF-αwas added; IL-10 group, 10μg/l IL-10 was added; IL-10+TNF-αgroup, cells precultured by 10μg/l IL-10 for 2 hours were cultured with 10μg/l TNF-α. After cells in four groups were cultured for 0 h, 6 h, 12 h, 24 h, 48 h, Cells were cultured by 10mg/L apolipoprotein A-Ⅰ(apoA-Ⅰ) for 12 h. Transmission electronic microscope and light microscope (using red oil O staining technique) was applied to appraise the morphology and change of foam cells. Cholesterol content of foam cells was measured by zymochemistry via-fluorospectrophotometer, which excitation wave is 325nm, and fluorescent light emission is 415nm.Results In TNF-αgroup,cholesterol contnt in THP-1 mactophage-derived foam cells increased markedly in 12 h,24 h and 48 h comparsing with 0h( p<0.05). In IL-10 group,there was no difference in cholesterol content of foam cells in various time (p>0.05). In IL-10+TNF-αgroup, cholesterol content in THP-1 macrophage-derived foam cells also increased in 24h and 48h. But in comparision with TNF-αgroup, the degree of increment was greatly lessened (p<0.05).Conclusion TNF-αincrease markedly cholesterol content in THP-1 macrophage-derived foam cells, and aggravate the extent of macrophage-derived foam cells. IL-10 partly inhibits the increment of cholesterol content by TNF-α, and slows the extent of macrophage-derived foam cells. PartⅡRegulations of TNF-αand IL-10 on ATP Binding Cassette Transporter A1 Expression in THP-1 Macrophage-derived Foam CellsBackground ATP binding cassette transporter A1 (ABCA1) is an important protein in promoting cholesterol effux from macrophage foam cells. Objective To investigate regulations of tumor necrosis factor-α(TNF-α) and interleukin-10(IL-10) on ABCA1 expressions as well as the relationships between ABCA1 expression and cholesterol content in THP-1 macrophage-derived foam cells.Methods Cultured THP-1 monocyte cells was induced into macrophages by 160nmol/l phorbol myristate acetate(PMA )for 72 h , then incubated with 50mg/l oxidized low density lipoprotein(ox-LDL)for 48 h,macrophages were induced to foam cells. Foam cells were randomly allocated into four groups: control group, culture medium had no other substances; TNF-αgroup,10μg/l TNF-αwas added;IL-10 group , 10μg/l IL-10 was added;IL-10+TNF-αgroup, cells precultured by 10μg/l IL-10 for 2 hours were cultured with 10μg/l TNF-α. After cells in four groups were cultured for 0 h, 6 h, 12 h, 24 h, 48 h, Cells were cultured by 10mg/L apolipoprotein A-Ⅰ(apoA-Ⅰ) for 12 h. ABCA1mRNA and its protein levels in THP-1 macrophage-derived foam cells were determined by RT-PCR and Western blot, respectively.Results In TNF-αgroup, ABCA1mRNA and its protein level in THP-1 macrophage-derived foam cells markedly reduced in 12 h,24 h and 48 h comparing with 0 h (p<0.05). In IL-10 group, there was no difference between ABCA1mRNA and its protein level in various time.In IL-10+TNF-αgroup, ABCA1mRNA and its protein levels also reduced in a time-dependent manner, but in comparision with TNF-αgroup,the degree of reduction in 12 h,24 h and 48 h were greatly lessened(p<0.05). ABCA1mRNA was obviously negatively correlated with cholesterol content in THP-1 macrophage-derived foam cells(r =-0.984, p<0.05).ABCA1 protein level was also obviously negatively correlated with cholesterol content in THP-1 macrophage-derived foam cells(r=-0.986, p<0.05).Conclusion Change of ABCA1 expression is negatively correlated with cholesterol content in THP-1 macrophage-derived foam cells. TNF-αdownregulates ABCA1 expression in THP-1 macrophage-derived foam cells , and increases cholesterol content, which promotes the development and progression of atherosclerosis(AS). IL-10 partly inhibits the downregulaton of ABCA1 expression and the increment of cholesterol content by TNF-αin THP-1 macrophage-derived foam cells,and slows the development and progression of atherosclerosis.