Effects of 3,5,4'-trimethoxystilbene on TNF-α Production and NF-κB Activity in Murine Macrophages

Objectives:To study the effects of 3,5,4'-trimethoxystilbene(BTM) on the TNF-αproduction and the NF-κB activity in murine macrophages activated by lipopolysaccharide(LPS).Methods:RAW264.7 cells,the murine macrophage cell line,were cultured in RPMI-1640 medium supplemented with 10%fetal bovine serum,penicillin(100U/ml),and streptomycin(100U/ml).Cells were then seeded at 3×10~5 cells/well in 24-well plate,and at the same time,every well of the plate was put in a cleaned cover glass.24 hours later,BTM and resveratrol were freshly added in the wells with different concentration(5,10,20,40,80μmol/L)and cultured with macrophages for 30 minutes,then LPS was added in the wells.After 6 hours reaction,we measured the TNF-αproduction in cell supernatant by staining L929 with crystal violet,and measured the NF-κB activity in macrophages by immunocytochemistry method.The cell groups added resveratrol and LPS were control groups,the cell groups added only LPS were negative control groups,the cell groups added neither LPS nor resveratrol were normal control groups.Results:1.①BTM and resveratrol had no direct cytotoxicity to L929 cells.②The cytotoxicity in supernatant from macrophages induced by LPS was 62.27%.③The TNF-αproduction in supernatant from macrophages caused by LPS and drugs decreased depending the concentration,the cytotoxicity rate were 41.72%、32.82%、25.77%、13.50%、4.29%for BTM and 54.91%、50.00%、45.40%、33.74%、21.78%for resveratrol respectively,and were lower than that of group LPS(P＜0.01),significant differences of the cytotoxicity rate were found among different concentration BTM groups(P＜0.05),and also among resveratrol groups(P＜0.05).④The inhibitory effect of BTM on the TNF-αproduction from macrophages was higher than that of resveratrol(P＜0.01), the inhibition rate were 33.00%VS11.82%、47.29%VS19.70%、58.62%VS27.09%、78.32%VS45.81%、93.11%VS65.02%respectively.2.①The positive rate of NF-κB expression in macrophages in group LPS was 98.55±1.36%.②The positive rate of NF-κB expression in macrophages were 63.17±2.22%、53.28±1.60%、40.97±2.11%、27.06±2.22%、7.05±0.92%for BTM and 88.42±4.51%、77.37±2.03%、69.22±3.31%、56.21±1.41%、35.83±2.56%for resveratrol respectively, and were lower than that of group LPS(P＜0.01),significant differences of the positive rate were found among different concentration BTM groups(P＜0.01),and also among resveratrol groups(P＜0.01).③The inhibitory effect of BTM on the NF-κB expression in macrophages was higher than that of resveratrol(P＜0.01),the inhibition rate were 35.90%VS10.28%、45.94%VS21.49%、58.43%VS29.26%、72.54%VS42.96%、92.85%VS63.64%respectively. 3.The cytotoxicity of TNF-αin supernatant and the positive rate of NF-κB expression in macrophages were all in negative correlation with the concentration of BTM and resveratrol(P＜0.01),and the cytotoxicity of TNF-αin supematant was in positive correlation with the positive rate of NF-κB expression in macrophages(P＜0.01).Conclusions:1.BTM and resveratrol inhibited the TNF-αproduction and the NF-κB activity in murine macrophages activated by LPS depending the concentration of 5-80μmol/L.2.BTM and resveratrol inhibited the TNF-αproduction in murine macrophages through inhibiting the NF-κB activity.3.The inhibitory effects of BTM on the TNF-αproduction and the NF-κB expression in macrophages were higher than that of resveratrol.