The Effects Of DFMG On The Proliferation And TLR4-MyD88 Signaling Of Marcrophages Treated With LPC

Objective:To explore the effects of 7-difluoromethyl-5,4’- dimethoxygenistein(DFMG) on the proliferation and Toll-like receptor 4(TLR4)- myeloid differentiation factor 88(My D88) signaling of marcrophages treated with lysophosphatidylcholine(LPC).Methods:THP-1 cells were cultured in vitro and induced to macrophages by PMA.Macrophages were treated with LPC(3.0、 10、30、100 μM) for 12 hours to establish the model of macrophages treated with LPC. Treated with DFMG(0.3、1.0、3.0 μM) of the model of macrophages treated with LPC for different time period(6h、12h、24h、48h),at the same time,established the model control group and solvent control group,then the proliferation was detected by CCK-8. The model of macrophages was treated with DFMG(0.3、1.0、3.0 μM)、lovastatin(50μM)、TLR4 antagonists(1μg/ml)、LPS(1μg/ml) for 12 h,respectively,then collecting cell culture medium to detect the expression of human tumor necrosis factor-α(TNF-α) by TNF-α ELISA Kit.The model of macrophages was treated with DFMG(3.0 μM) 、lovastatin(50μM)、TLR4 antagonists(1μg/ml)、LPS(1μg/ml) for 12 h, respectively,then collecting cells to detect the expression of TLR4 and My D88 by western blotting.Results:1.The cells differentiation percentage was(39.67±0.05) % after inducing THP-1 cells by 150 n M PMA which was significantly higher than that of control group( 0.55±0.19) %(P < 0.05).Thus,150 n M PMA was choosed as the induced concentration of marcrophages which derived from THP-1 cell lines.2.After dealing with different concentrations of LPC(3.0、10.0、30.0、100.0μM),the survival rate of marcrophages were(95.83±2.08)% 、(86.17±2.62)% 、(52.43±1.59)% 、(38.19±1.20)% by Typan Blue Exclusion.The difference was statistically significant between cell control group and treatment group..(P<0.05).3.CCK-8 kits results showed: Compared with solvent control group,30.0μM LPC cansignificantly inhibited cell growth,the difference was statistically significant(P<0.05).In conclusion,we choose 30.0μM as the concentration of the model of macrophages treated with LPC.4.DFMG can increase the cell proliferative activity in a dose and time-dependent manner.5.ELISA results showed:Different concentrations of DFMG(0.3、1.0、3.0μM) can decrease concentrations of TNF-α in the model of macrophages in a dose-dependent manner.6.Western blotting results showed:LPC(30.0μM) and LPS(1μg/ml) can up-regulate the proteins expression of TLR4,My D88 in macrophages treated with LPC,the difference was statistically significant( P<0.001).7.Western blotting results showed:3.0μM DFMG and TLR4 antagonists can inhibit the effect of up-regulating the poteins expression of TLR4,My D88 in macrophages treated with LPC( P <0.001).Conclusion:1.Treatment with LPC can inhibit the proliferation activity of macrophages、increase the secretion of TNF-α and up-regulate the poteins expression of TLR4, My D88.2.DFMG can against the effect of LPC on the inhibiting proliferation of macrophages adn antagonism the effect on the secretion of TNF-α in model of macrophages.3.DFMG can against the effect of LPC on the inhibiting proliferation of macrophages.DFMG can antagonism the secretion effect of TNF-α in macrophages induced by LPC which may be associated with Toll-like receptor 4-myeloid differentiation factor 88 signal transduction.