The Effect Of TLR4-MyD88 Signal Transduction In DFMG On The Injury Of Endothelial Cells Induced By LPC

Objective:Assessment the effect of 7-difluoromethoxy-5,4'-dimethoxygenistein on Toll-like receptor 4(Toll-like receptor 4,TLR4)and myeloid differentiation factor 88,MyD88-dependent signal transduction pathway,and explore the mechanisms of DFMG anti-vascular endothelial injury.Method:Cultured HUVE-12 cells in vitro,using LPC 10?mol/L incubated cells 24h to make inflammatory injury model and collecting cells,which refer to relevant literature and the preliminary research results of mentor,treated with 0.3?1.0?3.0 u mol/L DFMG 6 hours,12 hours,24 hours and 48 hours respectively then collecting cells and using MTT assessment the proliferation of cells.Using 0.3?1.0?3.0 ?mol/L DFMG?GEN?lovastatin?TLR4 antagonists?TLR4 antagonist+DFMG incubated inflammatory injury model and using LPS 10?mol/L incubated cells 24h,collecting cell culture supernatants and cells.Using ELISA detection of the expression of human tumor necrosis factor-a(TNF-a),western blotting to detect of the expression of TLR4,MyD88 and TRAF-6 protein.Results:1?MTT results showed that:10 ?mol/L LPC can impact on cell viability,significantly inhibited cell growth compared with the vehicle control group,the difference was statistically significant(P<0.005).0.3,1.0,3.0?mol/L DFMG+LPC in a dose and time-dependent antagonism LPC-induced cell growth inhibition,compared with 10 ?mol/L LPC group,the difference was statistically significant(P<0.005).2?ELISA results showed that:10 ?mol/L LPC,LPS effect on HUVE-12 compared with the vehicle control group?3.0?mol/L DFMG,cell culture medium concentrations of TNF-? were significantly increased,the difference was statistically significant(P<0.005).0.3,1.0,3.0?mol/L DFMG+LPC concentration-dependent decrease in the concentration of TNF-a,compared with 10?mol l L LPC group,the difference was statistically significant(P<0.005);3.0 ?mol/L DFMG+LPC?lovastatin+LPC group and GEN+LPC group had no significant difference(P>0.005).Meanwhile TLR4 antagonist+LPC,TLR4 antagonist+DFMG+LPC group TNF-a concentration is reduced,compared with 10 ?mol/L LPC group,the difference was statistically significant(P<0.005).3?Western blotting results showed that:10 ?mol/L LPC,LPS effect on HUVE-12 compared with the vehicle control group?3.0?mol/L DFMG,TLR4,MyD88,TRAF-6 protein expression was significantly increased.0.3,1.0,3.0 ?mol/L DFMG+LPC concentration-dependent decrease the expression of TLR4?MyD88?TRAF-6;the expression of TLR4?MyD88?TRAF-6 in 3.0 ?mol/L DFMG+LPC and lovastatin+LPC group?GEN+LPC group had no difference.Meanwhile TLR4 antagonist+LPC?TLR4 antagonist+DFMG+LPC group can reduce TLR4,MyD88,TRAF-6 protein expression.Conclusion:DFMG can play a role in anti-vascular endothelial inflammatory injury,which may be associated with inhibition of Toll-like receptor 4 and downstream myeloid differentiation factor 88-dependent transduction.