Modulation Of Soyasaponins On The Expression Of Molecules In Toll-Like Receptor 4 Inflammatory Signaling Pathway

BackgroundNon-communicable chronic diseases(NCDs)seriously threats human’s health,which makes its prevention and control urgent.Accumualted studies have shown that food-derived phytochemicals can prevent and control NCDs through their anti-inflammatory bioactivities.Soyasaponins(SS)are phytochemicals widely existing in soybean and its products.Recent studies indicate that SS have anti-inflammatory bioactivities.However,its underlying molecular mechanisms are not fully understood.Studies from our and other labs suggest that SS’s anti-inflammatory bioactivites is associated with Toll-like receptor 4(TLR4)signaling pathway.However,the underlying mechanisms of how SS modulate TLR4 signaling pathway is unclear.ObjectivesIn this study.lipopolysaccharides(LPS)and palmitic acid(PA)-induced macrophage inflammatory models were applied to investigate the effects of three different types of SS(A1,A2 and I)on the expression of molecules in TLR4 signaling pathway.The aim of this study is to understand the modulation of SS on TLR4 inflammatory signaling pathway and to clarify the underlying molecular mechanisms of SS’s anti-inflammatory bioactivities.Results from this study will provide potential new clues and scientific basis for the prevention and control of NCDs.Methods1.Experimetnal groups(1)LPS-or PA-stimulated RAW264.7 murine macrophages:cells were stimulated with 1 μg/mL of LPS or 200 μmol/L of PA for different time(0 min,10 min,30 min,lh,3h,6h,12hand24h).(2)SS intervention in LPS-or PA-stimulated RAW264.7 murine macrophages:cells were preincubated with graded concentrations(10,20 and 40 μmol/L)of SS(A1,A2 and I)for 2 h and then stimulated with 1 μg/mL of LPS or 200 μmol/L of PA for suitable times as determined by previous results.At the same time,negative(no LPS or PA stimulation)and positive(1 μg/mL of LPS or 200 μmol/L of PA)control were applied.2.Western blotting for analyzing protein expressionCells were collected and lysated to prepare purified protein samples.The protein expression of molecules(TLR4,MD-2,CD14,TIRAP,MyD88,TRAM,TRIF,IRAK4,IRAK1 and TRAF6)in TLR4 signaling pathway was assessed by WB.The grey value of protein bands was analyzed by using Image J software.Experiments repeated at least three times.Data were analyzed by ANOVA of SPSS 19.0 statistical software.Results were shown as mean±SEM.P<0.05 indicated significantly statistical differences.Results1.The expression of molecules in TLR4 inflammatory signaling pathway in LPS-stimulated murine macrophagesMyD88 levels were increased in cells stimulated by LPS for 1 h and 3 h(P<0.05),When LPS stimulated for 3 h,the phosphorylation levels of IRAK4 were rised(P<0.05).The phosphorylation levels of IRAK1 were increased in LPS-stimulated cells for 3 h,6 h and 12 h(P<0.05).TRAF6 levels were increased for 30 min,1 h,3 h and 6 h,but decreased for 12 h and 24 h in LPS-stimulated cells(P<0.05).With the increase of LPS stimulation time,TRAF6 showed a trend of increasing at the beginning and then decreasing.2.Modulation of SS(A1,A2 and I)on the expression of molecules in TLR4 inflammatory signaling pathway in LPS-stimulated murine macrophagesThe 40 μmol/L of SS-A2 and 10μmol/L of SS-I suppressed LPS-induced increase of MyD88(P<0.05).LPS increased p-IRAK4 and p-IRAK1 levels(P<0.05),whereas 10～40 μmol/L of SS(A1,A2 and I)blocked LPS-induced increase of p-IRAK4 and p-IRAK1(P<0.05).LPS decreased TRAF6 levels in stimulated 12 h,whereas 10-40pmol/L of SS-A1 and 10～20 pmol/L of SS-A2 inhibit LPS-induced suppression of TRAF6 levels(P<0.05).3.The expression of molecules in TLR4 inflammatory signaling pathway in PA-stimulated murine macrophagesMyD88 levels were increased(P<0.05)in cells stimulated by PA for 10 min,30 min,1 h and 3 h.The phosphorylation levels of IRAK4 were increased in PA-stimulated cells for 10 min to 24 h(P<0.05).When PA stimulated for 30 min,1 h,and 3 h,p-IRAKl levels were rised(P<0.05).TRAF6 levels were increased for 10 min,but decreased for 12 h in PA-stimulated cells(P<0.05).4.Modulation of SS(A1,A2 and I)on the expression of molecules in TLR4 inflammatory signaling pathway in PA-stimulated murine macrophagesThe 10～40 μmol/L of SS(A1,A2 and I)suppressed PA-induced increase of MyD88(P<0.05).The 20～40μmol/L of SS-A1 and 10～40 μmol/L of SS(A2 and I)suppressed PA-induced increase of p-IRAK4(P<0.05).PA increased p-IRAKl levels(P<0.05),whereas 10～40 μmol/L of SS(A1,A2 and I)blocked PA-induced increase of p-IRAK1(P<0.05).PA increased TRAF6 levels in stimulated 10 min,whereas 10 μmol/L and 40 μmol/L of SS-I inhibited PA-induced increase of TRAF6 levels(P<0.05).10～40 μmol/L of SS(A1 and A2)could inhibit the decrease of TRAF6 induced by PA-stimulated for 12 h.Conclusion1.The protein levels of MyD88 and the phosphorylation of IRAK4 and IRAKI were increased in LPS-or PA-stimulated RAW264.7 murine macrophages,but the protein levels changed times were different.2.In LPS-or PA-stimulated RAW264.7 murine macrophages,the protein level of TRAF6 could be up-regulated in a short time,but prolonged stimulation could inhibit the expression,showing a trend of increasing first and then decreasing.3.SS-A2 and SS-I can inhibit the protein levels of MyD88 in LPS-stimulated RAW264.7 murine macrophages.SS-A1,SS-A2 and SS-I can suppress the phosphorylation of IRAK4 and IRAK1.SS-A1,SS-A2 can inhibit the decrease of TRAF6 caused by prolonged stimulation of LPS.4.SS-A1,SS-A2 and SS-I can inhibit the protein levels of MyD88 and the phosphorylation of IRAK4 and IRAKI in PA-stimulated RAW264.7 murine macrophages.SS-A1 and SS-A2 can inhibit the decrease of TRAF6 induced by prolonged stimulation of PA,and SS-I can suppress the increase of TRAF6 caused by short-term PA stimulation.