ABSTRACT
Objective To study the effect of candesartan (Can) on the LPS‐induced proinflammatory cytokines release in VSMCs ,and to explore the role of TLR4‐mediated signaling pathway in this process .Methods Rat VSMCs were primarily cul‐tured ,and the effect of different concentrations of Can on VSMCs activity was observed by M TT assay .The cells were divided into 5 groups :A(control) ,B(LPS intervention) ,C(LPS + 10 - 7 mol/L Can) ,D(LPS + 10 - 7 mol/L Can ) and E (LPS + Can 10 - 5 mol/L Can) .mRNA and protein levels of Toll‐like receptor‐4(TLR4) ,myeloid differentiation primary response gene 88(Myd88)and nu‐clear factor‐κB(NF‐κB) nuclear translocation were determined by real‐time PCR and Western blot ,respectively ;IL‐1β and TNF‐αconcentration were detected by ELISA .The production of intercellular reactive oxygen species(iROS) was measured by the DCFH‐DA assay .Pretreating VSMCs with the inhibitors against TLR4 ,NADPH oxidase ,and NF‐κB ,or a combination with candesartan and these inhibitors ,and then the expression of IL‐1β and TNF‐α was measured by ELISA .Results Can in the concentration range of 10 - 8 - 10 - 3 mol/L had no significant effect onVSMCs activity .Compared with th control group ,Can could effectively inhibit LPS‐induced VSMCs ,IL‐1β and TNF‐α release ,decreased the mRNA and protein expression of TLR4 ,Myd88 ,reduced the produc‐tion of iROS ,inhibited the NF‐κB(p65) nuclear translocation with a concentration‐dependent manner(P< 0 .05) .Anti‐TLR4 anti‐body ,DPI ,and PDTC all inhibited LPS‐induced inflammatory cytokines release in VSMCs ,and the combination of Can and these blockers showed stronger anti‐inflammatory effect .Conclusion Can can decrease the releaseof inflammatory cytokines IL‐1β and TNF‐α in VSMCs stimulated by LPS ,which is realized by inhibiting the signaling pathway of TLR4/Myd88‐iROS‐NF‐κB .
ABSTRACT
ObjectiveTo study the impact of quercetin on LPS-stimulated macrophage inflammatory cytokine release.MethodsDetermined by MTT assay with different concentrations of quercetin on the activity of RAW 264.7 cells.Cells were divided into five groups:A (control group),B group (LPS intervention group),C (LPS+quercetin 50 μmol/L),D group (LPS+quercetin 150 μmol/L) and E group (LPS+quercetin 250 μmol/L).Cells expression of toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (Myd88) and NF-kappa B (p65) mRNA in each group was determined by real-time determination; Supematant IL-6 and TNF-α secretion level was tested by ELISA determination.ResultsThe quercetin at 0 to 250 μmol/L concentration range on the activity of RAW 264.7 cells did not significantly affect.Compared with the control group,quercetin can effectively reduce the level of LPS-induced TLR4 and of MyD88 and of NF-κB (p65) of rmRNA expression and supematant IL-6 and TNF-α secretion,and dose-dependent manner(P＜0.01).ConclusionQuercetin may reduce macrophage inflammatory cytokine secretion by inhibiting the activation of TLR-Myd88-NF-κB pathway.