Inhibitory Effect and Mechanism of Riligustilide from Danggui(Angelica sinensis)on LPS-induced Inflammatory Response in RAW 264.7 Cells
Objective To investigate the inhibitory effect and mechanism of riligustilide(DG2),a phthalide dimer from Dang-gui(Angelica sinensis)on lipopolysaccharides(LPS)-induced inflammatory response in RAW 264.7 macrophages.Methods The inflammation model of RAW 264.7 cells was established by LPS induction.The effect of DG2 on the survival rate of RAW 264.7 cells was detected by MTT method,and the effect of DG2 on the levels of inflammatory mediator nitric oxide(NO)in LPS-induced RAW 264.7 cells was determined by Griess method.Enzyme-linked immunosorbent assay(ELISA)was used to investigate the effect of DG2 on the secretion of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)in LPS-in-duced RAW 264.7 cells.Western blotting was used to detect the effects of DG2 on inducible nitric oxide synthase(iNOS)and cyclooxygenase-2(COX-2)in RAW 264.7 cells induced by LPS,as well as mitogen-activated protein kinase(MAPK),nu-clear factor-κB(NF-κB)and signal transducer and activator of transcription(STAT)signaling pathways.Immunofluorescence was used to observe the effect of DG2 on the nuclear translocation of STAT3 in RAW 264.7 cells induced by LPS.Results DG2 at the concentration of 64 µmol/L had no effect on the survival rate of RAW 264.7 cells.DG2 significantly reduced the level of NO released by LPS-induced RAW 264.7 cells(P＜0.001)[IC50=(26.13±5.75)μmol/L],which was similar to that of pos-itive control quercetin[IC50=(26.06±2.28)μmol/L].It also significantly inhibited the production of inflammatory factors IL-6 and TNF-α(P＜0.01,P＜0.001).In addition,DG2 significantly inhibited the LPS-induced expressions of iNOS and COX-2 proteins(P＜0.01,P＜0.001),as well as phosphorylation STAT3(p-STAT3),phosphorylation protein kinase B(p-AKT)and p-p38 proteins(P＜0.05,P＜0.01).Moreover,it inhibited the nuclear translocation of STAT3 in LPS-induced RAW 264.7 cells.Conclusion DG2 could inhibit LPS-induced inflammatory response in RAW 264.7 cells,and the mechanism may be related to the down-regulation of STAT,NF-κB and MAPK signaling pathways.
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