The Effect And Underlying Mechanism Of Cucurbitacin Ⅱa On Inflammatory Reponses Of Raw264.7Macrophages Activated With Lipopolysaccaride

Objective:This study aimed to explore the potential anti-inflammatory activity andunderlying action mechanism of cucurbitacin IIa (CuIIa) using lipopolysaccaride(LPS)-activated RAW264.7macrophages as a model.Methods:The effect of CuIIa on cell proliferation was measured by MTS assay. Transwellmigration assay was used to observe the effect of CuIIa on cell migration ability. Thelevels of G-actin and F-actin in CuIIa-treated cells were measured by Western blotanalysis and SDS-polyacrylamide gel electrophoresis (PAGE) analysis. Theexpression levels of proinflammatory cytokines TNF-were evaluated byintracellular cytokine staining and flow cytometry. Phase contrast microscopy wasused to observe the morphology changes of cells while the morphology of cellnucleus was revealed by Hoechst33342staining under fluorescence microscope. Sub-G0/G1(apoptotic) peak was identified by flow cytometry. Immunoblotting wasused to evaluate the levels of MAPKs, NF-B, cleaved capase-3, survivin andLC3B-II. The distribution of actin cytoskeleton and autophagy-related protein LC3Bin cells was observed by immunofluorescence analysis.Result:The results showed that CuIIa inhibited the proliferation and migration of RAW264.7cells in a dose-dependent manner. CuIIa disrupted actin cytoskeleton viainducing G-actin aggregation into F-actin. However, neither the synthesis of tumornecrosis factor (TNF)-, nor the activation of the mitogen-activated protein kinases(MAPKs) and NF-B pathways in LPS-stimulated cells was suppressed by CuIIatreatment. CuIIa treatment caused cell shrinkage and disappearance of pseudopodiaprotrusions of LPS-activated cells, and led to significant actin aggregation within cells,indicating a disruption of microfilament structures. The hypodiploid peaks (apoptoticpeaks) were increased in LPS-activated RAW264.7cells treated with increased dosesof CuIIa. But CuIIa couldn’t induce cell apoptosis in the absence of LPS. Western blotanalysis indicated that these effects were caspase-3dependent and associated withdownregulation of survivin. Furthermore, LPS induced autophagy in RAW264.7cellsand this effect was further enhanced by CuIIa as evidenced by increased levels ofLC3B-II and enhanced formation of LC3B puncta.Conclusion:CuIIa inhibited the proliferation and migration of RAW264.7cells in adose-dependent manner and induced actin aggregation in cells. Although CuIIa didnot inhibit the secretion of proinflammatory cytokine TNF-and the activation ofinflammation signaling pathway, it could effectively induce cell apoptosis andautophagy in LPS-activated macrophage cells. This may be one of the mechanisms for CuIIa against inflammation responses.