Abstract:

Objective To discuss the effects of different concentrations of apigenin （API） on the inflammatory and polarization response of the mouse mononuclear macrophage cells RAW264.7 induced by oxidized low-density lipoprotein （ox-LDL），and to clarify their possible mechanisms. Methods The RAW264.7 cells were divided into RAW264.7 group （without any treatment）and RAW264.7+API group （2， 4， 8， 16 and 32 μmol·L^－1 API）.After the cells were treated for 24 h.CCK-8 assay was used to detect the proliferation rates of the cells in various groups.The experimental concentration of API was selected.The RAW264.7 cells were divided into RAW264.7 group（normal RAW264.7 cells）， RAW264.7+ox-LDL group（induced with 0.08 g·L^－1 ox-LDL for 24 h）， RAW264.7+ox-LDL+low dose of API group（induced with 2 μmol·L^－1 API and 0.08 g·L^－1 ox-LDL for 24 h） and RAW264.7+ox-LDL+high dose of API group. Oil red O staining was used to observe the morphology of the foam cells in various groups. The levels of tumor necrosis factor-α（TNF-α），interleukin-1β（IL-1β）， interleukin-4（IL-4）， and interleukin-10（IL-10） in the cell culture supernant were detected by enzyme-linked immunosorbent assay（ELISA） method. The expression levels of neclear factor-κB（NF-κB） p65， phosphorylated NF-κB（p-NF-κB） p65，inducible nitric oxide synthase（iNOS）， signal transducer and activator of transcription 6（STAT6）， phosphorylated STAT-6（p-STAT6）， and arginase-1（Arg-1） in the cells in various groups were detected by Western blotting method. Results The results of CCK-8 assay showed that the proliferation rates of the cells were significantly decreased with the increasing of the concentrations of API （P<0.05）.The non-toxic low concentration （2 μmol·L^－1） and high concentration （8 μmol·L^－1） of API were selected to use in the follow-up experiment.The Oil red O staining results showed that few RAW264.7 cells were stained with Oil red O； a lot of RAW264.7 cells in RAW264.7+ox-LDL group were stained as dark red，and the lipids in the cells were increased，indicating the foam cell model was successfully established； a little of RAW264.7 cells in RAW264.7+ox-LDL+low dose of API group and RAW264.7+ox-LDL+high dose of API group were stained with Oil red O.The ELISA results showed that compared with RAW264.7 group， the levels of TNF-α and IL-1β in the cell culture supernatant in RAW264.7+ox-LDL group were significantly increased （P<0.05）， while the levels of IL-4 and IL-10 were significantly decreased （P<0.05）； compared with RAW264.7+ox-LDL group， the levels of TNF-α and IL-1β in the cell culture supernatant in RAW264.7+ox-LDL+low dose of API group，the levels of IL-4 and IL-10 were significantly increased （P<0.05）.The Western blotting results showed that compared with RAW264.7 group， the expression levels of iNOS and p-NF-κB p65 proteins in the RAW264.7 cells in RAW264.7+ox-LDL group were significantly increased （P<0.05）， and the expression levels of Arg-1 and p-STAT6 proteins were significantly decreased （P<0.05）； compared with RAW264.7+ox-LDL group， the expression levels of iNOS and p-NF-κB p65 proteins in the RAW264.7 cells in RAW264.7+ox-LDL+low dose of API group and RAW264.7+ox-LDL+high dose of API group were decreased（P<0.05），and the expression levels of Arg-1 and p-STAT6 proteins were increased（P<0.05）；compared with RAW264.7+ox-LDL+low dose of API group，the expression levels of iNOS and p-NF-κB p65 proteins in the RAW264.7 cells in RAW264.7+ox-LDL+high dose of API group were decreased（P<0.05），and the expression levels of Arg-1 and p-STAT6 proteins were increased（P<0.05）. Conclusion API can inhibit the foam cell formation of RAW264.7 cells induced by ox-LDL and improve the inflammatory response by regulating the polarization from macrophages into M2， and their mechanisms may be related to NF-κB and STAT6 pathways.

Key words: Apigenin, Atherosclerosis, Nuclear factor-κB, Signal transducer and activator of transcription 6, Macrophage polarization