(P18-009-25) Apigenin Suppressed M1 Macrophage Polarization but Did Not Promote M2 Macrophage Polarization in Raw 264.7 Cells

Objectives: M1 and M2 macrophages are responsible for maintaining tissue homeostasis for inflammatory responses. M1 macrophages exhibit proinflammatory responses while M2 macrophages exhibit anti-inflammatory responses. The imbalance between M1 and M2 macrophages is closely related to inflammation-associated disease development. Apigenin (API), a flavonoid present in apiaceous vegetables, is known for its anti-inflammatory properties, however, there is a paucity of data on its potential role in macrophage polarization.

Methods: Raw 264.7 cells were pre-treated with API for 2 hr prior to applying lipopolysaccharide (LPS) and interferon γ (IFNγ) for M1 macrophage differentiation or interleukin-4 (IL-4) for M2 macrophage differentiation. After 22-hr incubation, culture media was used for nitric oxide (NO) quantification, and the cells were used for qPCR. Data were analyzed by one-way ANOVA with Duncan’s post-test.

Results: LPS and IFNγ induced NO production (1.7 µM to 15.0 µM, P< 0.05) while various concentrations of API (1.6, 3.1, 6.3, 12.5, 25 µM) decreased NO production dose-dependently (14.7, 14.1, 13.7, 11.5, 10.52µM, respectively, P<0.05). LPS/IFNγ induced M1 macrophage markers, TNFα (12-fold), IL-1β (39-fold), CD86 (12-fold), iNOS (40-fold), IL-6 RNA (4-fold) when compared to vehicle control, while pretreatment with API (25µM) suppressed TNFα, CD86, and IL-6 RNA by 38 – 48% (P < 0.05) but not IL-1β and iNOS. In addition, M2 markers, CD206, CD163, and IL-10, were reduced by 76%, 50%, and 15%, respectively (P < 0.05) in M1 macrophages, however, API had no effect in reversing this reduction, indicating that API did not have potential to repolarize M1 macrophage toward M2 macrophage. In line with this, API suppressed M2 macrophage polarization in IL-4-stimulated cells as evidenced by the decreased Arg-1, CD206, and IL-13 (72%, 80%, and 69%, respectively, P< 0.05).

Conclusions: API suppressed M1 macrophage-related gene expression but had no effect on polarizing macrophages toward M2. Hence, further investigation is warranted to elucidate the mechanism by which API regulates M1 macrophage polarization.

Funding Sources: 21st Century Endowed Chair in Human Environmental Sciences, University of Arkansas.