Background: Interleukin-6 (IL-6), an inflammatory cytokine, plays a majoy role in the pathogenesis of inflammation and serves as a key marker in the diagnosis and treatment of inflammation and related diseases. Formononetin (FMN), an isoflavone ingredient in various Chinese herbal medicines, inhibits the production of IL-6 in some cells. However, its mechanism of action has not been clearly established. In this study, we aimed to identify whether FMN inhibits IL-6 production in Lipopolysaccharide (LPS)-stimulated macrophages via AMPK activation.
Methods: In the current study, the anti-IL-6 mechanism of FMN was evaluated using a macrophage model with LPS stimulation. An inverted microscope was utilized to obtain images of cells. Nuclear staining assay and CCK-8 assay were used to identify the viability of ANA-1 cells. The expression of IL-6 in cells was investigated by Enzyme-linked immunosorbent (ELISA) and Western blotting. The expression of AMP-activated protein kinase (AMPK) was determined by Western blotting and phosphorylation of AMPK was determined by Western blotting and Immunofluorescence assay. Bioinformatic analysis was used to predict potential targets of FMN.
Results: we found that FMN reduced the expression of IL-6 in ANA-1 cells and increased the phosphorylation of AMPK. The effect of FMN was similar to that of acadesine, an AMPK activator, which also reduced IL-6 expression in LPS-induced ANA-1 cells and increased AMPK phosphorylation. Its combination with dorsomorphin (an AMPK inhibitor), however, reversed the effects of FMN on AMPK phosphorylation and IL-6 expression. The target of FMN was identified as the cAMP-dependent protein kinase inhibitor alpha, as searched in Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform and further verified with the STRING database.
Conclusions: In conclusion, our findings suggest that FMN effectively inhibits IL-6 production by activating AMPK in LPS-stimulated macrophages.