Apigenin alleviates inflammation as a natural IRAK4 inhibitor

Uncontrolled inflammation is a key factor in the development of many diseases, and targeting pivotal kinases involved in the inflammatory response, such as interleukin-1 receptor-associated kinase 4 (IRAK4), holds promise for the treatment of inflammatory conditions. Apigenin (Api) is a popular element in numerous plants, possessing anti-inflammatory properties. Many studies have shown that Api modulates NF-κB signaling and MAPK cascade to reduce inflammation, but the exact mechanisms by which Api regulates these pathways remain unclear.

The aim of this study was to investigate the role of Api on acute inflammation and its specific mechanism in mediating inflammation.

The dextran sulfate sodium (DSS) induced ulcerative colitis (UC) model and LPS induced acute inflammation mouse model were established to investigate the anti-inflammatory effects of Api. Subsequently, the anti-inflammatory activity of Api was validated in vitro and vivo by RNA-seq, qPCR, Western blot, cytokine ELISA, immunofluorescence and histological analysis. A series of experiments were performed to study the effects of Api on IRAK4, including ADP-Glo™ kinase assay, surface plasmon resonance (SPR), cellular thermal shift assay (CETSA), and molecular docking simulation. The targeting of Api to IRAK4 was verified by IRAK4 inhibitor and siRNA.

Oral administration of Api significantly ameliorated inflammatory conditions in the LPS induced acute inflammation and DSS induced UC mouse models. Furthermore, Api inhibited the expression of interleukin and chemotactic factor genes and downregulated the immune response of macrophages at the transcriptome level. Mechanistically, Api acted as a novel IRAK4 inhibitor, inhibiting kinase activity by direct binding to IRAK4 (Kd = 4.78 μM) with an IC₅₀ of 1.74 μM, interfering with extracellular signaling to the NF-κB and MAPK pathways, and reducing the expression of pro-inflammatory factors in an IRAK4 dependent manner.

In this study, Api was identified for the first time as a natural IRAK4 inhibitor that suppresses cytokine signaling pathways and modulates the immune response at the level of the transcriptome. The results provided valuable insights into the specific mechanism of Api inhibition of inflammatory activation and shed light on opportunities for the development of novel IRAK4 inhibitors based on Api, which is found in various plants.