Supplementary file 1_Apigenin accelerates fracture healing by enhancing endochondral differentiation and inflammation resolution via targeting NF-κB signaling.pdf

Quantitative PCR, Western blotting, RNA sequencing, and flow cytometry were used to evaluate inflammatory responses and chondrogenic and osteogenic differentiation. Ikbkb knockdown and BAY 11–7082 treatment were used to examine the involvement of IKKβ/NF-κB signaling. Molecular docking and molecular dynamics simulations were performed to investigate the potential interaction between apigenin and IKKβ, which was examined by cellular thermal shift assay (CETSA).</p>Results<p>Apigenin significantly improved fracture healing when treatment was initiated on postoperative day 5, whereas treatment initiated on day 1 showed no clear therapeutic effect.

Apigenin reduced inflammatory responses in the fracture callus and suppressed NF-κB signaling. BAY 11–7082 produced effects similar to those of apigenin in the femoral fracture model, and combined treatment did not produce a clear additional benefit. In BMSCs, apigenin promoted chondrogenic differentiation and attenuated the suppression of chondrogenic markers under inflammatory conditions, whereas its effects on osteogenic differentiation were less consistent.

Ikbkb knockdown also enhanced chondrogenic differentiation, and addition of apigenin after Ikbkb knockdown produced no further clear increase. Molecular docking, molecular dynamics simulations, and CETSA provided supportive evidence for a potential interaction between apigenin and IKKβ.</p>Conclusions<p>Through modulation of IKKβ/NF-κB signaling, apigenin acts on both inflammatory responses and MSC differentiation to enhance fracture healing.

The therapeutic effect was most evident when treatment was initiated on postoperative day 5, during the transition from inflammation to cartilage formation. These findings suggest that appropriately timed apigenin treatment may represent a potential strategy for improving fracture repair.</p>