In silico and in vitro investigation of the safety and immunomodulatory mechanisms of 7-hydroxyflavone

7-Hydroxyflavone (7HF) is a natural flavonoid with recognized antioxidant and anti-inflammatory properties, but its immunomodulatory potential and underlying molecular mechanisms remain poorly defined. This study investigated the immunomodulatory activity of 7HF in human peripheral blood mononuclear cells (PBMCs) and murine splenocytes through in silico and in vitro approaches. Pharmacokinetic and toxicity predictions were obtained with ProTox 3.0. Cytotoxicity and hemolysis assays evaluated compound safety, whereas lymphoproliferation and cytokine secretion (IL-2 and IFN-γ) were analyzed in PHA- or ConA-stimulated cells with or without the glucocorticoid-receptor (GR) antagonist RU-486. The expression of inflammatory mediators and signaling molecules was quantified by RT-qPCR, and molecular docking assessed the affinity of 7HF for calcineurin and GR. 7HF showed favorable pharmacokinetic predictions, low acute toxicity, and moderate CC 50 values in murine splenocytes (74.2 ± 6.1 μM) and human PBMCs (60.1 ± 1.9 μM), with no cytotoxic or hemolytic effects at immunomodulatory concentrations. The compound significantly inhibited lymphocyte proliferation and cytokine production in activated PBMCs and splenocytes, effects partially reversed by RU-486, indicating GR involvement. Gene-expression analysis revealed downregulation of calcineurin subunits (PPP3CA/B/C), PLCG1, NFAT1/2, and pro-inflammatory mediators, with additional modulation of PI3K/AKT, GR, NF-κB/AP-1, and JAK/STAT pathways. Molecular docking confirmed stable interactions of 7HF with both calcineurin and GR, with ChemPLP scores comparable to dexamethasone. Overall, 7HF exhibited selective immunosuppressive activity by modulating multiple signaling pathways involved in lymphocyte activation, supporting its potential as a safe lead compound for developing novel therapeutic agents. • 7-Hydroxyflavone (7HF) shows a favorable pharmacokinetic and safety profile. • 7HF selectively suppresses lymphocyte proliferation and cytokine release. • Glucocorticoid receptor antagonist RU486 partially reverses 7HF effects. • 7HF downregulates calcineurin/NFAT and modulates GR and PI3K/AKT signaling. • Molecular docking confirms stable 7HF interactions with calcineurin and GR.