Hexane extract of Pittosporopsis kerrii Craib mitigates LPS-driven inflammatory responses via JAK/STAT1 pathway inhibition in RAW 264.7 macrophages.

BACKGROUND: Pittosporopsis kerrii Craib is traditionally used to relieve inflammatory conditions, but its molecular mechanisms remain unclear. This study investigated the anti-inflammatory effects of the hexane extract of P. kerrii (PK-H) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages, with emphasis on STAT1 signaling.

METHODS: Cell viability was evaluated using the MTT assay. Anti-inflammatory activity was assessed by measuring nitric oxide (NO) production, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6). Activation of NF-κB, MAPKs, and STAT1 was examined by Western blotting and immunofluorescence. Phytochemical profiling was performed by LC-MS/QTOF, and putative constituents were analyzed by molecular docking against STAT1, JAK1, JAK2, and IFNGR1.

RESULTS: PK-H showed low cytotoxicity (CC₅₀ ≈ 155.4 μg/mL) and significantly inhibited NO production (IC₅₀ ≈ 15.7 μg/mL). The extract reduced iNOS expression and proinflammatory cytokine release, while not markedly reducing NF-κB phosphorylation but partially preserving IκB and limiting NF-κB nuclear accumulation, despite negligible effect on MAPK activation. In contrast, PK-H markedly suppressed STAT1 phosphorylation and nuclear translocation. LC-MS/QTOF analysis revealed diterpenoids dominated by icacinlactone and icacinol derivatives. Docking studies identified 12-hydroxyicacinlactone A and icacinlactone A as consistent binders to JAK-STAT pathway targets.

CONCLUSIONS: PK-H exerts anti-inflammatory effects predominantly through selective inhibition of the JAK-STAT1 pathway. Its diterpenoid constituents represent promising leads for STAT1-targeted anti-inflammatory agents.