Ostruthin Targets IL-6 and Downregulates Arid5a Expression to Inhibit Cytokine Production Under TLR3-TLR4 Cotriggering: In vitro and in silico Studies

Objectives This study aimed to investigate the anti-inflammatory properties and underlying mechanisms of ostruthin, a natural coumarin isolated from the roots of Paramignya trimera . Using an in vitro model of co-stimulation with bacterial and viral mimetics in RAW264.7 macrophages, we examined the effects of ostruthin on the expression of key signaling molecules: Myd88 and Nf-κb1 (mediators of Toll-like receptor signaling), and Arid5a (a stabilizer of IL-6 mRNA). We also assessed the production of inflammatory cytokines, including IL-6, TNF-α, and IL-10. Methods Ostruthin was isolated from P. trimera roots collected in Khanh Hoa Province, Vietnam. RAW264.7 cells were pre-treated with various concentrations of ostruthin before being stimulated with lipopolysaccharide (LPS) and poly(I:C). The mRNA expression of Myd88 , Nf-κb1 , and Arid5a was quantified at 3, 6, and 24 h using qRT-PCR. Cytokine levels were quantified at 24 h using ELISA. Additionally, molecular docking and molecular dynamics simulations were performed to predict direct interactions between ostruthin and the inflammation-related target, IL-6. Results Ostruthin significantly and dose-dependently reduced IL-6, TNF-α, and IL-10 secretion in co-stimulated RAW264.7 cells, with IC 50 values of 3.0 ± 1.2 µM, 4.8 ± 1.5 µM, and 11.3 ± 0.9 µM, respectively. Treatment with ostruthin (3-60 μM) also markedly downregulated Myd88 (by 36%-79%), Nf-κb1 (by 49%-97%), and Arid5a (by 29%-83%) mRNA expression relative to untreated controls. Docking simulations revealed favorable binding affinities of ostruthin with IL-6 (−5.381 kcal/mol), comparable to or exceeding those of known inhibitors like tocilizumab. Conclusions Ostruthin exhibits potent anti-inflammatory activity in RAW264.7 macrophages stimulated with bacterial and viral mimetics, primarily by suppressing pro-inflammatory cytokines and modulating both TLR signaling and IL-6 mRNA stability pathways. These findings suggest that ostruthin may serve as a promising lead compound for the development of anti-inflammatory therapies targeting infections of mixed microbial origin.