Synergistic anti-inflammatory potential of andrographolide and hesperetin for psoriasis treatment: Molecular docking, molecular dynamics simulations, and in vitro validation in LPS-stimulated RAW264.7 cells

Psoriasis is a chronic immune-mediated inflammatory skin disorder characterized by keratinocyte hyperproliferation and elevated levels of pro-inflammatory mediators. The pursuit of effective and safe treatments at affordable costs remains an ongoing challenge. Natural compounds show promise due to their antioxidant, anti-inflammatory, and immunomodulatory effects. Phytochemicals andrographolide (AG) and hesperetin (HST) exhibit excellent anti-inflammatory properties in cells; however, their low oral bioavailability in the blood compromises their therapeutic potential. This study investigates the synergistic anti-inflammatory potential of AG and HST as a novel therapeutic strategy for psoriasis. This study combined computational docking and molecular dynamics simulations with in vitro macrophage assays to evaluate the synergistic anti-inflammatory potential of AG and HST. Molecular docking analyses revealed that AG and HST exhibit strong binding affinities toward key inflammatory targets within the TLR4 signaling cascade, including TLR4/MD-2, TAK1, NF-κB, AP-1, iNOS, COX-2, and NLRP3. AG showed the highest affinity for TLR4/MD-2, TAK1, and NF-κB (−8.3 kcal/mol), while HST demonstrated potent interactions with AP-1 (-7.5 kcal/mol), iNOS (-9.5 kcal/mol), COX-2 (-8.7 kcal/mol), and NLRP3 (-8.7 kcal/mol). Molecular dynamics simulations of 100 ns depict that AG and HST form stable, compact complexes with TLR4/MD-2 and iNOS, respectively, exhibiting lower structural deviations, sustained hydrogen bond interactions, and preserved protein integrity compared to control ligands. In vitro validation using LPS-stimulated RAW264.7 macrophages confirmed that AG and HST, individually and in combination, significantly inhibited nitric oxide (NO), IL-1β, and IL-6 production without cytotoxicity. Synergy analysis using CompuSyn software revealed a combination index (CI) < 1 for NO inhibition, indicating a synergistic interaction, with the most pronounced effect observed at AG (20 μM) and HST (40 μM). These findings suggest that AG and HST target complementary nodes within the inflammatory signaling network, and their combination offers enhanced anti-inflammatory efficacy. These findings provide preliminary mechanistic evidence of anti-inflammatory synergy, warranting further validation in keratinocyte models, animal studies, and clinical trials to establish translational relevance.