Anti-inflammatory, antioxidant, and protective effects of methanolic extract of Paederia foetida and keracyanin in carrageenan-induced inflammation in rats: Integrated in silico, in vitro and in vivo mechanistic evidence targeting MAPK and NF-κB pathways

ETHNOPHARMACOLOGICAL RELEVANCE: Paederia foetida L. is traditionally used in Northeast India and Southeast Asia to treat inflammatory disorders. This study validates its traditional use and bridges ethno-medicinal knowledge with modern pharmacological evidence by providing mechanistic support for the anti-inflammatory and antioxidant activities of P. foetida and its bioactive constituent keracyanin. AIM OF THE STUDY: This study aimed to evaluate the anti-inflammatory, antioxidant, and protective effects of P. foetida extract and its bioactive phytoconstituent, keracyanin, through integrated in silico, in vitro, and in vivo approaches, and to elucidate the underlying molecular mechanisms with particular emphasis on the MAPK and NF-κB signaling pathways. MATERIALS AND METHODS: Thirty-six polyphenolic compounds previously identified from the methanolic extract of P. foetida (PFME) by HR-LC-MS were retrieved from PubChem and evaluated as phytoligands. Molecular docking and 100 ns molecular dynamics simulations were performed against key inflammatory targets, including iNOS, MAPK, and TNF-α. The pharmacokinetic, toxicity, and biological activity profiles of the identified compounds were predicted using ADMET and PASS analyses. Based on the docking and simulation results, keracyanin was selected for further investigation. The in vitro anti-inflammatory and antioxidant activities of PFME and keracyanin were assessed using lipoxygenase (LOX), cyclooxygenase-2 (COX-2), and nitric oxide (NO) inhibition assays, with quercetin and celecoxib as reference compounds. In vivo anti-inflammatory activity was evaluated in a carrageenan-induced paw edema model in Wistar albino rats orally administered PFME (250 and 500 mg/kg) or keracyanin (25 and 50 mg/kg). Paw edema and percentage inhibition were recorded at different time points, and histopathological changes were examined using hematoxylin and eosin staining. Levels of inflammatory mediators, including CRP, NF-κB, iNOS, TNF-α, IL-1β, IL-6, p38-MAPK, MPO, COX-2, NO, and PGE2, the anti-inflammatory cytokine IL-10, oxidative stress markers (MDA and NO), and antioxidant parameters (TAOC, CAT, SOD, GPx, and GSH), were quantified using ELISA and biochemical assay kits. The expression of TNF-α, p38-MAPK, and NF-κB signaling proteins was further evaluated by Western blot analysis. Statistical significance was determined using one-way ANOVA followed by Tukey's post hoc test (p < 0.05). RESULTS: Molecular docking and molecular dynamics simulations demonstrated that keracyanin exhibited strong and stable interactions with key inflammatory targets, including iNOS, MAPK, and TNF-α, showing binding affinities superior to those of the standard inhibitors. PASS prediction suggested a high probability of anti-inflammatory activity, while ADMET analysis indicated favorable pharmacokinetic properties, good drug-likeness, and low toxicity. In vitro studies showed that keracyanin significantly suppressed LOX and COX-2 activities as well as NO production, with efficacy comparable to PFME and greater than that of the reference compounds quercetin and celecoxib. In vivo, PFME (250 and 500 mg/kg) and keracyanin (25 and 50 mg/kg) significantly attenuated carrageenan-induced paw edema in a dose-dependent manner, achieving 53.5-64.2% and 54.5-65.8% inhibition, respectively, at 5 h (p < 0.05), compared with 69.5% inhibition by diclofenac. Histopathological examination confirmed marked protection against carrageenan-induced tissue injury, evidenced by reduced inflammatory cell infiltration, diminished epithelial hyperplasia, minimal edema, and preservation of normal tissue architecture. Furthermore, PFME and keracyanin significantly decreased the levels of inflammatory mediators, while enhancing IL-10 production and restoring antioxidant defenses. These effects were accompanied by reduced lipid peroxidation and significant downregulation of the MAPK and NF-κB signaling pathways, as confirmed by ELISA and Western blot analyses. CONCLUSION: The integrated in silico, in vitro, and in vivo findings demonstrate that PFME and its bioactive constituent keracyanin exert potent anti-inflammatory and antioxidant effects through modulation of MAPK and NF-κB signaling pathways. These results provide mechanistic support for the traditional use of P. foetida in the management of inflammatory disorders and identify keracyanin as a key contributor to its therapeutic activity.