Unveiling the mechanism of amelioration of adjuvant-induced rheumatoid arthritis by Drynaria quercifolia rhizome extract using network pharmacology and gene expression-based studies.

Rhizomes of Drynaria quercifolia have long been traditionally used to manage rheumatic pain. However, there is limited research supporting this traditional practice and insufficient evidence demonstrating the molecular mechanisms of action of plant-derived bioactives in rheumatoid arthritis (RA). The current study aims to identify the effective components in Drynaria quercifolia methanol rhizome extract (DME) and their probable pharmacological mechanisms in alleviating Rheumatoid Arthritis (RA) using network-pharmacology, molecular docking, molecular-dynamics simulations, and gene expression-based validation. Gas chromatography-mass spectrometry (GC-MS) based screening identified 41 volatile phytocomponents from DME having drug-like potentiality. Network pharmacology-based screening revealed 117 therapeutic targets for RA of which 11 have been identified as core targets. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that key target genes were mostly enriched in the inflammatory response associated with multiple signalling pathways. Molecular docking and molecular dynamics studies revealed that key target proteins like serine/threonine-protein kinase (AKT1), peroxisome proliferator-activated receptor alpha (PPARA), and peroxisome proliferator-activated receptor gamma (PPARG), exhibited strong binding affinity and stable interactions with multiple phytocomponents present in DME. For experimental verification FCA (Freund's complete adjuvant)-induced chronic arthritis model employed for further molecular investigation. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) results validated that DME significantly (p ≤ 0.05) regulate the expression of key identified target genes AKT and PPARG in experimental RA model. Moreover, this study further confirmed that DME significantly (p ≤ 0.05) downregulated pro-inflammatory mediators like COX-2, IL-6 and TNF-α at gene and protein levels and also normalized (p ≤ 0.05) different oxidative stress parameters in both the low and high dose groups of DME-treated arthritic animals. In conclusion, the network-based in silico approach indicated that the phytocomponents present in DME probably act in a synergistic way to modulate key identified targets associated with RA, which was further validated by experimental studies. Therefore, DME could be a potential alternative in immunomodulatory therapies to combat RA and related chronic inflammatory conditions.