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

Abstract

Background: Fronds and rhizomes of Drynaria quercifolia have long been used to manage rheumatic pain. This study aimed to identify the effective components in Drynaria quercifolia methanol rhizome extract (DME) and their roles in alleviating Rheumatoid Arthritis (RA) using network-pharmacology, molecular docking, molecular-dynamics simulations, and gene expression validation. Result: Gas chromatography–mass spectrometry (GC-MS) based screening identified 41 volatile phytocomponents from DME having drug-like potentiality. Database mining and network analyses revealed 117 potential targets of which 11 have been identified as core targets. In addition, the analysis of the herb-compound-target-pathway network investigation showed n-Hexadecanoic acid, octadecanoic acid, Ergost-5-en-3-ol, and (3 beta 24r) as potential key phytocomponents for the treatment of RA. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) 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. Moreover, the influence of DME on regulating the expression of essential gene targets, as identified in silico, has been verified through experimental validation using RT-Q-PCR. Freund's Complete Adjuvant (FCA)-induced chronic RA model animals further validated that DME could significantly ameliorate RA in experimental Wistar rat model. Quantitative RT-PCR results demonstrated that DME significantly (p<0.05) downregulated the mRNA expression of the core target gene AKT1, while enhancing the expressions of PPARG and Iκβ genes in the experimental animal groups compared to that in arthritic control group animals. Furthermore, our investigation revealed a noteworthy (p<0.05) downregulation of pro-inflammatory mediators such as COX-2, TNF-α, and IL-6, both at the gene and protein levels, suggesting noteworthy mitigation in the severity of inflammatory arthritis in the experimental animal groups. Conclusion: In conclusion, the phytocomponents present in DME seem to act in a synergistic way to modulate key targets of inflammatory pathways associated with RA and could provide an immunomodulatory therapy for RA and associated chronic inflammations.