Unraveling the Anti-Arthritic Potential of Bioactive Compounds From Melaleuca alternifolia Oil: Integrated In Vivo, Molecular Docking, and Network Pharmacology Insights.

Rheumatoid arthritis (RA) is a chronic inflammatory condition that affects millions of people globally, often leading to morbidity. Recent research highlights the potential of natural products in managing arthritis symptoms and modifying disease progression. Among these, Melaleuca alternifolia oil (MAO) has emerged as a promising therapeutic option due to its anti-inflammatory and analgesic properties. A total of 36 Sprague-Dawley rats weighing between 200 and 220 g were divided into six groups (n = 6). On day 0, arthritis was induced by CFA. On day 8, treatment started and lasted till day 28. Paw edema and arthritic score were measured. Moreover, hematological, histological, and biochemical parameters were evaluated. The mRNA expression levels of anti-inflammatory cytokine IL-4 and pro-inflammatory cytokines such as TNF-α, IL-6, NF-κB, and IL-1β were measured using real-time polymerase chain reaction (qPCR). ELISA was used to detect PGE2 levels. Network pharmacology was used to evaluate the hub genes between the phytocompounds and rheumatoid arthritis. Molecular docking and scoring calculations were done for the top matched hub genes for moderate confirmations between phytocompounds and targets. GC-MS analysis was also performed to identify potential anti-inflammatory constituents present in the plant oil. Treatment with MAO effectively reduced paw edema and swelling. Histopathological examinations demonstrated significant improvement in the treatment groups. Biochemical parameters such as creatinine, urea, alanine aminotransferase (ALT), and alkaline phosphatase (ALP) did not significantly alter, and hematological parameters such as red blood cells (RBC), white blood cells (WBC), hemoglobin (Hb), and platelet count were restored in the treated group. The mRNA expression of anti-inflammatory cytokines, including IL-4, was upregulated, and pro-inflammatory biomarkers such as TNF-α, IL-1β, IL-6, and NF-κB were significantly downregulated in the arthritic control group. Furthermore, treatment with MAO significantly reduced PGE2 levels. Network pharmacology indicated the hub genes and several biological processes, molecular functions, cellular components, and KEGG pathways that are involved in rheumatoid arthritis. Molecular docking was performed against hub genes such as AKT1, BCL-2, MAPK3, and MMP9 that are involved in various pro-inflammatory cytokine formation and pathophysiology of rheumatoid arthritis. GC-MS analysis of the plant's oil revealed the presence of numerous anti-inflammatory and antirheumatic phytochemicals. MAO possesses considerable anti-arthritic properties by downregulating pro-inflammatory biomarkers such as IL-1β, NF-κB, IL-6, TNF-α, and upregulating anti-inflammatory IL-4. Further investigations are needed to explore its potential as a natural therapeutic option for treating RA.