Targeting NF-κB pathway for the anti-inflammatory potential of Bhadradarvadi kashayam on stimulated RAW 264.7 macrophages

Macrophage-arbitrated inflammation is associated with the regulation of rheumatoid arthritis (RA). Low risk and better efficiency are steered herbal drugs more credible than conventional medicines in RA management. Bhadradarvadi (BDK) concoction has been traditionally used for rheumatism in Ayurveda. However, the mechanisms at the molecular level are still elusive. This study was designed to inspect the process of immunomodulation and anti-inflammatory properties of BDK in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages for the first time. BDK concoction was prepared and evaluated with the stimulated murine macrophage-like RAW 264.7 cell lines. TNF-α, IL6, and PGE₂ were quantified by ELISA. The normalization of the fold change in the expression of the target gene mRNA was done by comparing the values of the β-actin housekeeping gene using the 2^-ΔΔCt comparative cycle threshold. The expression of TNF-α, IL6, iNOS, and COX-2 in the RAW 264.7 macrophage cells was analyzed using flow cytometry. Our results showed that BDK (150-350 μl/ml) treatment significantly decreased the inflammatory cytokines (TNF-α, and IL-6) and inflammatory mediators (PGE₂) in LPS-stimulated RAW 264.7 macrophage cells. The pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) expression, inflammatory enzymes (iNOS and COX-2), and NF-κBp65 were significantly downregulated at transcriptome level in LPS-stimulated RAW 264.7 macrophage cells. The flow cytometry analysis revealed that BDK treatment diminished the TNF-α, IL-6, iNOS, and COX-2 expression at the proteome level, as well as obstruction of NF-κB-p65 nuclear translocation was observed by immunofluorescence analysis in LPS-stimulated RAW 264.7 macrophage cells. Collectively, BDK can intensely augment the anti-inflammatory activities via inhibiting the NF-κB signaling pathway trigger for treating autoimmune disorders including RA.