Evaluation of synergistic anti-inflammatory activity of selected natural products in combination with teriflunomide in LPS stimulated RAW 264.7 cells for rheumatoid arthritis treatment

Abstract

Rheumatoid arthritis (RA) is an immune-mediated disorder that involves joints and synovial membrane, and requires combination therapy for effective management. Teriflunomide (TFD) belongs to Synthetic Disease-modifying antirheumatic drugs (DMARDs), prescribed either alone or in combination therapy. The combination of clinical drugs with natural products offers a promising strategy to enhance therapeutic efficacy and reduce the dose and thus ameliorate side effects. This study aims to evaluate the synergistic anti-inflammatory efficacy of TFD with selected natural products such as Andrographolide (ANG), Quercetin (QCN), Resveratrol (RES), Rutin (RUT) and Tanshinone IIA (TAN) in LPS-stimulated RAW264.7 cells. The initial screening was performed using nitric oxide (NO) assay to determine the IC50, with prior determination of the safest concentration range using the MTT assay. The study utilized a constant ratio design to assess synergy, employing the NO assay and calculating the combination index (CI), isobologram analysis, and dose reduction index (DRI). Among the tested combinations, QCN, RES, and TAN with TFD displayed low CI values and were evaluated for proinflammatory cytokines using ELISA. Notably, the combination of QCN and TFD showed significant synergistic activity with CI values of 0.470 and 0.607 (at Fa = 0.5) against TNF-α and IL-6, respectively. Intracellular reactive oxygen species (ROS) measurement with this combination revealed even greater synergistic activity, suggesting a promising approach for RA treatment.