Synergistic anti-inflammatory effects of silibinin and thymol combination on LPS-induced RAW264.7 cells by inhibition of NF-κB and MAPK activation

Background: Combination drug therapy has become an effective strategy for inflammation control. The anti-inflammatory capacities of silibinin and thymol have each been investigated on its own, but little is known about the synergistic anti-inflammatory effects of these two compounds. Purpose: This study aims to investigate the synergistic anti-inflammatory effects of silibinin and thymol when administered in combination to lipopolysaccharide (LPS)-induced RAW264.7 cells. Methods: RAW264.7 cells were pre-treated with silibinin and thymol individually or in combination for 2 h before LPS stimulation. Cell viability was detected by the MTT assay. Nitric oxide (NO) production was measured by Griess reagent. Reactive oxygen species (ROS) was evaluated by 2',7'-dichlorofluorescein-diacetate. ELISA was used to detect tumour necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6). Western blot was performed to analyse the protein expression of LPS-induced RAW264.7 cells. Results: We observed a synergistic anti-inflammatory effect of silibinin and thymol when administered in combination to LPS-induced RAW264.7 cells. Silibinin combined with thymol (40 mu M and 120 mu M respectively, with the molar ratio 1:3) had more potent effects on the inhibition of NO, TNF-alpha, and IL-6 than those exerted by individual administration of these compounds in LPS-induced RAW264.7 cells. The combination of silibinin and thymol (40 mu M and 120 mu M respectively, with the molar ratio 1:3) strongly inhibited ROS and cyclooxygenase-2 (COX-2). More importantly, the combination of silibinin and thymol (40 mu M and 120 mu M respectively, with the molar ratio 1:3) was also successful in inhibiting nuclear factor-kappa B (NF-kappa B) and mitogen-activated protein kinase (MAPK) activities. Our results suggest that the synergistic anti-inflammatory effects of silibinin with thymol were associated with the inhibition of NF-kappa B and MAPK signalling pathways. Conclusion: The combination of silibinin and thymol (40 mu M and 120 mu M, respectively, with the molar ratio 1:3) could inhibit inflammation by suppressing NF-kappa B and MAPK signalling pathways in LPS-induced RAW264.7 cells.