Unraveling the Dual Anti-Inflammatory and Antioxidant Mechanisms of Acteoside: Computational Insights and Experimental Validation.

Acteoside (ACT) is one of the primary bioactive ingredients in Cistanche tubulosa (Schenk). Its remarkable efficacy in treating immune-related and inflammatory disorders has garnered significant interest among scientific circles. However, the anti-inflammatory and antioxidant effects of ACT and its underlying molecular mechanisms require further investigation. In this study, pharmacophore-based reverse docking and molecular dynamics simulations identified potential anti-inflammatory targets in silico. Studies conducted in vitro with lipopolysaccharide (LPS)-induced RAW264.7 cells validated the anti-inflammatory properties of ACT. Methyl thiazolyl tetrazolium (MTT) and lactate dehydrogenase (LDH) assays indicated ACT's non-toxic and growth-promoting effects on cells. ACT significantly reduced nitric oxide (NO) and reactive oxygen species (ROS) production and restored levels of antioxidant enzymes. It also decreased pro-inflammatory cytokines. Western blotting assays indicated that ACT inhibited p38, TNF-α, PI3 K/AKT, and NF-κB signaling pathways. These findings underscore ACT's ability to mitigate acute inflammation in RAW264.7 cells by modulating key signaling pathways and provide the scientific basis for enhancing the medicinal value of ACT and future drug development.