Evaluation of the anti-inflammatory potential of atorvastatin targeting TNF-α, IL-6, and IL-1β using integrated in vitro and in silico approaches

Inflammation is a complex biological response driven by excessive activation of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6), which play pivotal roles in chronic inflammatory diseases. Given the safety and wide clinical use of atorvastatin, this study aimed to explore its potential repurposing as an anti-inflammatory agent through the inhibition of these cytokines. Molecular docking was performed to predict the binding affinity and interaction profile of atorvastatin with TNF-α, IL-1β, and IL-6, followed by 200 ns molecular dynamics (MD) simulations, principal component analysis (PCA), and energy calculations to evaluate the stability and energetics of the complexes. Furthermore, the anti-inflammatory effects of atorvastatin (5–20 µM) were evaluated in LPS-stimulated RAW 264.7 macrophages using MTT cytotoxicity, cytokine assays (TNF-α, IL-6, IL-1β), and nitric oxide quantification. Docking study revealed strong binding affinity toward TNF-α (− 7.9 kcal/mol) through hydrogen bonding, supported by stable MD trajectories. The MM-GBSA binding energy (− 72.90 ± 4.38 kcal/mol) confirmed a favorable and stable interaction predominantly driven by hydrophobic and van der Waals forces. In -vitro study revealed, that atorvastatin showed no cytotoxicity and produced a strong dose-dependent inhibition of TNF-α, IL-6, IL-1β, and nitric oxide levels, with maximal anti-inflammatory activity observed at 20 µM. These findings suggest that atorvastatin may directly inhibit TNF-α and related cytokines, thereby modulating NF-κB–mediated inflammatory signaling. This study provides molecular-level insight supporting the repurposing of atorvastatin as a promising multi-target anti-inflammatory therapeutic.