Network-guided identification and in vitro/in vivo validation of withaferin A as an inhibitor of nuclear factor kappa B for spinal cord injury repair

Virtual screening and experimental validation identified Withaferin A as a candidate therapy for spinal cord injury. • Withaferin A attenuates NF‑κB signaling by reducing p65 phosphorylation and promoting an anti‑inflammatory macrophage phenotype. • Molecular modeling and experimental validation confirm covalent binding of Withaferin A to p65 Cys160 via Michael addition. • Withaferin A enhances neuronal regeneration, restores motor function, preserves axonal integrity, and promotes angiogenesis in SCI mice. Background Spinal cord regeneration after injury remains a major clinical challenge due to the persistent inflammatory microenvironment associated with immune cell infiltration. Withaferin A (WA), a natural anti-inflammatory steroidal lactone with potential NF-κB-modulating activity, was identified by network-based screening as a candidate for spinal cord injury (SCI) and selected for further study. Purpose This study aimed to identify and evaluate potential anti-inflammatory small-molecule therapeutics for SCI using a network-based drug screening strategy. Study Design Experimental study combining computational drug screening, in vitro macrophage assays and in vivo SCI mouse models. Methods Network pharmacology identified 296 candidate drugs targeting 113 SCI-related genes. Five top candidates, selected for chemical diversity and accessibility, were tested in LPS-stimulated in vitro models for effects on macrophage polarization and cytokine release. Molecular docking was used to predict drug–target interactions. The lead compound, WA, was then evaluated in SCI mice for inflammation, angiogenesis, neuroregeneration and motor recovery. Results WA showed the strongest anti-inflammatory activity, dose-dependently inhibiting LPS-induced M1 polarization and TNF-α/IL-6 secretion while promoting M2 polarization and IL-4/IL-10 secretion. Integrated computational and experimental analyses identified Cys160 in a hydrophobic pocket of NF-κB p65 as the covalent binding site of WA. In vivo , WA modulated macrophage polarization, reduced inflammatory mediator secretion, increased VEGF immunoreactivity and promoted neuroregeneration and motor recovery. Conclusion WA exerts significant anti-inflammatory and neuroregenerative effects in SCI models. It also increases VEGF immunoreactivity, suggesting a potential pro-angiogenic effect after injury. These findings support WA as a promising therapeutic candidate for SCI. Download: Download high-res image (272KB) Download: Download full-size image