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
- Phytomedicine. 2026 Aug:158:158380. doi: 10.1016/j.phymed.2026.158380.
- 1. Department of Spine Surgery, Jining NO.1 People's Hospital Affiliated to Shandong First Medical University & Shandong Academy of Medical Sciences, Jining, 272011, China; Medical Integration and Practice Center, Cheeloo College of Medicine, Shandong University, Jinan, 250012, China; Shandong Provincial Key Medical and Health Laboratory of Neuroinjury and Repair, Jining, 272011, China.
- 2. Department of Spine Surgery, Jining NO.1 People's Hospital Affiliated to Shandong First Medical University & Shandong Academy of Medical Sciences, Jining, 272011, China; Shandong Provincial Key Medical and Health Laboratory of Neuroinjury and Repair, Jining, 272011, China.
- 3. Department of Spine Surgery, Jining NO.1 People's Hospital Affiliated to Shandong First Medical University & Shandong Academy of Medical Sciences, Jining, 272011, China; Medical Integration and Practice Center, Cheeloo College of Medicine, Shandong University, Jinan, 250012, China; Shandong Provincial Key Medical and Health Laboratory of Neuroinjury and Repair, Jining, 272011, China. Electronic address: [email protected].
- 4. Department of Spine Surgery, Jining NO.1 People's Hospital Affiliated to Shandong First Medical University & Shandong Academy of Medical Sciences, Jining, 272011, China; Shandong Provincial Key Medical and Health Laboratory of Neuroinjury and Repair, Jining, 272011, China. Electronic address: [email protected].
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.