Columbianadin improves M1 polarization of microglia after spinal cord injury by stabilizing PTEN and inhibiting the PI3K/AKT pathway.

BACKGROUND

Inhibiting the polarization of microglia toward the M1 pro-inflammatory phenotype to alleviate neuroinflammation and improve neuronal survival is a critical strategy for promoting spinal cord injury (SCI) repair. PTEN, a core regulator of cellular inflammatory responses, exerts its function by negatively modulating the PI3K/AKT signaling pathway. Columbianadin (CBN), a coumarin derivative isolated from the roots of Angelica sinensis (Oliv.) Diels, possesses multiple biological activities, including potent anti-inflammatory effects.

OBJECTIVE

This study aimed to evaluate the regulatory effect of CBN on microglial polarization post-SCI and elucidate its underlying molecular mechanisms.

METHODS

A combination of experimental techniques, including Western blotting, quantitative real-time PCR (qPCR), immunofluorescence, ubiquitination assays, co-immunoprecipitation (Co-IP), live/dead staining, molecular docking, cellular thermal shift assay (CETSA), bioluminescence imaging (BLI), differential scanning fluorimetry (DSF), and network pharmacology analysis, was employed in this study. Additionally, a rat SCI model was established to evaluate the impact of CBN on motor function recovery.

RESULTS

CBN inhibited microglial M1 polarization, mitigated neuroinflammation, and improved neuronal survival, thereby facilitating SCI repair. Mechanistically, CBN directly targeted PTEN, by suppressing the ubiquitin-mediated degradation of PTEN while spatially promoting the interaction between PTEN and the PI3K regulatory subunit p85α. These dual effects synergistically enhanced PTEN protein stability and biological activity, jointly inhibiting PI3K/AKT signaling pathway activation.

CONCLUSION

Our study identifies CBN as a potential candidate drug for SCI treatment, as it enhances PTEN stability to inhibit the PI3K/AKT pathway, thereby regulating microglial polarization and protecting neurons from neuroinflammatory damage.