Sappanone A Promotes Motor Function Recovery in Spinal Cord Injury Mice by Inhibiting Microglial <scp>M1</scp> Polarization via Activation of the Keap1/Nrf2 Pathway

Spinal cord injury (SCI), a central nervous system disease resulting in significant functional impairment, neuroinflammation and oxidative stress are its core pathological features. Sappanone A (SA), a naturally occurring flavonoid compound predominantly derived from Caesalpinia sappan heartwood, demonstrates dual functionality encompassing both anti-inflammatory and antioxidant effects. The specific function of SA in SCI and its associated mechanisms constitute uncharted territory. This study examined the underlying neuroprotective mechanisms of SA in spinal cord injury using a T9 spinal cord contusion mouse model and lipopolysaccharide (LPS) + interferon-gamma (IFN-γ) -stimulated BV2 microglia. The Basso Mouse Scale (BMS), footprint analysis, and swimming tests were employed to comprehensively evaluate motor function in mice. Spinal cord lesion areas were quantified via Hematoxylin-Eosin (HE) staining, surviving neurons were counted using Nissl staining, and myelin integrity was assessed with Luxol Fast Blue (LFB) staining. Western blot, Enzyme-Linked Immunosorbent Assay (ELISA) and Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) detected inflammatory and anti-inflammatory factor expression, whereas immunofluorescence and flow cytometry analyzed microglial activation and polarization states. Oxidative stress levels were quantitatively assessed using an oxidative stress assay kit in conjunction with the DCFH-DA fluorescent probe. Investigate the underlying mechanisms by analyzing the changes in nuclear factor erythroid 2-related factor 2 (Nrf2) expression through Western blot and immunofluorescence. Results demonstrated that SA treatment significantly improved motor function in SCI mice, reduced the lesion area in SCI, increased residual myelin area, and enhanced neuronal survival. In both SA-treated SCI mice and LPS + IFN-γ-stimulated BV2 cells, oxidative stress responses were attenuated, accompanied by a reduction in the number of M1-type microglia and reduced expression of inflammatory factors. Mechanistic investigations reveal that SA potentially alleviates oxidative stress via Keap1/Nrf2 pathway activation, inhibition of M1 polarization in microglia. Rescue experiments confirmed that the Nrf2 inhibitor ML385 reversed SA's neuroprotective effects. In conclusion, SA may modulate oxidative stress responses through the Keap1/Nrf2 pathway, thereby regulating microglial polarization and neuroinflammation, providing insights for clinical therapeutic strategies targeting SCI.