LEP Knockdown Improves Functional Recovery After Spinal Cord Injury and Potentially Regulates Microglial Polarization...

Neuroinflammation following spinal cord injury (SCI) is primarily driven by abnormal microglial activation and represents a major barrier to neurological recovery. This study aimed to investigate the regulatory role of leptin (LEP) in SCI, with a particular focus on its effects on the JAK-STAT signaling pathway, microglial polarization, and neuronal injury. Transcriptome sequencing of spinal cord tissues was performed to identify differentially expressed genes (DEGs), followed by enrichment, immune cell infiltration, and protein-protein interaction (PPI) analyses to determine key pathways involved in SCI. A mouse SCI model was established, and LEP expression was silenced using shRNA. Motor function recovery and pathological changes were assessed by Basso-Beattie-Bresnahan scoring, rotarod testing, Nissl staining, and hematoxylin-eosin staining. qRT-PCR, Western blot, and immunofluorescence were used to examine inflammatory mediators, microglial polarization markers, and JAK-STAT signaling. LPS-stimulated BV2 and primary microglia were used to evaluate the effects of LEP on inflammatory activation and polarization, and interferon gamma (IFN-γ) was applied to verify the involvement of JAK-STAT signaling. A microglia-PC12 co-culture system was used to determine the effects of LEP intervention on neuronal apoptosis and oxidative stress. Transcriptomic profiling revealed a marked enhancement of inflammation- and immunity-related pathways after SCI, with the JAK-STAT pathway identified as a major regulatory axis strongly associated with upregulated LEP expression. Functional analyses showed that LEP knockdown significantly improved motor recovery after SCI. LEP silencing suppressed the phosphorylation of JAK2 and STAT3, promoted the shift of microglia from the M1 to the M2 phenotype, reduced iNOS, TNF-α, and IL-6, and increased IL-4 and IL-10 expression. IFN-γ partially reversed these effects, confirming that LEP regulates microglial polarization through the JAK-STAT pathway. LEP knockdown in microglia also attenuated neuronal apoptosis and oxidative stress, decreasing cleaved caspase-3, Bax, MDA, and ROS levels while restoring Bcl-2 and enhancing SOD activity. LEP may function as a modulator associated with JAK-STAT signaling activation during neuroinflammation after SCI. Integrating transcriptomic and mechanistic evidence, we demonstrate that LEP silencing is associated with suppressed activation of JAK-STAT pathway, promotes M2 microglial polarization, reduces neuronal apoptosis and oxidative damage, and ultimately enhances spinal cord repair and functional recovery.