Multi-omics analysis of ketogenic diet-mediated neural repair in spinal cord injury: Targeting of lysosomal autophagy through CTSB/LAMP2 regulation.

Spinal cord injury (SCI) initiates secondary pathologies characterized by dysregulated autophagy and neuroinflammation Although the ketogenic diet (KD) has shown potential in promoting functional recovery after SCI, the mechanisms underlying KD-mediated neural repair remain unclear. We employed an integrated multi-omics approach combining 4D proteomics, transcriptomics, and single-cell RNA sequencing in a C5 hemi-contusion mouse model. This was combined with in vitro validation using β-hydroxybutyrate (β-OHB)-treated BV2 microglia cells to investigate KD's effects on lysosome-mediated autophagy and microglial dynamics. Behavioral assessments and histopathological analyses were conducted over acute to chronic phases, spanning from 0 to 8 weeks post-injury. KD attenuated maladaptive lysosomal activation by downregulating cathepsin B (CTSB) and lysosomal-associated membrane protein 2 (LAMP2). This suppression concurrently reduced pro-inflammatory cytokines levels (IL-1β, TNF-α, IL-6) while facilitating M2 microglia polarization. Proteomic analysis identified 73 proteins responsive to KD that are associated with endoplasmic reticulum stress and chaperone-mediated autophagy. Single-cell transcriptomics revealed co-upregulation of CTSB and LAMP2 in injury-associated microglia subpopulations. Importantly, β-OHB partially replicated the effects of KD in vitro, reducing autophagy hyperactivity and enhancing M2 polarization. By targeting CTSB/LAMP2 axis, KD orchestrates dual neuroprotective mechanisms: lysosomal homeostasis restoration and immunomodulatory reprogramming. This coordinated action reconciles proteostatic regulation with microglial M1/M2 polarization dynamics, establishing KD as a multimodal metabolic intervention capable of simultaneously addressing autophagy dysregulation and neuroinflammation following SCI. These findings hold significant translational potential for neurotrauma management.