Inflachromene Blocks Neuronal HMGB1 Translocation to Mitigate Hippocampal TLR4-NF-κB-Mediated Neuroinflammation and Depression-Like Behavior After Spinal Cord Injury.

Growing evidence suggests that spinal cord injury (SCI) can result in chronic brain damage, potentially leading to depression and cognitive deficits. High mobility group box-1 (HMGB1) plays a pivotal role in the pathogenesis of central nervous system (CNS) disorders, yet the mechanism by which SCI induces depression remains unclear. This study aims to explore the key mechanisms by which HMGB1 contributes to depression-like behaviors following SCI. A rat model of SCI-induced depression was established to assess the therapeutic effects of ICM on neurobehavioral outcomes, microglial activation, and neuronal damage. Molecular docking was performed to predict the interaction between ICM and HMGB1. Hippocampal RNA-seq profiled ICM-responsive transcriptional changes, with DEGs identified and KEGG enrichment highlighting TLR4-NF-κB signaling, followed by Western blot validation of selected targets. In vivo, immunofluorescence, Western blotting, qRT-PCR, and ELISA were utilized to evaluate HMGB1 translocation, TLR4-NF-κB pathway activation, and inflammatory cytokine production in the hippocampus. In vitro, a microglia-neuron co-culture system was used to investigate the role of neuronal HMGB1 in microglia-mediated neuroinflammation, with suppression of HMGB1 achieved via ICM pretreatment or siRNA knockdown. Our findings showed that inflachromene (ICM) effectively suppressed microglial activation, thereby reducing hippocampal neuronal injury in the SCI-induced depression model. ICM alleviated neuroinflammatory responses by directly preventing the nuclear-to-cytoplasmic translocation of HMGB1, a critical damage-associated molecular pattern released from injured neurons that triggers microglial activation and inflammatory cytokine expression. Bulk RNA-seq of hippocampal tissue from the Sham, SCI, and SCI + ICM groups revealed significant enrichment of Toll-like receptor and NF-κB signaling pathways following SCI, highlighting the HMGB1-TLR4-NF-κB axis as a central inflammatory mechanism. Consistent with the transcriptomic prediction, both ICM treatment and neuronal HMGB1 knockdown markedly inhibited TLR4-NF-κB signaling, reduced reactive-oxygen-species accumulation, and diminished pro-inflammatory cytokine production in microglia. Notably, the combination of ICM and siHMGB1 produced no additive effect, confirming neuronal HMGB1 as the upstream driver of TLR4-NF-κB-dependent neuroinflammation. SCI-induced release of hippocampal neuronal HMGB1 activates microglial TLR4-NF-κB signaling, driving neuroinflammation and contributing to depressive-like behaviors. Targeting HMGB1 nuclear translocation with ICM represents a promising therapeutic approach for depression following SCI.