Intervention of Rutin and Ochnaflavone in the Attenuation of Neuroinflammation and Neuronal Apoptosis in Spinal Cord Injury.

Study DesignA multimethod experimental study.ObjectivesSpinal cord injury (SCI) has devastating neurological consequences, mainly through secondary injury mechanisms. Chinese medicine-derived flavonoids, including rutin (RUT) and ochnaflavone (OCE), have shown potential in modulating these processes, although their molecular mechanisms are not understood. This study aims to elucidate the neuroprotective mechanisms of RUB and OCE in SCI, with an emphasis on their regulatory function in microglia and the PI3K/AKT and NF-κB signaling pathways.MethodsSingle-cell RNA sequencing (scRNA-seq) of SCI mouse models was used to identify the inflammatory microglial subtype and define its molecular signature. Network pharmacology predicted that RUB and OCE targets overlap with SCI pathology. Their anti-inflammatory and anti-apoptotic effects were tested by vitro assays using LPS-stimulated BV2 microglia and a microglia-neuron co-culture system. In vivo validation was conducted using a murine SCI model to test protein expression by western blotting, immunofluorescence, and Enzyme-linked immunosorbent assay (ELISA).ResultsRUB and OCE synergistically inhibited LPS-induced microglial activation with significant downregulation of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) and neuronal apoptosis markers (Bax and cleaved Caspase-3) and upregulation of anti-apoptotic B. Mechanistically, the combination therapy suppressed the phosphorylation of PI3K, AKT, IKKβ, and NF-κB p65 without affecting their protein levels. These molecular effects were parallel in vivo, significantly reducing microglial hyperactivation and apoptotic signaling.ConclusionOur findings suggest that targeting the PI3K/AKT and NF-κB pathways may be an effective strategy for inhibiting secondary damage post-SCI, offering a novel therapeutic approach to reshape the post-injury environment and restore neural homeostasis.