Kakkalide promotes spinal cord injury repair by regulating microglial M2 polarization via mitophagy.

Abstract

BACKGROUND: Secondary inflammatory cascades after spinal cord injury (SCI) drive progressive neurological deterioration, with microglial activation as a key determinant of lesion progression. Kakkalide, a naturally occurring isoflavone, exhibits antioxidant and anti-inflammatory activities; however, its efficacy and mechanism of action in SCI remain insufficiently defined. PURPOSE: To evaluate the therapeutic effects of kakkalide in SCI and delineate the molecular pathway through which it modulates microglia-driven neuroinflammation and functional recovery. METHODS: SCI was induced in mice to assess the effects of kakkalide on locomotor function and secondary inflammation. In vitro, LPS-stimulated BV2 microglia were used to examine phenotypic polarization. Proteomic profiling and molecular docking were performed to identify candidate targets of kakkalide. Mechanistic dependence was tested using the SIRT3-selective inhibitor 3-TYP in both cell and animal models. Immunofluorescence, western blotting, qPCR, and assays of mitochondrial membrane potential and mitochondrial reactive oxygen species (mtROS) were used to interrogate microglial phenotypes, mitochondrial homeostasis, and related signaling pathways. RESULTS: Kakkalide treatment significantly reduced secondary inflammation and improved locomotor recovery in SCI mice. In BV2 microglia, kakkalide promoted a shift from a pro-inflammatory M1-like state toward an anti-inflammatory M2-like phenotype. Mechanistically, kakkalide restored mitochondrial homeostasis by activating BNIP3/NIX-dependent mitophagy, thereby suppressing mtROS accumulation. Proteomics and docking analyses identified sirtuin 3 (SIRT3) as a putative direct target of kakkalide. Consistently, kakkalide increased SIRT3 expression and activity in microglia, and SIRT3 activity was required for kakkalide-induced mitophagy. Notably, co-administration of 3-TYP abrogated the neuroprotective and functional benefits of kakkalide in vivo. CONCLUSION: Kakkalide mitigates SCI by directly engaging SIRT3 and activating BNIP3/NIX-mediated mitophagy, which stabilizes mitochondrial function, limits oxidative stress, and biases microglia toward an M2-like protective phenotype. These findings define a previously unrecognized mechanism for kakkalide in SCI and nominate the SIRT3-mitophagy axis as a tractable therapeutic target for neuroinflammatory disorders.