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Kakkalide promotes spinal cord injury repair by regulating microglial M2 polarization via mitophagy
Tao You1, Bin Dai1, Xintian Ding2
1Department of Orthopedics, Centre for Leading Medicine and Advanced Technologies of IHM, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, 230001, China.
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.
Insights
Kakkalide, an isoflavone, improves spinal cord injury recovery by activating SIRT3 and mitophagy, reducing inflammation and oxidative stress. This study reveals a novel therapeutic pathway for neuroinflammatory disorders.
Area of Science:
- Neuroscience
- Pharmacology
- Cell Biology
Background:
- Secondary inflammation post-spinal cord injury (SCI) exacerbates neurological damage, with microglial activation being a key factor.
- Kakkalide, a natural isoflavone, possesses antioxidant and anti-inflammatory properties, but its role in SCI is not well-understood.
Purpose of the Study:
- To investigate the therapeutic potential of kakkalide in SCI models.
- To elucidate the molecular mechanisms underlying kakkalide's effects on microglia-mediated neuroinflammation and functional recovery.
Main Methods:
- SCI was induced in mice to evaluate kakkalide's impact on locomotor function and inflammation.
- In vitro studies used LPS-stimulated BV2 microglia to assess phenotypic changes.
- Proteomic profiling, molecular docking, and mechanistic studies with a SIRT3 inhibitor (3-TYP) were employed.
- Techniques included immunofluorescence, western blotting, qPCR, and assays for mitochondrial function.
Main Results:
- Kakkalide treatment improved locomotor function and reduced inflammation in SCI mice.
- Kakkalide shifted microglia from a pro-inflammatory M1 to an anti-inflammatory M2 phenotype.
- The compound restored mitochondrial homeostasis via BNIP3/NIX-dependent mitophagy, suppressing mitochondrial reactive oxygen species (mtROS).
- Sirtuin 3 (SIRT3) was identified as a direct target; kakkalide increased SIRT3 activity, which was essential for mitophagy and neuroprotection.
Conclusions:
- Kakkalide mitigates SCI by activating SIRT3 and mitophagy, stabilizing mitochondrial function, and reducing oxidative stress.
- This mechanism promotes a protective microglial phenotype, offering a novel therapeutic strategy for SCI.
- The SIRT3-mitophagy pathway represents a promising target for neuroinflammatory diseases.
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