Methylnissolin mitigates microglia-mediated neuroinflammation and ischemic brain injury through PI3K/AKT and MAPK pathways

Background: Neuroinflammation driven by activated microglia is a major contributor to secondary brain damage following ischemic stroke. Targeting microglial activation has thus emerged as an important therapeutic strategy. Methylnissolin (ML), a pterocarpan-type isoflavonoid with documented anti-inflammatory activity and predicted ability to cross the blood-brain barrier, represents a potential modulator of microglial responses.

Methods: Primary microglia subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) and mice subjected to transient middle cerebral artery occlusion (tMCAO) were treated with ML. Microglial inflammatory responses were measured by RT-qPCR, Western blotting, Enzyme-linked immunosorbent assay, and immunofluorescence. Brain pathological alterations were assessed by 2,3,5-triphenyltetrazolium chloride (TTC) staining, Evans blue extravasation, and brain water content measurement. Behavioral changes was evaluated using grip strength, rotarod, modified neurological severity score (mNSS), and foot-fault tests. RNA Sequencing (RNA-seq) and targeted protein-level analyses were performed to investigate signaling pathways.

Results: ML markedly reduced the OGD/R-induced expression of pro-inflammatory mediators (IL-1β, IL-6, TNF-α, iNOS, COX-2) and suppressed activation of the PI3K/Akt and MAPK pathways in microglia. In tMCAO mice, ML treatment lessened infarct volume, brain edema, and blood-brain barrier leakage, preserved neuronal morphology, and improved neurological performance. RNA Sequencing and biochemical analyses in primary microglia showed modulation of the PI3K/Akt and MAPK signaling cascades by ML.

Conclusion: ML mitigates microglia-mediated neuroinflammation and protects against ischemic brain injury by downregulating the PI3K/Akt and MAPK pathways, suggesting that ML may be a promising therapeutic candidate for ischemic stroke.