Obacunone Inhibits Microglia-Mediated Neuroinflammation and Ischemic Injury by Targeting Mitogen-Activated Protein Kinase 1.

OBJECTIVE

Obacunone (OB) possesses anti-inflammatory, antioxidant, and anticancer properties. This study aimed to investigate the neuroprotective effects of OB in ischemic stroke, and to elucidate the underlying mechanisms.

METHODS

Primary microglia were preincubated with OB for 2 h, followed by lipopolysaccharide (LPS) stimulation for either 3 or 24 h. The levels of inflammatory cytokines in primary microglia were assessed via real-time PCR, enzyme-linked immunosorbent assay (ELISA), and Western blot. The activation of the mitogen-activated protein kinase/nuclear factor kappa B (MAPK/NF-κB) signaling pathway was evaluated via immunofluorescence staining and Western blot. For in vivo experiments, 8-week-old male C57BL/6J mice were randomly assigned to 4 groups: the sham-operated group, the middle cerebral artery occlusion (MCAO) model group and the MCAO group treated with OB (5 mg/kg/day and 10 mg/kg/day), and the sham-operated and MCAO model groups received an equivalent volume of vehicle. The neurological deficits and memory functions were evaluated by a cassette of behavior tests. 2,3,5-Triphenyl tetrazolium chloride (TTC) staining and Evans blue staining were performed to evaluate infarct size and blood-brain barrier permeability. Additionally, network pharmacology and molecular docking predicted mitogen-activated protein kinase 1 (MAPK1) as a potential target of OB, and this interaction was validated via surface plasmon resonance (SPR), cellulase thermal shift assay (CETSA), and drug affinity responsive target stability (DARTS) experiments.

RESULTS

OB effectively inhibited the activation of the MAPK/NF-κB pathway and reduced microglia-mediated inflammatory cytokine production both in vitro and in vivo. In addition, OB attenuated ischemic brain injury in MCAO mice and improved memory function 30 days after MCAO. Moreover, OB directly bound to MAPK1, with ARG-146 as the critical binding site.

CONCLUSION

Our findings suggest that OB binds to MAPK1 and alleviates neuroinflammation and ischemic injury, making it a potential therapeutic agent for ischemic stroke.