What does this research mean for the field?

Obacunone alleviates neuroinflammation and ischemic injury by binding to mitogen-activated protein kinase 1 (MAPK1) in a model of ischemic stroke. Novelty: ClaimNovelty.NOVEL_FINDING. Consensus alignment: ConsensusAlignment.SUPPORTS_CONSENSUS.

What question did this study set out to answer?

The aim is to explore the neuroprotective effects of obacunone in ischemic stroke and the mechanisms involved.

March 5, 2026Journal of Inflammation Research0 citationsOpen Access

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

JHJing HuangNanjing University of Chinese Medicine ZHZuobin HuNanjing University of Chinese Medicine JZJie ZhangNanjing University of Chinese Medicine

Key Points

The aim is to explore the neuroprotective effects of obacunone in ischemic stroke and the mechanisms involved.
Preincubation of primary microglia with obacunone (OB) followed by LPS stimulation.
Assessment of inflammatory cytokines via real-time PCR, ELISA, and Western blot.
Evaluation of MAPK/NF-κB pathway activation using immunofluorescence and Western blot.
In vivo experiments with male C57BL/6J mice using MCAO model and behavior tests.
Predictions of OB's target through network pharmacology and validation using SPR, CETSA, and DARTS.
OB significantly inhibited MAPK/NF-κB pathway activation in both vitro and in vivo assessments.
Reduced inflammatory cytokine production was observed resulting from OB treatment.
Ischemic brain injury was alleviated in MCAO mice receiving OB.
Memory function improved 30 days post-MCAO in the OB-treated group.
OB was found to directly bind to MAPK1 at ARG-146 as the critical binding site.

Abstract

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. Keywords: obacunone, ischemic stroke, microglia, neuroinflammation, mitogen-activated protein kinase 1

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