Formononetin attenuates cerebral ischemia-reperfusion injury by regulating microglial glycolysis through AMPK.

ETHNOPHARMACOLOGICAL RELEVANCE: Astragalus membranaceus (Fisch.) Bunge is a fundamental herb in traditional Chinese medicine. It has a long history of use in clinical practice for treating stroke and its associated sequelae. Notably, Formononetin (FN) has been identified as a primary bioactive isoflavone in this herb. However, its specific function in cerebral ischemia‒reperfusion injury remains unclear, especially regarding the area of immunometabolic regulation.

AIM OF STUDY: Ischemic stroke remains a major cause of neurological disability worldwide. Although reperfusion is crucial for salvaging the ischemic penumbra, it also contributes to secondary injury driven by microglial activation. Emerging evidence suggests that targeting metabolic immune interactions may help mitigate postreperfusion neuroinflammation. This study aimed to investigate whether FN attenuates cerebral ischemia‒reperfusion injury by modulating microglial metabolism and polarization.

MATERIALS AND METHODS: A transient middle cerebral artery occlusion (tMCAO) model was established in mice. Formononetin (FN) was administered immediately after reperfusion. Neurological deficit scores were assessed daily for three days. Twenty-four hours after reperfusion, pathological injury was evaluated by hematoxylin and eosin (H&E) staining and a TUNEL assay. Microglial polarization markers were analyzed using immunofluorescence and Western blotting. Inflammatory cytokines were measured by real-time quantitative polymerase chain reaction (RT‒qPCR) and enzyme-linked immunosorbent assay (ELISA). RNA sequencing was performed on peri-infarct cortical tissue to identify affected pathways. Glycolytic protein levels in the same region were measured by western blotting. Molecular docking was employed to predict key bioactive compounds and their potential targets. These predictions were further validated by Western blot analysis. For in vitro studies, BV2 cells were subjected to oxygen‒glucose deprivation and reperfusion (OGD/R). Polarization markers were subsequently detected by flow cytometry and RT‒qPCR. Cellular ATP and lactate levels, glucose uptake, the extracellular acidification rate (ECAR) and the oxygen consumption rate (OCR) were measured. An AMPK inhibitor was used for intervention in both in vivo and in vitro experiments. Key indicators related to AMPK signaling, microglial polarization, and glycolysis were examined via immunofluorescence and Western blotting.

RESULTS: FN treatment improved neurological function scores, reduced infarct volume, and attenuated pathological injury in tMCAO mice. Both in vivo and in vitro, FN treatment inhibited glycolysis and enhanced mitochondrial oxidative metabolism. It also decreased M1-like polarization while increasing M2-like polarization markers, leading to a reduction in proinflammatory cytokine production. RNA sequencing and molecular docking analysis revealed the AMPK/mTOR/HIF-1⍺ pathway, which was subsequently confirmed at the protein level. An AMPK inhibitor reversed the FN-induced changes in both metabolic and microglial phenotypes.

CONCLUSIONS: FN suppresses glycolytic metabolism in microglia through AMPK signaling, thereby influencing microglial polarization and attenuating neuroinflammation.