Formononetin Protects Against Cerebral Ischemia-Reperfusion-Induced Lung Injury: Insights from Animal and Cell Models

Abstract

Background Cerebral ischemia-reperfusion (CIR) affects multiple organs, with lung injury being particularly prevalent. Formononetin, derived from legumes, has multiple pharmacological effects and demonstrated potential for the treatment of neurological diseases. However, its effects on CIR-induced lung injury are unknown. Objective To investigate the protective effect of formononetin in CIR-induced lung injury (in the Sprague-Dawley (SD) rats’ model). Methods Sprague-Dawley rats (n=27) underwent middle cerebral artery occlusion (MCAO) to establish a CIR model. Formononetin was then administered at specific time points. Longa neurological deficit score, histological staining, enzyme-linked immunosorbent assay (ELISA), and flow cytometry were subsequently used to assess lung injury. In addition, rat microglia were exposed to oxygen-glucose deprivation/re-oxygenation (OGD/R) to mimic CIR in vitro, and exosomes were then extracted from these cells for testing. A lipopolysaccharide (LPS)-induced lung epithelial cell injury model was subsequently used to investigate the protective effects of formononetin. Results ELISA revealed that formononetin exerted broad-spectrum anti-inflammatory effects, reducing TNF-α, IL-1β, IL-6, and IL-10 by 46.7%, 46.2%, 40.1%, and 54.6%, respectively, in bronchoalveolar lavage fluid after MCAO (P<0.05). In lung tissues, superoxide dismutase (SOD) expression was increased, while malondialdehyde (MDA) was decreased (22.8%). Additionally, formononetin reduced interferon-gamma positive (INF-γ⁺), IL-4⁺, IL-17A⁺, forkhead box P3 protein positive (Foxp3⁺), cluster of differentiation 4 positive (CD4⁺), and CD8⁺ by 72.3%, 76.7%, 66.1%, 67.7%, 78.4%, and 80.4%, respectively, all to sham levels (P < 0.05). The in vitro experiments also revealed a downward trend in the expression of inflammatory factors in lung epithelial cells, with increased SOD expression (26.2%) and decreased MDA levels (21.7%, P<0.05). Conclusion Formononetin protects against lung injury by inhibiting inflammatory cytokines, reducing oxidative stress, and regulating immune cells. Notably, exosomes derived from formononetin-treated microglia were shown to attenuate LPS-induced lung epithelial cell injury, highlighting a novel intercellular communication pathway. Together, these in vivo and in vitro studies offer new insights into the clinical application of formononetin in cerebrovascular diseases for secondary lung injury.