Abstract: Ischemic stroke represents a significant global health challenge, frequently associated with intricate pathophysiological alterations. During ischemic stroke, the generation of reactive oxygen species markedly increases, leading to direct neuronal damage as well as initiating a cascade of inflammatory responses. This oxidative stress can also disturb the equilibrium of the gut microbiota, resulting in dysbiosis. In turn, an imbalance in gut microbiota can further exacerbate the production of reactive oxygen species and contribute to a pro-inflammatory environment within the body. This creates a vicious cycle that not only promotes the progression of stroke but also leads to adverse functional outcomes. The neuroinflammation and intestinal microbiota dysbiosis that occur following ischemic stroke are critical contributors to stroke progression and adverse functional outcomes. We previously developed manganese-ferric Prussian blue nanozymes, characterized by a multi-enzyme structure and a porous design, that exhibit strong antioxidant properties. However, the therapeutic effects of manganese-ferric Prussian blue nanozymes on ischemic stroke and their mechanisms of action remain have not been fully elucidated. To investigate this, we constructed a mouse model of middle cerebral artery occlusion and administered manganese-ferric Prussian blue nanozymes via gastric gavage. Our results demonstrated that these nanozymes substantially reduced infarct volume, improved neurological function, restored gut microbiota balance, and increased levels of short-chain fatty acids in the mouse model. Treatment of lipopolysaccharide-treated BV-2 cells with short-chain fatty acids markedly decreased the expression levels of components of the Toll-like receptor 4/nuclear factor kappa B signaling pathway, including Toll-like receptor 4, inhibitor of nuclear factor kappa-B kinase subunit alpha, and pp65. These findings suggest that manganese-ferric Prussian blue nanozymes can correct gut microbiota dysbiosis and increase short-chain fatty acid production by modulating the Toll-like receptor 4/nuclear factor kappa B signaling pathway, thereby providing therapeutic benefits in the context of ischemic stroke. 

Key words: BV-2 cells, gut microbiota, inflammatory response, ischemic stroke, manganese-iron Prussian blue nanozymes, neurological function, nuclear factor-κB, oxidative stress, short-chain fatty acids, Toll-like receptor 4