Prussian Blue Nanozymes For The Treatment Of Ischemia-reperfusion In Mice

Ischemic stroke was one of the leading causes of death and severe disability.Effective recanalization methods after ischemia include intravenous thrombolysis and mechanical thrombectomy.After vascular recanalization,a large number of destructive reactive oxygen species and reactive nitrogen species were produced.Excessive accumulation of reactive oxygen species and reactive nitrogen species may led to the inactivation and excessive consumption of endogenous antioxidant enzymes,and can induced mitochondrial damage and caspase-mediated apoptosis.In addition,excessive RONS can act as a key signaling molecule to trigger the activation of microglia,induced the infiltration of peripheral blood leukocytes,and stimulate inflammatory cells to secrete cytokines,further aggravating brain tissue damage.However,the effect of current clinical drug treatment was not ideal.Therefore,ROS scavengers capable of scavenging excess ROS production had great therapeutic potential.Nanozymes had potent anti-oxidative stress and anti-inflammatory properties,and we believe that they had the ability to treat ischemic stroke.Therefore,we investigated the therapeutic effect of Prussian blue nanozyme（PBzyme）on ischemic stroke.We examined the scavenging effect of PBzyme on ROS in vivo and in vitro,and we found it can inhibit the activation of macrophages and the release of inflammatory factors in the brain,promote the polarization of microglia to M2,inhibit the apoptosis of nerve cells,and improve of neurological function.This study may provide a promising application of nanoenzymes in the treatment of brain diseases.Objective:（1）To investigate the anti-oxidative stress effect of Prussian blue nanozymes in vitro and in vivo.（2）To investigate the effects of Prussian blue nanozyme on inflammation and neuronal apoptosis in acute phase after ischemia-reperfusion（I/R）in mice.（3）To investigate the effect of Prussian blue nanozyme on long-term neurological function recovery in mice after ischemia-reperfusion.Methods:（1）Prussian blue nanozymes were synthesized by one-step hydrothermal method and characterized.（2）The superoxide dismutase and catalase properties of Prussian blue nanozymes were detected in vitro using kits.（3）The experimental animals were randomly divided into three groups:Sham group,MCAO+Saline group and MCAO+PBzyme group.At 60min of cerebral ischemia,the thread was pulled out,that can simulate reperfusion injury.Normal saline and Prussian blue nanoenzyme were injected into the tail vein of the normal saline group and Prussian blue nanoenzyme group,respectively.The sham operation group was not injected with any drug.24 hours after surgery,the experimental animals were scored using the Longa score,and mice with a score of 2-3 were included in the trial.（4）72 hours after successful modeling of ischemia-reperfusion,the mice were decapitated and the brain tissue was obtained directly.And the brain tissues were tested for superoxide anion（O₂·-）and nitric oxide（·NO）to determine the oxidative stress level after I/R injury.Western Blot was used to analyze the expression of inflammatory factor IL-1β,pro-apoptotic protein Bax and anti-apoptotic protein BCL-2 in the brain.（5）At 72h after surgery,mice were in deep anesthesia and perfused with PBS/PFA from their heart.The PFA fixed brain was removed from skull for IF（immunofluorescen）.The activation of microglia（Iba1）and astrocytes（GFAP）and neuronal apoptosis（Caspase-3）in the injured side were observed.（6）5 days after I/R injury,the mice in each treatment group were divided into two parts.One part of the mice were decapitated directly to obtain the brain tissue,and the brain tissues were used to analyze the activation levels of M1 and M2 microglia by western blot.The other part of the brain was perfused and sectioned,and the polarization of M1 and M2 microglia was analyzed by immunofluorescence staining.（7）The sensory,motor,and cognitive functions of the mice were assessed by rotarod,foot slip,paper stick,turn,and water maze tests for 28 days after I/R injury.（8）After 28 days,the brains of the mice in the different treatment groups were perfused and removed.The loss of brain tissue in the different treatment groups was observed by weighted glucose section and immunofluorescence MAP2 staining.Results:（1）Prussian blue nanozymes have anti-oxidative stress effects both in vitro and in vivo.（2）Prussian blue nanoenzyme alleviates inflammatory response and inhibits neuronal apoptosis after ischemia-reperfusion injury in mice.（3）Prussian blue nanozyme can promote long-term neurological function recovery and reduce brain tissue loss in mice with ischemia-reperfusion injury.Conclusion:In this study,we investigated the neuroprotective effect of Prussian blue nanozymes on mice with ischemia-reperfusion injury.This paper expounds the important role of Prussian blue nanozyme in anti-oxidative stress,scavenging reactive oxygen species,reducing inflammatory response,inhibiting cell apoptosis and promoting long-term neurological function recovery in ischemia-reperfusion injury.This study provides an important theoretical basis for the treatment of brain diseases with nanomaterials.