Study Of Protective Effect And Mechanisms Of Novel Composite Nanozyme Fe₂NC@Se NPs On Cerebral Ischemic Reperfusion Injury

Stroke is the second leading cause of death and a major cause of disability worldwide.Ischemic stroke,mainly due to the sudden blockage of blood supply to the brain,accounts for approximately 87%of all stroke types.Currently,efficient and prompt restoration of blood supply via intravenous application of thrombolysis agent or intra-arterial thrombectomy is recognized as effective therapeutic regimens for ischemic stroke.During the reperfusion therapy with the recovery of cerebral blood flow,an explosively increasing amount of reactive oxygen species（ROS）are generated and mediate cerebral ischemic reperfusion injury（CIRI）via participating a series of pathophysiological processes and aggravating neurological deficits.Thus,antioxidant therapy has become one of the therapeutic strategies for ischemic stroke.Whereas,exogenous supplement of natural antioxidant enzymes has disadvantages such as limited sources,poor in vivo stability and high costs for production.Therefore,potent,stable and safe antioxidant enzyme mimics are urgently to be developed.Nanozyme is one kind of nanomaterial with catalytic activity similar to natural enzyme,which combines the advantages of nanomaterial and natural enzyme with stable physical and chemical properties,high efficiency and tunable catalytic activity and easy to large-scale production.Previous studies proved single-atom iron nanozyme with excellent antioxidant properties comparable to natural enzymes.Moreover,compared with single-atom nanozyme,the potential synergistic cooperation of dual-atom nanozyme between two atoms would further improve the catalytic performance.Metal-organic framework（MOF）is one kind of porous nanomaterial coordinated by organic ligands and metal ions with the advantage of large specific surface area,stable structure,potent loading capacity and biosafety,which enables MOF as nanocarrier of drugs or other nanomaterials.Herein,our current study intends to integrate the advantages of dual-atom iorn nanozyme and MOF to construct a nitrogen-doped carbon based Fe-Fe nanozyme（Fe₂NC nanozyme）and designed a selenium-imidazole containing MOF shell for encapsulation to form composite Fe₂NC@Se MOF nanoparticles（Fe₂NC@Se NPs）with multi-enzyme activities.Thereafter,we verified the neuroprotective effects of Fe₂NC@Se NPs on cellular and animal level after CIRI.Apoptosis signal-regulating kinase 1（ASK1）/c-Jun N-terminal protein kinase（JNK）signaling pathway plays a significant role in oxidative stress and cell neural apoptosis after CIRI.Studies indicated that ROS were an effective ASK1activator and activated ASK1 could further active downstream signaling pathways by phosphorylation of JNK,which participated in the regulation of cell apoptosis.Thus,on the basis of the construction of the composite nanozyme and the validation of protective effects,we hypothesized that Fe₂NC@Se NPs might exert the protective effects by inhibiting the activation of ASK1/JNK pathway after CIRI and further verified our hypothesis,for shedding lights on the protective effects of Fe₂NC@Se NPs on molecular level.This study laid a solid foundation for the clinical translation of nanozyme for treating CIRI.PartⅠ:The synthesis,characterization and catalytic activity of Fe₂NC@Se NPsObjective:To synthesis,characterize and investigate the antioxidant enzyme-mimicking activities of Fe₂NC@Se NPsMethod:The Fe₂NC nanozyme was synthesized by using the“precursor-preselected”wet-chemistry carbonization strategy and then used a wet-chemistry method to grow a Se MOF shell outside Fe₂NC to form Fe₂NC@Se NPs.Multiple antioxidant enzyme-mimicking activities were measured.Results:The synthesized Fe₂NC@Se NPs presented a uniformed core-shell construction with rhombic dodecahedral morphology as core encapsulated with a20nm shell outside.The size andζ-potential of Fe₂NC@Se NPs were 220nm and33.7m V with good stability.Fe₂NC@Se NPs exhibited superoxide dismutase（SOD）-like,catalase（CAT）-like and glutathione peroxidase（GPx）-like activity cooperative for ROS scavenging.PartⅡ:The in vitro protective effect and mechanisms of Fe₂NC@Se NPs on oxygen glucose deprivation/reoxygenation（OGD/R）induced cell injuryObjective:To explore the in vitro protective effect and the underlying mechanisms of Fe₂NC@Se NPs on OGD/R induced PC12 cell lineMethods:The cellular uptake of Fe₂NC@Se NPs in OGD/R induced PC12 cell line was examined by confocal laser scanning microscopy.For functional assessment,the cells were divided into six groups:Control group,Control+Fe₂NC@Se NPs（Control+NPs）group,OGD/R group,OGD/R+Fe₂NC@Se NPs（OGD/R+NPs）groups with three different concentrations（1,2.5,5μg/ml）of Fe₂NC@Se NPs.Cellular ROS molecular probe,cell counting kit-8,lactate dehydrogenase（LDH）measurement,cell apoptosis detection by flow cytometry,mitochondrial membrane potential assay,western blot analysis etc.were used to explore the in vitro cellular protective effects and the molecular mechenisms of Fe₂NC@Se NPs after OGD/R injury.Results:Cellular uptake of Fe₂NC@Se NPs by OGD/R induced PC12 cell line was in a time and dose dependent manner.Compared with Control group,intracellular ROS level,LDH level,the proportion of apoptotic cell and JC-1monomers were significantly increased,cell viability was significantly decreased in OGD/R group（P﹤0.001）;Compared with OGD/R group,intracellular ROS level,LDH level,the proportion of apoptotic cell and JC-1 monomers were significant decreased and cell viability was significantly increased in OGD/R+various concentration of NPs groups（P﹤0.05）.Compared with Control group,Bcl-2 protein expression was significantly decreased and Bax,cleaved-Caspase-3,p-ASK1 and p-JNK proteins expression were significantly elevated in OGD/R group（P﹤0.001）;Compared with OGD/R group,Bcl-2 protein expression was significantly elevated and Bax,cleaved-Caspase-3,p-ASK1 and p-JNK proteins expression were significantly decreased in OGD/R+various concentration of NPs groups（P﹤0.05）.Part III:The in vivo neuroprotective effect and mechanisms of Fe₂NC@Se NPs of middle cerebral artery occlusion（MCAO）ratsObjective:To explore the in vivo neuroprotective effect and the underlying mechanisms of Fe₂NC@Se NPs on MCAO rats and to preliminary evaluate the in vivo biosafetyMethods:88 healthy adult Sprague-Dawley rats were randomly assigned into four groups:Sham+Na Cl group,MCAO+Na Cl group,MCAO+Fe₂NC@Se NPs（MCAO+NPs）group,Sham+Fe₂NC@Se NPs（Sham+NPs）group.CIRI was induced by MCAO model with ischemia for 1.5h and reperufusion for 24h.Fe₂NC@Se NPs（0.25mg/ml,10μl）or equivalent volume of 0.9%Na Cl was administrated by cerebral lateral ventricles injection before the MCAO procedure.By evaluation of neurological deficits with Longa’s scale,2,3,5-triphenyltetrazolium chloride（TTC）staining,haematoxylin and eosin staining,immunofluorescence staining,western blot analysis etc.to explore the in vivo protective effect and the molecular mechanism.Besides,to evaluate the biosafety of the Fe₂NC@Se NPs,15 healthy SD rats were administered different concentrations of nanozyme and the main organs were harvest for haematoxylin and eosin staining.Results:Compared with Sham+Na Cl group,the neurological score,brain infarct volumes and the extent brain edema,the content of brain tissue MDA and the apoptotic index were significantly increased in MCAO+Na Cl group（P﹤0.001）;Compared with MCAO+Na Cl group,those indicators were significant decreased in MCAO+NPs group（P﹤0.001）.Compared with Sham+Na Cl group,the Bcl-2protein expression was significantly decreased and Bax,cleaved-Caspase-3,p-ASK1and p-JNK proteins expression were significantly increased in MCAO+Na Cl group（P﹤0.001）;Compared with MCAO+Na Cl group,Bcl-2 protein expression was significantly elevated（P﹤0.01）and Bax,cleaved-Caspase-3,p-ASK1 and p-JNK proteins expression were significantly decreased in MCAO+NPs group（P﹤0.001）.Besides,all the aboved indicators were nonsignificant between Sham+Na Cl and Sham+NPs groups.Furthermore,no detectable pathological injury was observed after Fe₂NC@Se NPs administration,suggesting the in vivo biosafety.Conclusion:（1）We successfully constructed composite nanozymes Fe₂NC@Se NPs containing multiple antioxidant enzyme-mimicking activities,including SOD-like,CAT-like and GPx-like activities.（2）After OGD/R injury in vitro,Fe₂NC@Se NPs could be effectively uptake by PC 12 cell line and potently scavenge intracellular ROS,ameliorate oxidative-stress induced injury and suppress cell apoptosis.（3）In rats MCAO model,Fe₂NC@Se NPs improved neurological function, decreased the brain infarct volumes,ameliorated oxidative-stress induced brain injury and suppressed the neural apoptosis with good in vivo biosafety.（4）Fe₂NC@Se NPs exerted protective effects possibly by suppressing the ASK1/JNK signaling pathway activation after CIRI.