Role Of Nanomolybdenum In Protecting Rat Heart Against Ischemia/Reperfusion Injury And The Underlying Mechanisms

Background:Oxidative stress is one of the main mechanisms of myocardial ischemia/reperfusion injury.As the level of reactive oxygen species（ROS）exceeds the antioxidant capacity,it directly causes oxidative damage or activates signaling pathways such as apoptosis,ultimately leading to myocardial cell death.Alleviating oxidative damage has become an effective way to combat myocardial ischemia/reperfusion injury.However,traditional antioxidants are largely limited by pharmacokinetics and routes of administration,including short half-lives,low stability,and low bioavailability.Therefore,the search for new antioxidant drugs with good targeting,high scavenging efficiency and low toxic and side effects is currently a research hotspot in this field.Recent advances in nanomedicine have made it possible to use various functional nanomaterials for the treatment of myocardial ischemia/reperfusion injury.Compared with traditional drugs,these nanodrugs targeting ROS scavenging can improve ROS scavenging efficiency and reduce side effects by improving pharmacokinetics and pharmacodynamics due to their size,shape,and properties.Therefore,the development of anti-oxidative nanomaterials with good biocompatibility and efficient targeting is a new idea for the treatment of oxidative damage in ischemia/reperfusion.Objectives:1.To synthesize a novel antioxidant nanomaterial—molybdenum nanomaterials（Mo-NPs）,and to evaluate its reactive oxygen species scavenging ability and acute toxicity.2.Assess the effect of nanomolybdenum on myocardial ischemia/reperfusion injury3.Detect the ability of nanomolybdenum to remove reactive oxygen species from myocardial tissue and myocardial cells.Methods:1.Synthesis and Characterization Experiment of Mo-NPsThe shape and particle size of Mo-NPs were detected by TEM;the surface ligand modification of Mo-NPs was detected by FT-IR;the absorption peaks of Mo-NPs at specific wavelengths were detected by UV-vis;the colloidal stability of Mo-NPs in solution was characterized by Zeta potential Elemental composition and molybdenum valence distribution of Mo-NPs was characterized by XPS.2.In vitro scavenging of reactive oxygen species by Mo-NPsThe scavenging effect of Mo-NPs on superoxide anion,hydrogen peroxide and peroxynitrite anion was detected by UV-vis;the scavenging effect of Mo-NPs on hydroxyl radical was detected by fluorescence spectrophotometer.3.Animal experimentThe myocardial ischemia/reperfusion model in SD rats was established by ligating the left anterior descending coronary artery:ischemia for 1 h,reperfusion for 3 h;Control,sham operation,ischemia/reperfusion,Mo-NPs low and high dose groups were set up（5mg/kg and 10 mg/kg Mo-NPs were administered to rats by sublingual vein 5 mins before reperfusion,respectively）;TTC staining was used to detect myocardial infarction volume in rats;creatine kinase assay kit（phosphocreatine substrate method）was used to detect serum CK activity in rats;HE staining was used to detect myocardial histomorphology Changes;inhibition and generation of superoxide anion free radical assay kit to detect cardiac tissue anti-superoxide anion free radical activity;hydroxyl radical test kit to detect cardiac tissue anti-hydroxyl radical activity;malondialdehyde（MDA）assay kit（TBA）method to detect the content of malondialdehyde in cardiac tissue;in situ terminal transferase labeling（TUNEL）staining was used to detect the apoptosis of rat cardiomyocytes and count the number of apoptotic cells;Caspase-3 activity detection kit for detection of Caspase-3 protein activity units in H9c2 cardiomyocytes;HE staining in the acute toxicity test of rats was used to detect the histopathological conditions of the heart,liver,spleen,lung and kidney in the control group and the experimental group respectively.4.Cell experimentThe rat H9c2 cardiomyocyte hypoxia 8h/reoxygenation 12h（H:8h/R:12h）model was constructed;different doses of Mo-NPs（1,5,10,20,40,80,160,320μg/m L）were administered;The activity of H9c2cardiomyocytes was detected by MTS;the injury of H9c2cardiomyocytes was detected by LDH release rate;the generation of ROS in H9c2 cardiomyocytes was detected by DCFH-DA method,and the fluorescence intensity of each group was calculated by Image J;Caspase-3 activity detection kit for detection of Caspase-3 protein activity units in H9c2 cardiomyocytes.Result:1.Synthesis and Characterization Experiment of Mo-NPsTEM shows that Mo-NPs are monodisperse spherical structures with an average particle size of 4 nm;FT-IR characterization:the peaks at1694.48cm^-1,1574.68cm^-1,1200.44cm^-1 in Mo-NPs are carbonyl,benzene ring and Characteristic peaks of phenolic hydroxyl groups;UV-vis spectrum shows that Mo-NPs have absorption peak at 560 nm;Zeta potential of Mo-NPs is-42.0m V,indicating good colloidal stability;XPS characterization shows that Mo-NPs contain carbon,oxygen and phosphorus,molybdenum four elements,of which molybdenum element accounts for 1.32%of the total,and molybdenum element is a mixed valence state of+5 and+6.2.In vitro scavenging of reactive oxygen species by Mo-NPsUV-vis results show that Mo-NPs can scavenge superoxide anion in a concentration-dependent manner;at the same time,Mo-NPs can react with H₂O₂ and ONOO^-to scavenge H₂O₂ and ONOO^-with strong oxidizing properties;fluorescence spectroscopy results show that concentration-dependent scavenge hydroxyl radicals.3.Animal experiment（1）Compared with the sham-operated group,the myocardial ischemia/reperfusion group showed a significant increase in myocardial infarction area and increased serum CK activity.HE staining results showed that the myocardial fiber texture was disordered and the myocardial cell space was widened.Doses of Mo-NPs can inhibit this phenomenon.（2）Compared with the sham-operated group,the myocardial tissue apoptotic cells appeared or increased in the myocardial ischemia/reperfusion group,and the apoptotic cells were scattered and distributed without obvious distribution regularity,and the activity of Caspase-3 increased.And giving different doses of Mo-NPs can reduce the above-mentioned apoptosis phenomenon.（3）Compared with the sham-operated group,the myocardial tissue anti-superoxide anion activity and the ability to inhibit hydroxyl radicals in the myocardial ischemia/reperfusion group decreased,and the MDA level increased;and different doses of Mo-NPs could significantly reverse the above situation.（4）HE staining results showed that Mo-NPs had no significant effect on the morphology of the heart,liver,spleen,lung,kidney and other major organs in the two groups of rats.4.Cell experiment（1）Compared with the control group,different doses of Mo-NPs from1μg/m L to 80μg/m L had no significant effect on the viability of H9c2cardiomyocytes,indicating that Mo-NPs has a good safety range within this concentration.（2）Compared with the control group,the LDH release rate of H9c2cardiomyocytes in the hypoxia/reoxygenation group was significantly increased,and this phenomenon could be inhibited after administration of different doses of Mo-NPs in the concentration of 1μg/m L to 40μg/m L,indicating that Mo-NPs are effective in this concentration range.（3）Compared with the control group,the Caspase-3 activity of H9c2cardiomyocytes in the hypoxia/reoxygenation group increased,while the Caspase-3 activity decreased after different doses of Mo-NPs were administered.（4）Compared with the control group,the intracellular ROS in the H9c2 cardiomyocytes in the hypoxia/reoxygenation group increased significantly,and the intracellular ROS was significantly reduced after administration of different concentrations of Mo-NPs（1,5,10,and 20μg/m L）.Conclusion:1.A new anti-oxidant nanomaterial nanomolybdenum was successfully synthesized,and it was confirmed that it has the ability to scavenge reactive oxygen species without obvious acute toxicity.2.Nanomolybdenum has anti-myocardial ischemia/reperfusion injury.3.Nano-molybdenum exerts anti-myocardial ischemia/reperfusion oxidative damage by scavenging reactive oxygen species.