WPO-NDs Protect The Rat Brain From Ischemia/reperfusion Injury Via Cleaning Reactive Oxygen Species

Background:Stroke is an acute cerebrovascular disease,including ischemic stroke（80%）and hemorrhagic stroke（20%）.The primary principle for treating ischemic stroke is to rebuild blood reperfusion as soon as possible to restore blood oxygen supply to the brain.In addition to ischemia,reperfusion can also cause nerve cell damage,which is called“ischemia/reperfusion”injury.At present,the main therapy for ischemic stroke is to utilize tissue plasminogen activator（t PA）for thrombolysis,but the narrow therapeutic window（<4.5h）limits its application.The ischemic area of the brain can be divided into the ischemic core and the ischemic penumbra.The ischemic penumbra is the area around the ischemic core.The blood flow is reduced but the cells are not necrotic and can be reversed in the case of reperfusion,so the repair of neurons after cerebral ischemia mainly depends on the residual neurons in the penumbra.After cerebral ischemia,reactive oxygen species increase significantly in the ischemic penumbra,which eventually leads to the death of nerve cells through cascade reactions.It is well accepted that oxidative stress plays a key role in cerebral ischemia/reperfusion injury.Nanoparticles are tiny particles with a particle size of 1-100 nm,including polymer nanoparticles,liposomes,dendrimer micelles,inorganic nanoparticles,etc.Nano antioxidants are increasingly used in stroke models.Nanoparticles can increase the solubility of poorly soluble drugs,improve drug stability,improve targeting ability through ligand modification,and control drug release.Therefore,the development and application of nanomaterials to therapy for stroke possesses great potential.Objectives:1.To synthesize a novel nano-antioxidant Tungsten oxyphosphorus nanodots（WPO-NDs）,and to evaluate its ability for scavenging reactive oxygen species（ROS）.2.To evaluate the protective effect of WPO-NDs on cerebral ischemia/reperfusion injury.3.To examine the ability of WPO-NDs to remove reactive oxygen species from brain tissue.4.To evaluate the toxicological effects of WPO-NDs on SD rats at therapeutic doses.Methods:1.Synthesis and characterization of WPO-NDs:the shape and particle size of WPO-NDs were detected by transmission electron microscopy;the surface ligand modification of WPO-NDs was detected by Fourier transform infrared spectroscopy;the stability of WPO-NDs in solution was detected by Zeta potentiometer;the elemental composition of WPO-NDs was detected by XPS and the valence distribution of tungsten element;UV-Vis spectrophotometer was used to detect the ability of WPO-NDs for scavenging superoxide anion（O₂·^-）,hydrogen peroxide（H₂O₂）,peroxynitrite anion（ONOO^-）;the scavenging effect of WPO-NDs on hydroxyl radical（·OH）was detected by spectrophotometer.2.Animal experiments:focal cerebral ischemia/reperfusion injury model was established in SD rats（MCAO model:ischemia 2h,reperfusion 24h）.The rats were divided into 5 groups:a control group（Control）,a sham group（Sham）,a ischemia/reperfusion group（I/R）,a low-dose WPO-NDs group（+0.2 mg/kg WPO-NDs）,and a high-dose WPO-NDs group（+0.5mg/kg WPO-NDs）.Neurological function score（Longa score method）was performed;infarction volume was measured（TTC staining）;HE staining was used to observe the morphological changes in brain tissue;TUNEL staining was used to observe the apoptosis rate in brain tissue;Caspase-3 enzyme activity was detected by a commercial kit;Western-blot was used to detect the Bax and Bcl-2protein levels in brain tissue;the anti O₂·^-activity,anti·OH activity and malondialdehyde（MDA）levels in brain tissue were detected.3.Toxicological experiments on WPO-NDs at therapeutic doses:SD rats were injected sublingually with 0.5 mg/kg WPO-NDs,and 24 hours later,serum liver and kidney functional indexes were detected by automatic biochemical analyzer,and blood routine indexes were detected by blood cell analyzer.The heart,liver,spleen,lung and kidney were taken for HE staining to evaluate the toxic effect of WPO-NDs on rats.Results:1.Characterization of WPO-NDs:1)Transmission electron microscopy results showed that WPO-NDs were spherical nanoparticles with a diameter of about 5 nm;Fourier transform infrared spectroscopy results showed that 1698cm^-1,1586cm^-1and 3354cm^-1were the characteristic peaks of carbonyl group,benzene ring and phenolic hydroxyl group of WPO-NDs,respectively;Zeta potential meter results showed that the Zeta potential of WPO-NDs was-43.9m V,indicating that it has good stability;XPS results showed that WPO-NDs contained carbon,oxygen and tungsten elements,of which the valence of tungsten was+6 and+5 price.2)UV-Vis spectroscopy results showed that WPO-NDs eliminated superoxide anion,hydrogen peroxide and peroxynitrite anion in a concentration-dependent manner;fluorescence spectroscopy results showed that WPO-NDs eliminated hydroxyl radicals in a concentration-dependent manner.2.Animal experiments:1)Compared with the sham group,the rats in the ischemia/reperfusion group had higher neurological function scores,obvious infarcts,and disordered brain tissue cells;administration of WPO-NDs can significantly reduce cerebral ischemia/reperfusion injury,including decreasing neurological scores,reducing the volume of cerebral infarction,and improving the patholog ical morphology of brain tissue.2)Compared with the sham group,the number of TUNEL-positive cells in the cerebral ischemia/reperfusion group was increased significantly;the level of pro-apoptotic protein Bax was significantly increased,and the level of anti-apoptotic protein Bcl-2 was significantly decreased;the activity of Caspase-3 and the level of MDA were increased significantly;the activity of anti-superoxide anion and anti-hydroxyl radical were decreased significantly;these phenomena were attenuated in the presence of WPO-NDs.3.Toxicological experiment of WPO-NDs at therapeutic doseHE staining showed that there was no significant difference in the morphology of the main organs of the heart,liver,spleen,lung and kidney between the control group and the WPO-NDs high-dose group;the results of serum biochemical indexes,liver and kidney functional indexes showed that WPO-NDs had no obvious toxic effect on SD rats.Conclusions:1.A new type of nanomaterial WPO-NDs was successfully synthesized with the ability to scavenge reactive oxygen species.2.WPO-NDs possess the anti-cerebral ischemia/reperfusion injury effects,and have no obvious toxic side effects under the therapeutic dose.3.WPO-NDs attenuate oxidative injury in rat brain following ischemia/reperfusion via scavenging reactive oxygen species.