Effects Of Gold Nanoparticles On Rat Cortical Neurons Exposed To Oxygen-glucose Deprivation/reperfusion Injury And Corresponding Mechanisms

Background:Stroke is the leading cause of severe,long-term disability and mortality worldwide and ischemic stroke accounts for approximately 80%of all strokes.It' s necessary for immediate restoration of the blood flow to the ischemic tissue,but the aftermath of pathology associated with the ischemic cascade causes additional damage and exacerbates neurocognitive deficits,which is called cerebral ischemic-reperfusion(I/R)injury.Although the precise mechanism of cerebral I/R injury is not fully understood,oxidative stress,excitotoxicity,inflammation,apoptosis,calcium overload and mitochondrial dysfunction during I/R injury are widely recognized as important contributors in the development of ischemic neuronal cell death.Gold nanoparticles(AuNPs)are being used for advanced applications such as bio-sensing,cancer therapy,targeted drug delivery and radiography imaging due to their unique physicochemical characteristics,good biocompatibility and the simplicity of the control of their synthesis.Moreover,emerging attention is focused on the intrinsic treatment of AuNPs.Regarding the anti-inflammatory and antioxidant effects of AuNPs exerted in a few researches,AuNPs hint to an underappreciated therapeutic potential of cerebral I/R injury.Our previous work indicated that 20-nm AuNPs significantly protected the cerebra from I/R injury in rats via anti-inflammation,anti-apoptosis and immune-regulation.In contrast,5-nm AuNPs aggravated neurologic deficits and enlarged infarction volumes compared withI/R group.Nevertheless,the precise mechanism has not been systemically evaluated.Objectrive:To investigate the potential effects of AuNPs(5 nm,20 nm)on primary rat cortical neurons exposed to oxygen-glucose deprivation/reperfusion(OGD/R)injury and elucidate the corresponding mechanismsMethod:We synthesized highly stable 5-nm and 20-nm AuNPs using the NaBH4 reduction method and citrate reduction method respectively.Transmission electron microscope(TEM)and Ultraviolet-visible(UV-Vis)spectra were obtained to characterize AuNPs.Immunofluorescence staining was used to indentify primary neurons.After culture for 72 h,primary cortical neurons subjected to OGD for 4 h were incubated with neuron culture medium with or without AuNPs(5 nm and 20 nm)for 48 h.TEM was used to investigate the celluar uptake and distribution of AuNPs;cell counting kit-8(CCK-8)was employed to evaluate cell viability;TUNEL assay,caspase-3 activity assayand mitochondrial membrane potential assaywas performed to analyze apoptosis;MDA,SOD and cytosolic-aconitase(c-Aconitase)were measured to assess oxidative stress;mitochondrial stress test by Seahorse Bioscience XF96 Extracellular Flux Analyzer was conducted to explore mitochondrial respiration rate.Results:AuNPs were homogeneous in size and shape and well dispersed in culture medium.The purity of primary neuroncells was 82.7 ± 2.3%.TEM pictures indicated that AuNPs(5 nm and 20 nm)were both mainly distributed in cytoplasm,lysosomes and vesicles,moreover,a few of 20-nm AuNPs were found scattered in the mitochondrion.Compared to the control group,the cell viability was found to decrease after 5-nm AuNPs treatment while increase after 20-nm ones as a function of concentration.Apoptotic assay outcome indicated 5-nm AuNPs increased neural apoptosis compared to OGD/R group while 20-nm ones ameliorated the OGD/R injury induced apoptosis possibly by decreasing caspase-3 activity.Futhermore,our results suggested pro-oxidant ability of 5-nn AuNPs and anti-oxidant property of 20-nm AuNPs which is supported by MDA,SOD and c-Aconitase data.At last,upregulated mitochondrial respiration rate and ATP production suggested that 20-nm AuNPs exhibited neuroprotection effect against OGD/R injury possibly by mitochondrial preservation.Conclusion:The results of this study indicatd that AuNPs may cause different effects on neurons exposed to OGD/R injury possibly depending on the size employed.5-nm AuNPs aggravated neuronal OGD/R injury via pro-oxidant and pro-apoptotic activities.In contrast,20-nm AuNPs exerted neuroprotective function against ischemic neuronal injury and suggested effects against OGD/R injury via anti-oxidant,anti-apoptotic as well as mitochondrial preservation.Our observations enriched the application extent of AuNPs and suggested that 20-nm AuNPs could be potentially developed as a promising medicine for ischemic stroke treatment.