Studies On The Toxicity And The Mechanisms Of Silver Nanoparticles In SH-SY5Y Cells And Rat Brain Cortical Neurons

Silver nanoparticles(AgNPs)has been widely used in more than 1,800 kinds of products due to its advantages of higher surface area,surface effect,and quantum size effect,which is the most commonly used nanomaterial.However,given the widespread application of AgNP products,it has become increasingly important that we understand their potential health and environmental risks.In recent years,a large number of studies have been conducted to evaluate the influences of AgNPs on the major target-organ toxicity:liver and kidney.However,more and more evidences have reported that although the central nervous system is highly protected from biological penetration through the blood-brain barrier,AgNPs can still enter the body through multiple pathways and transfer across the blood-brain-barrier to the brain.Since AgNPs is cleared longer in the brain and neurons are more sensitive to stimuli,which may cause much greater damage to the body.In addition,there are still many limitations and controversies in the study of the effects of AgNPs on the central nervous system,including the method of exposure,the dose of exposure,and the time of exposure.Therefore,our study evaluates the safety of AgNPs on the nervous system both in vivo and in vitro,and explores the specific mechanisms of neurotoxicity,providing theoretical basis and preliminary experimental data for the safe use of AgNPs.Firstly,we selected three kinds of different size AgNPs and explored its potential mechanism of action on human neuroblastoma cells(SH-SY5Y).We found that AgNPs decreased the viability of SH-SY5Y cells in a time-and concentration-dependent manners.At the same time,we found that AgNPs in the range of 5-100 nm,smaller sized AgNPs showed greater toxicity,indicating that the cytotoxicity of AgNPs was nano-size dependent.In addition,AgNPs activated endoplasmic reticulum(ER)stress,resulting in the dissociation of the chaperone glucose-regulated protein 78(GRP78)from the ER transmembrane protein stress sensors:inositol-requiring enzyme(IRE),PKR-like endoplasmic reticulum kinase(PERK),and activating transcription factor-6(ATF-6).Activation of these three protein sensors induced the unfolded protein response(UPR),and down-regulated unfolded accumulated or misfolded proteins in the ER to protects cells against ER stress.Long-term ER stress activates C/EBP homology protein(CHOP)and the ER-specific cysteine protease caspase-12 and induces apoptosis.In addition,ER stress disrupted cellular calcium(Ca2+)homeostasis and increased the length of mitochondria-associated ER-membranes(MAMs).Furthermore,AgNPs increased the expression of PTEN and altered Inositol 1,4,5-trisphosphate receptor(IP3R)function by increasing the level of tensin homologue deleted on chromosome 10(PTEN),which enhanced Ca2+ transfer from ER to mitochondria and thus contributed to sensitization of the cells to apoptosis.Finally,Ca2+ overload and disruption of mitochondrial homeostasis triggered cell apoptotic.The above results indicate that the apoptosis of SH-SY5Y cells induced by AgNPs may be mediated by endoplasmic reticulum stress-triggered cell death and endoplasmic reticulum stress-induced mitochondrial apoptotic pathway.Further studies have shown that in addition to inducing apoptosis of SH-SY5Y cells,AgNPs induced autophagy.We focused on the potential role of AgNPs-induced autophagy and found that AgNPs-induced ER stress induced an increase in intracellular Ca2+ levels,which in turn activated calmodulin-dependent protein kinase kinase beta(CaMKKβ)and its downstream regulator adenosine 5’-monophosphate-activated protein kinase(AMPK),activated AMPK activate the ULK1-ATG13-FIP200 complex,ultimately resulting in activation of Beclin-1 and autophagy.In addition,inhibiting the occurrence of ER stress can significantly reduce the activation of autophagy,suggesting that AgNPs-induced ER stress participates in the regulation of autophagy.More importantly,we found that AgNPs induced protective autophagy,and inhibition of autophagy can aggravate apoptosis induced by AgNPs.This may be due to the fact that autophagy can in turn inhibit ER stress and promotes cell survival.In conclusion,we reporte for the first time that AgNPs induce protective autophagy via the Ca2+/CaMKKβ/AMPK/mTOR pathway.Furthermore,after oral administration of AgNPs for 28 continuous days,we found that there was a large of AgNPs in the brain tissues.Furthermore,we observed that neuron morphological changes in the cerebral cortex through Nissl’s staining,exhibiting loss of cell integrity,shrunken cytoplasm,and indistinct nuclei.Transmission electron microscope technique(TEM)also showed that the neurons exhibited typical apoptotic features including chromatin condensation,chromatin marginalization after AgNPs treatment,at the same time we could observe that neuronal mitochondrial bilayer membranes were blurred,the number of baboons decreased,and nearly vacuolization-like lesions formed.The rough ER was swollen and the ribosomal degranulation.Furthermore,the cells exhibited typical autophagy features including autophagosomes after AgNP treatment.Western blot results further demonstrated that AgNPs activated ER stress and mitochondrial pathways,which resulted in apoptosis and autophagy.The above results showed that AgNPs can penetrate the blood-brain barrier and be taken up by neurons,then activated the mitochondrial pathway and ER stress cause apoptosis,meanwhile induce the occurrence of autophagy.The results of in vivo and in vitro experiments indicate that there is potentially dangerous to the nervous system when useing AgNPs,suggesting that public exposure to AgNPs should arouse concerns regarding environmental safety and human health.However,at present,the research on the effect and mechanism of AgNPs on neurotoxicity is still limited,which is at a preliminary stage of exploration.The use of AgNPs safely remains to be further explored.