| BackgroundSilver nanoparticles(AgNPs)because of its bactericidal properties,AgNPs-related products are widely used in many fields.However,AgNPs will continue to release into the environment during mass production and widespread use.AgNPs released into the environment will migrate and transform in the atmosphere,soil,surface water and groundwater,and accumulate into the food chain.AgNPs in the environment can enter human life through a variety of pathway,the biosafety and toxicity of AgNPs have attracted considerable attention.Studies have shown that AgNPs enter the body in a variety of ways,and enter the brain through the blood-brain barrier and along the olfactory nerve pathway,and continues to accumulate in the brain.So,it is crucial to evaluate the toxic effects of AgNPs on the brain and its mechanism of action.Neuroinflammation is a common characteristic of most central nervous system diseases.Nanoparticles entering the brain as an exogenous substance induce neuroinflammation and also cause cellular autophagy activation.Autophagy activation is also one of the key mechanisms for toxicity effects of AgNPs.Mitochondria and lysosomes are vulnerable to damage by AgNPs and are sensitive organelles for toxic effects of AgNPs.The selective degradation of damaged organelles can be enabled by the autophagic pathway.Although some studies have reported the potential toxicological mechanisms of neurotoxicity induced by AgNPs,the molecular mechanisms of the regulatory relationships among the autophagic pathway,lysosomes and mitochondria in the neuroinflammatory responses induced by AgNPs are not clear.Therefore,the present study was conducted to explore the relationship between the three pathways and to find the key molecules in the mutual regulatory relationship,in order to provide valuable basic information for the wide application and safety evaluation of AgNPs.ObjectivesThe study aims to determine the mechanisms by which AgNPs cause increased polarization of pro-inflammatory phenotypes in mouse microglia in vivo and in vitro.To investigate the role of autophagy-lysosomal and autophagy-mitochondrial pathways in the activation of AgNPs-induced neuroinflammation;Study of the interrelation between autophagic pathways,lysosomes and mitochondria.And identified the critical proteins and exposure doses,provided a reference for evaluation the toxic effects of AgNPs.Contents and methods1.Characterization of AgNPs.Using transmission electron microscopy(TEM),the morphology of AgNPs coated with polyvinylpyrrolidone(PVP)was observed,the absorption spectrum was determined by UV-Visible Spectrophotometer(UV-Vis).The hydration particle size and zeta potential of AgNPs dispersed in ultrapure water and DMEM+10% FBS were determined by Malvern laser particle size analyzer,respectively.2.In vivo neuroinflammatory effects induced by AgNPs.Male ICR mice were exposed to AgNPs by intranasal injection once daily(5 mg/kg bw)for 1,2,4,8 Weeks and after 8 Weeks of exposure persistently observe 4 Weeks,respectively.The Open Field Test(OFT)and the morris water maze(MWM)were assessed using to test the effects on the voluntary locomotor and learning memory abilities of mice from a behavioral perspective.The distribution and timecumulative effects of AgNPs exposure in various organ tissues were assessed by Inductively Coupled Plasma-Mass Spectrometry(ICP-MS)technique.The toxicity of AgNPs to mice was detected by body weight,organ coefficients and blood routine.Observed the pathological damage of brain tissue by HE stains.The inflammatory of brain tissue was observed by immunohistochemical F4/80 staining.The microglia were detected by immunohistochemical microglia-specific protein antibody(Ionized calcium binding adapter molecule 1,Iba-1).Western blotting was used to assess the expression of phenotype-related proteins(iNOS and CD206)and inflammation-related proteins(p65 and IL-1β).3.BV2 cells were used as a study model to examine the alteration of inflammatory phenotype of microglia by AgNPs.We observed the effect of exposure AgNPs(1.25,2.5,5,10,25,50 and 100 μg/mL)24 h on BV2 cell morphology by light microscopy.The intracellular concentrations of AgNPs were detected by flow cytometry assay after exposure of cells to AgNPs(5 μg/mL)for 2,6,12,and 24 h and AgNPs(5,25 and 50 μg/mL)for 24 h.CCK-8(Cell Counting Kit-8)was used to detect the effect of survival after AgNPs(1.25,2.5,5,10,25,50,100 and 200 μg/mL)treatment on BV2 cells for 12,24 and 48 h.The changes of various inflammatory factors at transcription level and protein level in BV2 cells by exposure to AgNPs at different doses at different times were determined using real-time quantitative PCR(RT-PCR),ELISA and Western blotting,respectively.The p65 nuclear translocation was detected by p65 cell immunofluorescence.Flow cytometry was utilized to determine the ratio of M1 and M2 type microglia by tagging iNOS and CD206.4.The role of autophagic pathway to induce neuroinflammation by AgNPs.TEM was used to observe the changes in the number of autophagosomes and autophagic lysosomes induced by AgNPs in microglia during in vitro and in vivo experiments.Western blotting was used to determine the levels in autophagic signature proteins LC3 II,p62,and Beclin1.In vivo,LC3 II and Iba-1 proteins were co-localized by immunofluorescence labeling to indicate microglia autophagy levels in brain tissue.The autophagy activation inhibitor 3-MA and autophagy lysosomal degradation inhibitor chloroquine(Cq),autophagy activator rapamycin(Rapa)and siRNA-p62 were used in vitro,respectively.The effect of autophagy on AgNPs exposure inducing increased p65 nuclear translocation,IL-1β levels abnormally elevated and M1 types of microglia was observed by immunofluorescence,ELISA and Western blotting.Analysis of the role of key processes of autophagy on the induced neuroinflammation by AgNPs.5.To investigate the role of lysosomal damage in AgNPs-induced neuroinflammation.The effect of AgNPs(5,25 and 50 μg/mL)on the autophagic flux in BV2 cells was examined by mCherry-GFP-LC3 fusion protein of BV2 cells.Acridine Orange(AO)staining was used to observe the integrity of the lysosome membrane.The lysosome probes were used Lyso Tracker Red DND-99 for lysosomal relative.In vitro and in vivo lysosomal hydrolase(Cathepsin B,CTSB),transcription factor EB(TFEB)and lysosome-associated protein(LAMP)were measured by Western blotting expression changes.Immunofluorescence was used to detect the altered expression and nuclear localization of TFEB protein after AgNPs exposure.Using siRNA-TFEB,we investigated the effects of TFEB on AgNPs exposure inducing increased p65 nuclear translocation,abnormal increase in IL-1β levels and increased M1 microglia by immunofluorescence and Western blotting.Analysis of the role of TFEB in the induced neuroinflammation by AgNPs.6.To investigate the effect of mitochondrial dynamic dysfunction in AgNPs-induced neuroinflammation.The impact of AgNPs on the mitochondrial ultrastructure in vitro and in vivo were examined using TEM.Mitochondrial division of microglia in brain tissue induced by AgNPs was indicated by immunofluorescent labeling of Drp1 and Iba-1 protein colocalization.The changes in mitochondrial-associated proteins after exposure to AgNPs were detected by Western blotting including mitochondrial biogenesis-associated protein(PGC1-α),mitochondrial autophagy-associated protein(PINK1 and Parkin1)and mitochondrial fusionassociated protein(OPA1,Drp1,p-Drp1,MFF,Mfn1,Mfn2 and FIS1).To investigate the effect of AgNPs(5,25 and 50 μg/mL)on mitochondria of BV2 cells after 24 h exposure,using the JC-1 probe,mitochondrial membrane potential(MMP)was identified.Mitochondrial morphology and number were marked with Mito-Tracker Red CMXRos.The intracellular ATP content was measured with the assay kit.LDH levels in the cell culture medium were measured to evaluate the level of cell membrane damage.Flow cytometry was used to detect apoptosis and necrosis rates in FITC-Annexin-V and PI-stained BV2 cells.The alterations of mitochondrial apoptosis-related proteins(BAX,BCL-2,caspase 3,caspase 9 and Cyt C)after exposure to AgNPs were identified using Western blotting.The changes of intracellular reactive oxygen species(ROS)and mitochondrial reactive oxygen species(mtROS)were detected by DCFH-DA and Mito-Tracker Red Cos probe,respectively.Changes in oxidative stress-related proteins(HO-1 and Hsp70)were using Western blotting to detect after exposure to AgNPs.Mitochondrial division inhibitor 1(Mdivi-1)was used to confirm the effect of mitochondria on mitochondria-dependent apoptosis and inflammation induced by AgNPs in BV2 cells.Results1.Characterization of AgNPs.TEM results show that AgNPs coated with PVP in this study are well dispersed,uniform and spherical,with an average diameter of 23.44 ± 4.92 nm.The results of ultraviolet absorption spectrum show that AgNPs has an absorption peak at 400-420 nm.The hydrated particle sizes of AgNPs in DMEM complete medium and ultrapure water were 60.11 ± 1.7 nm and 50.16 ± 1.2 nm,respectively,and AgNPs were well dispersed in DMEM complete medium and ultrapure water with only slight agglomeration.2.Exposure to AgNPs in mice caused raised levels of inflammation and increased percentage of M1 type microglia in brain tissue.Mice were exposed to AgNPs by repeated nasal drips once daily(5 mg/kg bw)for various durations(1,2,4,and 8 Weeks)and for 4 Weeks after recovery from the 8-Week exposure(12-Week groups).Body weight inhibition were observed only in 12-Week groups.ICR mice exposed to AgNPs for 1 Week and 2 Weeks showed a decrease in lung weight.Behavioral experiments showed that mice in the 4 Week group showed anxious behavior and mice in the 12 Week group showed a slight impairment in learning memory.The in vivo distribution study of mice exposed to 5 mg/kg AgNPs intranasal drip revealed that the silver content in blood,brain,lung,heart,liver,spleen and kidney tissues increased with increasing exposure time,and there was a slight decrease in the silver content in blood,brain,heart,spleen and kidney tissues in the 12-week recovery period,however,it continued to increase in lung and liver tissues.The silver levels in the olfactory bulb,cortex and hippocampus increased with increasing exposure time,and a significant increase in silver levels was detected in the olfactory bulb and hippocampus after 2 weeks of AgNPs exposure and remained high after the recovery period.Blood routine examination only revealed a decrease in the number of WBCs in the blood of mice in the 1 Week and 2 Week groups,and no significant effects were observed in the blood routine indexes of mice in other exposure groups.AgNPs exposure caused an abnormal increase in serum levels of inflammatory markers IL-6 and TNF-α with prolonged exposure time in mice.although AgNPs exposure did not cause significant pathological damage to mouse brain tissue,increased brain inflammatory response and microglia activation were observed.At the same time,AgNPs exposure caused activation of NF-κB pathway in brain tissue,resulting in the release of pro-inflammatory factors IL-1βand IL-6.These results suggest that AgNPs inhalation exposure has the tendency to accumulate in the brain of mice and induce inflammatory effects in both the organism and the brain.3.AgNPs exposure induced an increased inflammatory response in BV2 cells and the proportion of M1 microglia.AgNPs exposure induced time-dose-dependent internalization of BV2 cells.AgNPs significantly reduced cell viability in a time-and concentration-dependent manner.5 μg/mL AgNPs exposure for 24 h resulted in prolonged BV2 cell morphology observed under light microscopy.AgNPs(5 μg/mL)treatment of BV2 cells for 12 or 24 h significantly increased the levels of pro-inflammatory-related m RNAs(Iba-1,IL-1β,TNF-α,NF-κB and MCP-1)and decreased the levels of anti-inflammatory-related m RNAs(TGF-β and IL-10).At concentrations that did not affect cell viability(5 μg/mL)resulted in an increase in BV2 cell TNF α and IL-6 pro-inflammatory factors increased over time.AgNPs(5,25 and 50μg/mL)treated BV2 cells for 24 h showed that IL-1β levels in the supernatant of BV2 cells increased with dose and activated the NF-κB pathway to release the inflammatory factor IL-1β.flow cytometry was used to detect the expression of iNOS and CD206,and the results showed that AgNPs induced microglia phenotypic changes,and the proportion of microglia proinflammatory phenotype increased with increasing time dose.These results indicated that AgNPs caused cellular inflammation and increased the proportion of M1 cells even at low concentrations that did not affect cell viability.4.AgNPs-induced increase in autophagic vesicles in microglia cells mediates AgNPsinduced neuroinflammation.In in vivo experiments,increased autophagic vesicles in glial cells of mice in the 1 Week group were observed by electron microscopy after repeated exposure to AgNPs(5 mg/kg bw),and the phenomenon was observed in both the AgNPs-exposed and recovery groups.Immunofluorescence results showed that increased co-localization of LC3 II and Iba-1 was observed at the 1 Week group and the fluorescence intensity increased with increasing exposure time.Brain tissue autophagy-associated proteins LC3 II and p62 also increased with increasing exposure time.Increased autophagy vesicles were observed in BV2 cells by electron microscopy after 24 h exposure to 5 μg/mL AgNPs.AgNPs(5,25 and 50μg/mL)exposure for 24 h increased expression of autophagy-related proteins(LC3II,Beclin1 and p62)in a dose-dependent manner,and induced blockade of autophagic in BV2 cells.Blocking autophagy production and blocking the autophagic degradation process using autophagy inhibitors(3-MA and Cq)both did not affect the increased p65 nuclear localization,M1-type cell ratio,and IL-1β levels in cell cultures induced by AgNPs.Whereas autophagy activator(Rapa)was able to reduce inflammatory factor levels in BV2 cells induced by AgNPs.The interaction between autophagy and inflammatory responses suggests that neuroinflammation is associated with elevated p62,p62 low expression partially relieves the increase in p65 and IL-1β inflammatory factors caused by exposure of AgNPs in BV2 cells,suggesting that p62-regulated autophagy is essential for the induction of neuroinflammation by AgNPs and that autophagy induction is a protective factor against neurotoxicity caused by AgNPs.5.AgNPs exposure induced lysosomal damage in microglia.mCherry-GFP-LC3 double fluorescent adenovirus indicated that AgNPs(5,25 and 50 μg/mL)caused autophagy-lysosome pathway blockage in BV2 cells.western blotting results showed that p62 levels increased continuously with increasing exposure time of AgNPs.The increase of p62 was mainly caused by the blockage of autophagic degradation pathway due to lysosomal damage.Therefore,we examined the lysosomal structure and function.AgNPs induced a significant decrease in LAMP1 lysosome-related protein in mouse brain tissue in the 8-Week and 12-Week groups,and a sustained increase in TFEB key protein expression after the 4-Week group.Immunofluorescence was also observed,24 h exposure of BV2 cells to AgNPs(5,25 and 50μg/mL)showed a decrease in red fluorescence of AO staining with increasing dose,suggesting damage to cellular lysosomal membranes.BV2 cells stained with Lyso Tracker Red DND-99 fluorescent labeling showed a decrease in lysosomal acidity with increasing dose.AgNPs(5,25 and 50 μg/mL)exposed to BV2 cells for 24 h decreased CTSB and LAMP1 lysosomeassociated protein significantly with dose increase.AgNPs caused TFEB activation and induced TFEB nuclear translocation.AgNPs caused lysosomal membrane damage and decreased lysosomal hydrolase p H in BV2 cells as key factors in the blockage of autophagic flow and diminished degradative clearance.Inhibition of autophagosome formation did not affect lysosomes;Cq increased lysosomal hydrolase activity but did not alleviate lysosomal membrane damage caused by AgNPs;while Rapa alleviated AgNPs-induced changes in lysosomal membrane proteins and lysosomal hydrolases.AgNPs caused lysosomal hydrolase inactivation,which in turn led to disruption of autophagic flow and subsequent abnormal degradation in BV2 cells,promoting microglia inflammation leading to a polarized proinflammatory phenotype.6.AgNPs exposure leads to mitochondrial damage in microglia involved in AgNPsinduced neuroinflammation.In in vivo experiments,microglia mitochondrial structural damage and loss of mitochondrial ridges in microglia of the 1 Week group were observed by electron microscopy after repeated exposure to AgNPs,and this phenomenon was observed in both the AgNPs-exposed and 12 Week groups.PGC-1α and MFF protein levels decreased with time of exposed,and Drp1,p-Drp1,MFN1 and MFN2 protein levels increased with time of exposed.The levels of BAX,caspase 3 and caspase 9 proteins were consistently increased with increasing exposure time after AgNPs treatment,BCL-2 protein expression did not change.In in vitro experiments,TEM observed that 5 μg/mL AgNPs exposure for 24 h induced structural damage to mitochondria in BV2 cells.AgNPs(5,25 and 50 μg/mL)exposure for 24 h decreased MMP,intracellular ATP content in BV2 cells in a dose-dependent manner.50 μg/mL AgNPs increased punctate mitochondria in BV2 cells.AgNPs exposure upregulated the expression of mitochondrial kinetics-related proteins Drp1,p-Drp1,Mfn1 and Mfn2,downregulated MFF and FIS1 expression in in BV2 cells.AgNPs exposure caused imbalance in mitochondrial kinetics.AgNPs exposure increased apoptotic cells in a dose-dependent manner.AgNPs exposure enhanced Cyt C,caspase 9,caspase 3 and BAX protein expression levels and decreased BCL-2 protein expression levels in a dose-dependent manner.AgNPs induced LDH release and ROS levels increased with increasing dose.AgNPs induced an increase in mtROS in BV2 cells,and oxidative stress-related proteins(HO-1 and Hsp70)expression increased with increasing dose.Mdivi-1 was able to reduce apoptosis,mtROS and inflammation levels in BV2 cells after 24 h exposure to 50 μg/mL AgNPs.These results indicated that AgNPs caused mitochondrial structural and functional damage and mitochondrial division/fusion disruption,which in turn led to increased mitochondria-dependent apoptosis,and that inhibition of mitochondrial division slowed down the damage of AgNPs in BV2 cells.Conclusion1.Inhalation exposed to AgNPs induces an increase in the level of inflammation in the organism and also induces inflammatory effects in the brain.AgNPs subacute inhalation exposure tended to accumulate in the brain of mice.The lowest adverse effect level(LOAEL)is 5 mg/kg bw for subacute inhalation exposure in mice.2.AgNPs exposure induced uptake of nanoparticles by BV2 cells and decreases in cell viability in a time-and dose-dependent manner.At lower concentrations(5 μg/mL)that did not affect cell viability,AgNPs activated microglia leading to an increase in the proportion of proinflammatory phenotype M1 cells and induced cellular inflammatory responses.3.AgNPs-induced autophagic lysosomal pathway damage in microglia caused by activation of NF-κB pathway in microglia led to the release of inflammatory factor IL-1β and increased M1-type cells;meanwhile,AgNPs induced imbalance of mitochondrial dynamics and altered mitochondrial autophagy in microglia caused mitochondrial damage and mediated mitochondria-dependent apoptotic pathway leading to neurotoxicity.4.AgNPs induced lysosomal damage in microglia and thus blocked autophagic lysosomal degradation process is a key factor leading to increased neuroinflammatory response.The neuroinflammation induced by AgNPs in microglia is engaged by the TFEB-p62-NF-κB pathway,and they are key targets to mitigate the potential neurotoxicity of AgNPs.In summary,AgNPs exposed can induce organismal,brain tissue and cell inflammation responses,and timely monitoring of organismal inflammation levels may be an effective means to prevent damage from AgNPs exposure;in microglia neuroinflammatory response induced by AgNPs,the autophagy,lysosomal and mitochondrial pathways are mutually regulated,and the TFEB-p62-NF-κB pathway is a potential neurotoxic mitigation of AgNPs.The results of the study provide valuable basic information for the safe application of AgNPs. |
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