| Nowadays,with the rapid development of nanotechnology,a variety of nano-products are emerging.Among the many nanomaterials,nanosilver ranks first in the application of nanomaterials due to its excellent antibacterial properties.The wide application of nanosilver in health care,food processing,industrial production and other aspects plaies an indispensable role in promoting the development of human society,but at the same time,it also increases the chance of contact between nanosilver and human,bringing potential safety risks.Many studies have shown that nanosilver can enter the body through the respiratory system,digestive system and skin contact,and can also reach the central nervous system along the olfactory and trigeminal nerves.The silver nanoparticles entering the body was distributed to the tissues and organs of the whole body through the blood circulation,which could cross the blood-brain barrier to the brain tissue and accumulated in it,and caused central nervous system toxicity.At present,the neurotoxic effects of silver nanoparticles mainly include brain histopathological effects,neurobehavioral changes and neurotransmitter level changes in the tested animals,and the toxic mechanism is related to oxidative stress and inflammatory response.The study on the neurotoxicity and the toxic mechanism of silver nanoparticles is not enough,which needs to be explored constantly,in order to provide experimental support and theoretical basis for the safety evaluation of silver nanoparticles.In this study,caenorhabditis elegans(C.elegans)and mouse hippocampal nerve cells(HT22 cells)were selected as the research objects.The neurotoxicity of silver nanoparticles was explored by investigating the effects of silver nanoparticles on thebehavior,learning and memory of caenorhabditis elegans to study the nerve damage effect of nanosilver from the perspective of in vivo experiment.The cytotoxicity of silver nanoparticles on HT22 cells was also evaluated and the role of oxidative stress and autophagy in cell injury was further explored,which provided support and evidence for the study of the mechanism of cytotoxicity of nanosilver.1.Characterization of nanosilver materials: It was found by TEM that the silver nanoparticles used in this study was spherical and the particle size was mainly concentrated at 13-21 nm,the average particle size was 20.14 nm.The results of Malvern particle size analyzer showed that the hydrated particle size of nanosilver was mainly distributed between 100-1000 nm,the average hydrated particle size was566.5 nm,and the Zeta potential value was 0.323 mV,indicating that the silver nanoparticles used in this experiment was slightly agglomerated in the medium,and the degree of dispersion was relatively better.2.The neurotoxicity of silver nanoparticles to caenorhabditis elegans: The frequency of head thrashes and body bending varied in different doses of nanosilver exposure group.Compared with the blank control group,the head thrashes frequency of the nematode decreased gradually with the increase of the concentration of nanosilver.The head thrashes frequency of the 50,100 and 200 μg/ml groups all decreased significantly(P<0.05)except for 25 μg/ml group.And the frequency of head swing in 25,50,100,200 μg/ml dose groups decreased by 5.56%,16%,25.47%,25.57% respectively compared with the blank control group.The body bending frequency of nematode decreased gradually with the increase of exposure dose.Compared with the blank control group,the head thrashesfrequency of 25,50,100,200 μg/ml dose group decreased significantly,and the difference was statistically significant(P<0.05).Compared with the blank control group,the body bending frequency of the 25,50,100,200 μg/ml groups decreased by 21%,37.50%,45.60%and 53%,respectively.It indicated that 20 nm silver nanoparticles could affect the head thrashes and body bending behavior of C.elegans,and the effect of nanosilver on the bending frequency of nematode body was greater than that of head thrashes frequency.In the experiment of the influence of nanosilver on the learning ability of C.elegans,it was found that the C.elegans without nanosilver exposure or with low doses exposure could better integrate the starvation signal and sodium chloride concentration signal.The number of C.elegans moving to the sodium chloride area increased with the increase of the dose,indicating that fewer C.elegans could integrate the starvation signal and sodium chloride concentration,and the C.elegans associative learning ability is constant.This phenomenon indicated that the damage of silver nanoparticles to the nervous system of C.elegans increased with the increase of exposure dose.In the experiment of the effect of silver nanoparticles on the memory ability of C.elegans,it was found that the number of C.elegans doing IT movement in the range of 20℃ declined as hunger time increased both in blank control group and each the experimental group.With the increase of the dose of nanosilver,the number of C.elegans doing IT movement in the range of 20℃ decreased gradually.And the blank control group had the highest number of C.elegans at each test point,followed by the 25 μg/ml group,the 50 μg/ml group,the 100 μg/ml group,and the200 μg/ml group.At the 0,1 and 3 h test time points,the difference between the exposed group and the blank control group was significant(P<0.05).At the 7 h and12 h test time points,the difference between the exposed group and the blank control group was small,indicating that in the first 3 hours of detection,different doses of nanosilver had a greater impact on the memory ability of C.elegans.The study showed that the memory ability of C.elegans was related to time.The longer the time,the worse the memory ability was;and as the dose of silver nanoparticles increased,fewer and fewer C.elegans could associate 20 ℃ temperature conditions with food signals by memory.The result suggested that the neurotoxicity of nanosilver to C.elegans gradually increased with the increase of the dose.3.Cytotoxicity of silver nanoparticles on HT22 cells: The results of MTT assay showed that the cell survival rate of HT22 cells was decreased in a dose-dependent manner after 24 h exposure to different doses of silver nanoparticles.There were statistically significant differences in the cell survival rate of all the experimental group compared with the blank control group(P<0.05).In 25,50 and 100 μg/ml dose group,the cells showed higher survival rate and survival rate were 94.25%,87.07%and 73.06% respectively,indicating that at doses below 100 μg/ml,the nanosilver had little impact on the survival rate of HT22 cells.When the concentration reached 200μg/ml,the survival rate of HT22 cells was as low as 39.90%,and the survival rates of HT22 cells at 400 and 800 μg/ml were 38.06% and 27.37%,respectively.The results of LDH assay showed that when the concentration was less than 400 μg/ml,the release rate of LDH in HT22 cells increased in a dose-dependent manner.Compared with the blank control group,the release rate of LDH was statistically different(P<0.05).However,when the concentration reached 800 μg/ml,the release rate of LDH decreased.The results of this experiment indicated that nanosilver could affect the release rate of LDH in HT22 cells and had cytotoxicity.Cell morphology study found that,compared with HT22 cells under normal culture conditions,the cell morphology changed to a certain extent after exposure to different concentrations of silver nanoparticles.The morphological changes of cells in the low-dose groups(25and 50 μg/ml)were not obvious,and there were not many floating cells.However,in the high-dose groups(100 and 200 μg/ml),there were more floating cells,larger cell spaces,less synaptic connections between cells,round cells,unclear cell membrane boundaries and other cellular damage phenomena.The results of the study indicated that different doses of silver nanoperticles could cause different degrees of damage to cell morphology.Low dose has little effect on cell morphology,and high-dose exposure has serious damage to cell morphology.4.Study on the toxic mechanism of silver nanoparticles of HT22 cells: In the experiment of the effect of nanosilver on cellular ROS levels,we found that the intracellular ROS level varied in each dose group after the HT22 cells were exposured by silver nanoparticles.In the lower dose group,the ROS level increased with the increase of concentration of silver nanoparticles,while the intracellular ROS level decreased in the higher dose group.The results showed that low dose exposure of silver nanoparticles could promote the generation of reactive oxygen species and enhanced oxidative stress in HT22 cells.However,when the toxic dose was too large,the ROS production capacity of cells was weakened.When the oxidative stress inhibitor NAC was added,the ROS level of HT22 cells did not change significantlywith the increase of the dose of silver nanoparticles.In the experiment of the effect of silver nanoparticles on the level of autophagy in HT22 cells,we observed autophagosome in both the blank control group and the experimental group,which proved that autophagy occurred in cells under normal culture conditions,and also after being poisoned by nanosilver.The expression levels of two autophagy-related genes,LC3 and Beclin-1,showed that there were no significant changes in the expression levels of LC3 at 25,50 μg/ml compared with the blank control group,while the expression levels were significantly increased in the high dose group at 100μg/ml.The expression level of Beclin-1 increased significantly in 25,50 μg/ml groups(P<0.05),while the gene expression level did not change significantly in the 100μg/ml group compared with the blank control group.The results of autophagy related protein expression test showed that the expression level of LC3-I protein was continuously decreased and the expression level of LC3-II protein was continuously increased after the HT22 cells were exposured by silver nanoparticles.The LC3-II/LC3-I values did not change significantly at the 25 μg/ml group,and increased significantly at the 50 and 100 μg/ml doses(P<0.05).The expression level of Beclin-1protein was significantly increased at the dose of 100 μg/ml(P<0.05),but not significantly in the 25 and 50 μg/ml groups.The results of the experiment showed that low concentration of nanosilver had little effect on the autophagy level of HT22 cells,while high concentration of nanosilver could increase the autophagy level of HT22 cells.After NAC pretreatment of cells,LC3-II/LC3-I values were not significantly changed in all dose groups,Beclin-1 protein expression levels were not significantly changed in all dose groups,suggesting that after inhibiting the oxidative stress level of cells,silver nanoparticle had no significant effect on the autophagy level of HT22 cells.In summary,the exposure of silver nanoparticles at different concentrations could affect the motor behavior,learning and memory ability of C.elegans,and caused damage to the nervous system of C.elegans.The greater the concentration of silver nanoparticles,the more serious the damage,indicating that silver nanoparticles had certain neurotoxicity.In addition,the study also found that silver nanoparticlescould destroy the normal morphology of HT22 cells,inhibited cell proliferation and reduced cell viability.As the concentration of nanosilver increased,the degree of damage gradually increased.In the study on the mechanism of toxicity,it was found that nanosilver could induce the increase of ROS production,enhanced oxidative stress,and initiated autophagy when exposed to medium and low doses,while reduced ROS production,excessive autophagy and accelerated cell death when exposed to high doses.After pretreatment of cells with oxidation inhibitors NAC,the effect of nanosilver on oxidative stress and autophagy levels were weakened.There was no significant difference in ROS level and autophagy level between the dose groups,indicating that autophagy was regulated by intracellular ROS.The moderate enhancement of ROS could induce autophagy initiation,but when the ROS level was unbalanced,the level of autophagy was also unbalanced.At this time,the damage of silver nanoparticles to cells would also be highlighted.This study provided theoretical support and experimental basis for the study of the neurotoxicity and toxic mechanism of silver nanoparticles. |
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