Effect Of CuInS₂/ZnS-PEG Quantum Dots On Neurons-like PC12 Cell And Its Mechanism

Background:In recent years,with the increasing application of quantum dots（QDs）in fields such as biomedicine,researchers have paid more and more attention to the possible adverse effects of QDs on the environment and humans.Previous studies have shown that QDs can cross the blood-brain barrier and enter the central nervous system,but its toxic effect on neuronal cells and its mechanism are not yet clear.In this study,PEGlayted CuInS2/ZnS QDs were selected as the research object.The purpose of this project is to study the toxic effects of PEGlayted CuInS2/ZnS QDs on neuron-like PC12 cells and to elucidate their mechanism.Methods:In this experiment,CuInS2/ZnS-PEG QDs were characterized by transmission electron microscopy,dynamic light scattering particle size analyzer,ultraviolet and visible light absorption spectrum,and fluorescence spectrum.Then the fluorescence microscopy and flow cytometry were used to observe the uptake of QDs by PC12 cells;the MTT method was used to detect the effect of QDs on the viability of PC12 cells;the effects of QDs on cell neurite outgrowth were observed using optical microscopy and immunofluorescence;Using full transcriptome sequencing technology combined with GO database and KEGG pathway informatics analysis,observe the effect of QDs on gene expression profiles and NGF related receptors and pathways,and use RT-PCR to verify;finally,at the protein level,the effect of QDs on the MAPK/ERK signaling pathway downstream of NGF was analyzed using a protein antibody chip,and verified by Western blotting.Results:CuInS2/ZnS-PEG QDs could be taken up by PC12 cells.At a concentration of 25μg/mL,the uptake rate reached 95%.When the concentration of QDs is less than 100μg/mL,there is no significant effect on cell viability.However,when the concentration is higher than100μg/mL,the QDs significantly inhibit cell viability,and the IC50 value of 48 hours is 256.2μg/mL.CuInS2/ZnS-PEG QDs significantly inhibited the length and number of neurites in PC12 cells stimulated by NGF,and significantly reduced their differentiation rate.Transcriptome sequencing and RT-PCR results showed that CuInS2/ZnS-PEG QDs significantly down-regulated the MAPK gene pathway downstream of NGF and reduced the transcription level of the low-affinity receptor,p75^NTR.Protein antibody chip and western blot analysis revealed that p75^NTR protein levels were significantly reduced,and ERK1/2 and AKT phosphorylation levels were also reduced.Conclusion:CuInS₂/ZnS-PEG QDs transmembrane into PC12 nerve cells,reduce the expression of the low-affinity receptor p75^NTR on the membrane,negatively regulate the downstream MAPK signaling pathway,and affect the exogenous NGF stimulation signal transduction through the NGF/p75^NTR/MAPK pathway,thereby affecting neurite outgrowth and neuronal differentiation.The development of this experiment is of great significance for correctly understanding the potential toxicity of QDs and optimizing the rational design and safe application of QDs.