The Mechanism Of Glycometabolic Changes And Cytotoxicity In Microglia Induced By CdTe/ZnS Quantum Dots

Aim:This study aimed to explore the mechanism of abnormal glucose metabolism and cytotoxicity in microglia exposed to CdTe/ZnS quantum dots(QDs)from the perspective of metabonomics,and to explore the toxicological significance of changes in the direction of glucose metabolism.Methods:1)CdTe/ZnS QDs were prepared by electrochemical synthesis.The fluorescence and particle size of CdTe/ZnS QDs were characterized and compared with bare core CdTe QDs.The cytotoxicity of the two kinds of QDs on microglia was compared by cell viability.2)CdTe/ZnS QDs were injected into the tail vein of mice.The distribution of CdTe/ZnS QDs in the brain and other organs were quantitatively analyzed by Cd element and fluorescence.The results showed that CdTe/ZnS QDs could penetrate the blood-brain barrier and produce neurotoxicity.3)According to the distribution of CdTe/ZnS QDs in vivo,the exposure concentration(1.25 ?M)and time(within 12 hours)were set.The metabolic pathways related to QDs neurotoxicity in microglia after CdTe/ZnS QDs exposure were screened by gas chromatography-mass spectrometry(GC-MS).Cell energy analysis was used to verify the differences in glucose metabolism and oxidative phosphorylation metabolism in microglia.Other differential metabolic pathways were verified by cytotoxic reactions such as reactive oxygen species(ROS),glutathione(GSH),DNA damage and organelle damage.4)Immunofluorescence analysis was used to verify the neurotoxicity of CdTe/ZnS QDs by measuring the activation of microglia and the number of neurons in the brain of miceexposed to CdTe/ZnS QDs.The secondary cytotoxicity of microglia activation on neurons was verified by microglia-neuron co-culture model in vitro.The expression of mTOR pathway-related proteins in hippocampus and microglia was detected in vivo and in vitro.To compare the effects of mTOR inhibitor pretreatment on microglia activation,inflammatory factor secretion,cell glycometabolism and neuronal secondary injury induced by CdTe/ZnS QDs exposure.Results:1)CdTe/ZnS QDs are spherical with an obvious lattice structure.The particle size of CdTe/ZnS QDs is 5.7± 0.8 nm,and the hydrated particle size is 8.57± 2.60 nm.The dispersion of CdTe/ZnS QDs is good.Compared with bare CdTe QDs(613 nm),CdTe/ZnS QDs fluorescence emission(655 nm)shifted to near the infrared region,which is more suitable for bioimaging.The toxicity of CdTe/ZnS QDs to microglia was significantly lower than that of naked CdTe QDs.2)The distribution level of CdTe/ZnS QDs in the brain reached its peak after exposure to CdTe/ZnS QDs via tail vein for 3 h,with the highest level in the hippocampus(1.25 ?M).CdTe/ZnS QDs can cross the blood-brain barrier and image in brain tissue.The effect of CdTe/ZnS QDs is better than that of nude CdTe QDs.The 12 h distribution of CdTe/ZnS QDs in mice showed that CdTe/ZnS QDs gradually entered various organs within 1 hour after entering the blood circulation,and the concentration of CdTe/ZnS QDs in liver and spleen was the highest.After 6 hours of exposure,CdTe/ZnS QDs in various organs gradually transferred to the kidney and excreted in vitro.After 12 h,most CdTe/ZnS QDs were discharged from the body,but there was still a small amount of accumulation in various organs.3)Based on the results of in vivo distribution,287 kinds of small molecule metabolites were identified by CG-MS detection platform after exposure of BV-2 cells to 1.25 ?M CdTe/ZnS QDs for 3 h,6 h,and 12 h.Finally,11 stable metabolic pathways related to the neurotoxic mechanism of CdTe/ZnS QDs were screened,which mainly involved glycometabolism,glutathione metabolism,nucleic acid metabolism,lipid metabolism,and active transport.It has been proved that after CdTe/ZnS QDs exposure,microglial cell glucose metabolism changes from aerobic respiration to glycolysis;intracellular reactive oxygenspecies level increases with exposure time,and reduced glutathione increases compensatively after exposure,and then gradually depletes;CdTe/ZnS QDs is genotoxic and can cause microglial DNA damage after exposure;cell electron microscopy observation shows that CdTe/ZnS QDs enter the cell body.It can cause subcellular structural damage such as endoplasmic reticulum,ribosome,and mitochondria.4)CdTe/ZnS QDs have neurotoxicity.After CdTe/ZnS QDs were exposed via tail vein,the polarization level of microglia in the hippocampus increased and the number of neurons decreased.Exposure to CdTe/ZnS QDs can activate microglia and increase the secretion of inflammatory factors.The indirect cytotoxicity caused by activation of microglia is additive to the direct cytotoxicity of CdTe/ZnS QDs.Exposure to CdTe/ZnS QDs can activate the mTOR pathway in microglia.After pretreatment with rapamycin,an mTOR inhibitor,and exposure to CdTe/ZnS QDs,the number of M1 microglia and M2 in the hippocampus of mice decreased.After pretreatment with rapamycin and exposure to CdTe/ZnS QDs in BV-2 cell model,the levels of aerobic glycolysis and aerobic respiration of glucose metabolism recovered.The secretion of extracellular pro-inflammatory factors NO,TNF-? and IL-1?decreased,while the levels of anti-inflammatory factors IL-6 and IL-10 had very limited changes.Conclusion:1.25 ?M CdTe/ZnS QDs can induce a series of damage and changes in microglia.Microglia can be polarized by CdTe/ZnS QDs,then the indirectly cytotoxic to neuronal cells can be induced by inflammatory factors.The glucose metabolism in the exposed microglia is transferred from aerobic oxidation to glycolysis,which is regulated by the mTOR pathway and is a necessary condition for the differentiation of microglia into M1 type.This study is an important supplement to the study of neurotoxicity of cadmium QDs.