The Mechanism Of Dorsal Root Ganglion Injury And Cytotoxicity Induced By Cdte Quantum Dot Exposure

Research objectives:In this study,we studied the toxic effects of MPA-modified CdTe QDs on the peripheral nerves,taking into account the safe application of QDs in neurological research,especially in tracing the peripheral nerve injury.The CdTe QDs used in subsequent chapters were all MPAmodified CdTe QDs.Methods:1)Preparation and characterization of CdTe QDs.The MPA-modified CdTe QDs were prepared by electrochemical synthesis,and characterized by Malvern size analyzer and TEM.2)Neurotoxic effects of CdTe QDs on zebrafish.The toxic effects of CdTe QDs on zebrafish embryos and their larvae in terms of peripheral motor neurotoxicity and motor behaviour toxicity were studied.Neuronal damage studies were performed using Tg(hb9:EGFP)transgenic zebrafish embryos.The neurobehavioural toxicity were analysed using the DanioVisionTM System.Changes in mRNA expression of neurodevelopment-related genes were measured by real-time fluorescence quantitative PCR to comprehensively assess the effects of CdTe QDs exposure on central and peripheral nerves and their mechanisms.3)The distribution of CdTe QDs in the major organs of rats and their neurotoxic effects on dorsal root ganglion.Twenty-five male Wistar rats,weighing 240-250 g,were divided into control,3h,6h,12h and 24h CdTe QDs injection groups,with five rats in each group.The rats were injected intraperitoneally with 2.5 mM CdTe QDs at 0.5 ml/100 g(QDs volume/rat body weight).The distribution of CdTe QDs in the main tissues and organs of rats,especially in dorsal root ganglion,was quantified by inductively coupled plasma mass spectrometry(ICP-MS)from 0 to 24h.The pathological results of the rats’ blood and dorsal root ganglion tissues were used to investigate the damage caused by QDs.The ability of CdTe QDs to penetrate the blood-nerve barrier and produce peripheral neurotoxicity remained to be verified.4)Toxic effects and mechanisms of CdTe QDs on ND7/23(rat dorsal root ganglion cell line)and RSC96 cells(rat Schwann cell line).The differences between the cytotoxic effects of ND7/23 cells and RSC96 cells exposed to CdTe QDs were analysed according to the distribution and specific physiological functions of these two cells.The mechanism of the neurocytotoxic effects due to CdTe QDs exposure was analysed specifically in terms of the different signalling pathways of apoptosis and cellular autophagy.Results:1)Preparation and characterization of CdTe QDs.The CdTe QDs were spherical,with a distinct lattice structure and uniform particle dispersion,with a particle size of 3.5±0.49 nm and a hydrated particle size of 6.346± 1.285 nm,and a zeta potential of-18.4±9.17 mV in aqueous solution.2)Neurotoxicity of CdTe QDs on zebrafish.CdTe QDs(100 nM and above)exposure caused a reduction in the hatching rate of 72 hours post-fertilization(hpf)zebrafish embryos and various malformations(yolk sac edema,small eyes,curved spine and tail deformities)in 24hpf-144hpf zebrafish.QDs(100 nM and above)exposure also led to a reduction in the axon length of motor neurons,reducing neurogenesis and the length and number of motor nerve branches in 144hpf zebrafish larvae.The mean swimming speed and total movement time of zebrafish in the 400 nM CdTe QDs treatment group were 24.7%and 17.1%of the control group,respectively.The genes related to neurogenesis(nrd)and axonal growth(mbp,gfap and gap43)were significantly affected by CdTe QDs exposure,and their expression was reduced in 72hpf zebrafish larvae exposed to 400 nM CdTe QDs.3)Neurotoxicity of CdTe QDs in rats.CdTe QDs were able to cross the blood-nerve barrier into the peripheral nerves and accumulate in the dorsal root ganglion neurons.After intraperitoneal injection of CdTe QDs,the Cd concentrations in the dorsal root ganglion tissues of rats at 3h(127.54±5.41 ng/g),6h(196.81±24.03 ng/g),12h(415.41±33.69 ng/g)and 24h(643.56±33.22 ng/g)exceeded those in the brain or spinal cord at the same time.The dorsal root ganglion cells of rats exposed to QDs for 6h and above showed a disorganized arrangement of neurons,a significant increase in cellular folds and cellular edema,and a significant consolidation of neuronal nuclei.24h exposure group showed a typical apoptotic cell morphology.4)Toxicity of CdTe QDs on ND7/23 cells.Based on CCK-8 results,ND7/23 cells were exposed to 10μM CdTe QDs for 0-24h.CdTe QDs exposure reduced the activity of ND7/23 cells and promoted apoptosis,with a significant dose-effect relationship.CdTe QDs exposure increased the release of cytochrome C and decreased the mitochondrial membrane potential,thereby promoting the flow of calcium ions from the extracellular to the intracellular compartment.The mitochondrial pathway was involved in CdTe QDs induced apoptosis of ND7/23 cells.Transmission electron microscopy results showed that CdTe QDs could cause damage to subcellular structures such as the endoplasmic reticulum,lysosomes and mitochondria.Further studies showed that CdTe QDs exposure triggered endoplasmic reticulum stress and activated the calpain2-caspasel2 signalling pathway,leading to mitochondrial independent apoptosis.5)Toxicity of CdTe QDs on RSC96 cells.After 24h exposure,0-80μM CdTe QDs induced apoptosis in RSC96 cells in a concentration-dependent manner,caused cytochrome C outflow,and increased the expression of Bax,caspase3 and cytochrome C proteins,and decreased the expression of Bcl-2 protein.CdTe QDs could be internalized by cells,and exposure and internalization of CdTe QDs could induce endoplasmic reticulum stress.Bip,CHOP and cleaved-caspase 12 proteins were increased in a concentration-dependent manner.Autophagyrelated proteins LC3Ⅱ,Beclinl and P62 were all increased after 24h CdTe QDs(40μM and above)exposure,indicating that QDs exposure both promoted autophagosome formation and inhibited autophagosome degradation.24h CdTe QDs(20μM and above)exposure led to oxidative stress and calcium overload in RSC96 cells.The endoplasmic reticulum and mitochondria were severely damaged morphologically and functionally by CdTe QDs exposure,as evidenced by swelling of the endoplasmic reticulum,blocked mitochondrial fission and a shift from normal rod-shaped to spherical mitochondria,along with loss of mitochondrial cristae,excessive mitochondrial reactive oxygen species production and a marked decrease in mitochondrial membrane potential.The toxic effects of CdTe QDs on these organelles were the primary cause of apoptosis in RSC96 cells.Pre-incubation of antioxidants or calcium chelators could alleviate apoptosis by improving organelle function.Conclutions:This thesis systematically investigates the peripheral neurotoxicity of CdTe QDs that have rarely been reported in current national and international laboratories.The conclutions are as follows.1)Exposure to CdTe QDs(100 nM and above)led to shortened axon lengths and reduced number of motor nerve branches in 24hpf-120hpf zebrafish embryos and larvae.CdTe QDs exposure also reduced zebrafish motility and decreased expression of genes related to neurogenesis and axon growth.Exposure to CdTe QDs of 100 nM and above resulted in reduced motility in 144hpf zebrafish larvae.The expression level of genes related to neurogenesis and axonal growth was significantly reduced in 72hpf zebrafish larvae after 400 nM QDs exposure.2)The present study also firstly proved that 3.5 nm CdTe QDs could cross the blood-nerve barrier into the peripheral nerves of rats and accumulate in dorsal root ganglion region,causing damage to dorsal root ganglion cells.3)In vitro experiments showed that exposure to CdTe QDs caused a series of damage and changes in ND7/23 cells and RSC96 cells.Both the exposed cells showed typical characteristics of apoptotic cells,and both mitochondria-dependent and mitochondria-independent signalling pathways were involved in the apoptotic process of these cells.ND7/23 or RSC96 cells exposed to CdTe QDs showed the occurrence of oxidative stress and overproduction of ROS.Meanwhile,the occurrence of cellular autophagy was detected in the RSC96 cell line.CdTe QDs exposure both promoted the formation and inhibited the degradation of autophagosomes,inducing defects in autophagy and ultimately apoptosis.This research fills the gap in the study of peripheral neurotoxicity of QDs and further extends and improves the study of QDs neurotoxicity.This study provides a theoretical basis for better investigation of the peripheral neurotoxicity of CdTe QDs,and also provides certain reference and help for the subsequent design optimization of QDs,the preparation of QDs and their safe use.