| Nanomaterials have attracted wide attention due to their special physical and chemical properties.Numerous studies have demonstrated that nano-TiO2 is a mass-produced material.Due to its small size,strong stability,large specific surface area,and ultraviolet absorption capacity,it is widely used in commercial,industrial production,and daily life.Nano-TiO2 in the environment can enter the body through the skin,digestive tract and respiratory system,damaging various tissues and organs of the body.Studies at home and abroad showed that nano-TiO2 can penetrate the blood-brain barrier and enter the central nervous system,affect the development of neurons,and cause damage to learning and memory.In addition,in vitro studies on the neurotoxicity of nano-TiO2 have shown that nano-TiO2 inhibits the development of hippocampal neurons and induces neuronal apoptosis.However,the effect of nano-TiO2 on the development of axons and synapses in hippocampal neurons related to learning and memory and its mechanism remain unclear.Therefore,this paper uses the primary cultured hippocampal neurons as a model to study the development of axons and synapses in neurons after exposure to anatase-type nano-TiO2,and further study its molecular mechanism.The results of this paper can provide theoretical and data support for neurotoxicity and impairment of learning and memory ability after nano-TiO2 exposure.The main results are listed as follows:(1)In this part of the study,in order to study the neurotoxic effects of nano-TiO2,hippocampal neurons isolated from 24-h-old Sprague-Dawley rats were exposed to 5,15 and 30 ?g/mL nano-TiO2 for 6 h,12 h and 24 h.The distribution of nano-TiO2 in neurons and the effect on the development of axons were studied.The results showed that nano-TiO2 enters the cytoplasm of primary cultured hippocampal neurons and accumulates around the nucleus in a concentration-and time-dependent manner.Increasing time in culture resulted in decreasing axon length by 32.5%,36.6%and 53.8%at 6 h,by 49.4%,53.8%and 69.5%at 12 h,and by 44.5%,58.2%and 63.6%at 24 h,for 5,15 and 30?g/mL nano-TiO2,respectively.Further research found that compared with the control group,the expression of Netrin-1 decreased by 9.27%,33.01%,and 44.47%,the expression of GAP-43 was down-regulated by 12.73%,20.65%and 33.33%;Neuropilin-1 protein expression decreased by 8.17%,22.14%and 36.9%when the nano-TiO2 concentration was 5,15,and 30 ?g/mL.And alterations in the following proteins were observed:increases of 0.08-,0.19-,and 0.29-fold for Sema3A;0.06-,0.24-and 0.29-fold for Nogo-A.These studies suggest that nano-TiO2 inhibited axonal development in rat primary cultured hippocampal neurons and this phenomenon is related to changes in the expression of axon growth-related factors.(2)In order to further discuss the neurotoxicity of nano-TiO2,the study of the synapse and plasticity of hippocampal neurons exposed to nano-TiO2 was studied.Specific research contents:Density spine density,which is closely related to synaptic growth,was detected by F-actin staining;the effect of nano-TiO2 on apoptosis of hippocampal neurons was analyzed by flow cytometry;F-actin and SYN I double immunofluorescence staining was used to determine the changes in synaptic density.Then the expression of SYN I and PSD95 in neuron synapse-derived genes was further tested.Finally,the BDNF-TkB signaling pathway was performed by ICC and Western blot analysis.The results showed that when the concentration of nano-TiO2 was 10,20,30,40,60,80,and 100 ?g/mL,compared with the control group,the dendritic spine density of neurons decreased by 18.06%,34.8%,33.33%,43.14%,53.65%,68.33%and 75%,which proves that nano-TiO2 inhibits the synaptic plasticity of hippocampal neurons.Studies on nano-TiO2 at lower concentrations(5,15 and 30 ?g/mL)found that the apoptosis rate of hippocampal neurons increased,neuronal synapse development was inhibited,and synaptic densities decreased by 24.29%,54.29%and 72.86%.In addition,the expressions of SYN I and PSD95 in neuron synapse were also significantly inhibited,of which SYN I decreased by 18.43%,37.2%and 51.6%and the PSD95 decreased by 16.02%,24.06%and 38.74%,respectively.Studies on the BDNF-TrkB signaling pathway found that nano-TiO2 inhibited the expression of key proteins in downstream MEK/ERK and PI3K/Akt signaling pathways by inhibiting the expression of BDNF.When the concentration of nano-TiO2 was 5,15,and 30 ?g/mL,the expression of BDNF decreased by 22.64%,33.3%and 53.58%compared with the control group.The downstream key proteins p-CREB/CREB ratios decreased by 3.03%,18.11%and 30.57%;p-ERK1/2/ERK1/2 ratios decreased by 19.11%,28.82%and 58.09%,and the ratios of p-Akt1/Akt1 decreased by 1.92%,27.79%and 41.33%,respectively.These results demonstrated that nano-TiO2 inhibits the normal function of the BDNF-TrkB signaling pathway,which is closely related to neuronal synapse.It can be speculated that the inhibition of neuronal synaptic growth by nano-TiO2 may be related to the inhibition of BDNF-TrkB signaling pathway. |
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