| Titanium dioxide nanoparticles(Ti O2 NPs) which exhibit novel physicochemical properties and functions are widely used in all kinds of consumer products such as environmental containmination management, cosmetics, antibacterial fiber, food additive, biomedical ceramic and paints. Its potential entry through dermal, ingestion, and inhalation routes exhibits an exposure risk to human beings. Moreover, Ti O2 NPs can enter the central nervous system via the olfactory pathway and damaged the brain neurons. Though there are some documents about neurotoxicity and impairment of spatial recognition memory caused by Ti O2 NPs worldwide, there are few studies on hippocampal neurons treated with Ti O2 NPs associated with learning and memory. In the present academic dissertation, we selected fetal rat primary hippocampal neurons which retain neurodevelopment characteristics and its growth is associated with learning and memory, to explore whether Ti O2 NPs would cause primary cultured hippocampal neuron apoptosis and activate signal pathways of apoptosis and influence neurite outgrowth of neurons. Our findings will provide an important theoretical basis for evaluating the neurotoxicity underlying effects of Ti O2 NPs on animals and human.The main results are listed as follows:(1) In this study, primary cultured hippocampal neurons from one-day-old fetal Sprague Dawley rats were exposed to 5, 15, or 30 μg/m L Ti O2 NPs for 24 h, we investigated cell viability, ultrastructure, and mitochondrial membrane potential(MMP), calcium homeostasis, oxidative stress, antioxidant capacity, apoptotic signaling pathway associated with the primary cultured hippocamal neuron apoptosis. Our findings showed that Ti O2 NP treatments decreased cell viability and increased LDH activities, suggesting 13.78%, 20.42% and 29.42% reductions and 19.54%, 36.13%, and 62.07% elevations, respectively, as compared with the control. With increasing Ti O2 NP concentration, primary neuron apoptotic rate were significantly increased, equal to 2.51-, 3.14-, and 4.25-fold of the control value, respectively. Furthermore, Ti O2 NPs led to [Ca2+]i elevation, and equal to 1.55-, 2.66-, and 3.76-fold of control value, and MMP reduction, and equal to 84.20%, 60.00%, and 38.00% of control value, respectively. Moreover, Ti O2 NPs upregulated protein expression of cytochrome c, caspase-3, Bax, caspase-12, glucose-regulated protein 78(GRP78) and C/EBP homologous protein(CHOP), equal to 1.54-, 2.22- and 3.77- fold, 2.15-, 2.85- and 4.15-fold, 1.33-, 2.17- and 2.83-fold, 1.10-, 1.36- and 1.62-fold, 1.15-, 1.27- and 1.37-fold and 1.07-, 1.25- and 1.40-fold of control value, respectively, and down-regulated bcl-2 expression, suggesting 8.63%, 19.76% and 34.70% reductions as compared with the control in the primary cultured hippocampal neurons. These findings suggested that hippocampal neuron apoptosis caused by Ti O2 NPs is associated with mitochondria-mediated signal pathway and endoplasmic reticulum-mediated signal pathway.(2) In order to further investigate the mechanism of neurotoxicity, rat primary cultured hippocampal neurons on the fourth day were exposed to 5, 15, and 30 ?g/m L Ti O2 NPs for 24 h, then Ti O2 NPs internalization, dendritic growth, glutamate metabolism, expression of N-methyl-D-aspartate(NMDA) receptor subunits(NR1, NR2 A and NR2B), calcium homeostasis, and sodium current(INa) and potassium current(IK) were examined. Our findings demonstrated that Ti O2 NPs easily crossed membrane into cytoplasm or nucleus, and greatly suppressed dendritic growth, e.g. length of neurite in neuron was inhibited by 6.92%, 28.91% and 55.54%, respectively, as compared with those of the control in primary cultured hippocampal neurons. Furthermore, Ti O2 NPs disturbed glutamic acid metabolism, showing great release of glutamate to extracellular and increased phosphate-activated glutaminase activity, suggesting 7.75%, 16.27% and 26.26%, 27.27%, 63.64% and 90.91% elevations as compared with the control, and decreased levels of glutamine and glutamine synthetase activity, suggesting 14.54%, 29.04% and 54.22%, 10.25%, 20.51% and 30.77% reductions as compared with the control. Moreover, Ti O2 NPs increased levels of nitric oxide and activity of nitric oxide synthesis, equal to 0.19-, 0.49- and 1.02-fold and 0.17-, 0.38- and 0.59-fold of control value. Furthermore, Ti O2 NPs led to [Ca2+]i elevation, equal to 1.35-, 2.75-, and 4.45-fold of control value, and down-regulation of the protein expression of NR1, NR2 A and NR2 B, showing 9.62%, 12.00% and 25.30%, 4.39%, 14.22% and 39.85%, 1.61%, 16.04% and 26.07% reductions as compared with the control. In addition, Ti O2 NPs attenuated the activities of Ca2+-ATPase and Na+/K+-ATPase, suggesting 9.46%, 29.04% and 45.32%,12.96%, 23.28% and 50.80% reduction as compared with the control, and increased the amplitudes of INa and IK in primary hippocampal neurons. Taken together, Ti O2 NPs inhibit neurite outgrowth of hippocampal neurons via interfering glutamate metabolism and impairing NMDA receptor function. |
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