Study On The Cell Injury Mechanism Of Silica Nanoparticles On Human Neuroblastoma SH-SY5Y Cells

Using human neuroblastoma SH-SY5Y cells as target cells, the effects andmechanism of silica nanoparticles on SH-SY5Y cells were studied. Influence ofsilica nanoparticles on SH-SY5Y cells vigor was determined by MTT test; HEstaining was used to observe cell morphology change; FCM and AO/EB stainingwere used to detect cell apoptosis, LDH activity experiment was used to test theeffects of silica nanoparticles on cell membrane integrity; Flow cytometry was usedto detect intracellular reactive oxygen species, cell cycle, intracellular Ca2+content,mitochondrial membrane potential change; Western blot was used to detect theexpression change of mitochondrial pathways apoptosis related proteins Cyt C、Apaf1、Caspese-9in SH-SY5Y cells.1. Characterization of silica nanoparticlesTEM images showed that the silica nanoparticles assumes circular, particleswere evenly dispersed, equally sized. Image J software are used to calculate theaverage particle size, the result was70.28±6.23nm. Zeta potential detection showedthat silica nanoparticles in serum-free DMEM medium and pure water wereuniformly dispersed, with stable potential.2. Effects of silica nanoparticles on SH-SY5Y cells vigorMTT results showed that after treated with silica nanoparticles for24h, withthe increase of exposure dose, SH-SY5Y cell viability decreased, compared withcontrol group,6.25,12.5,25,50μg/mL SiO2group cell survival rate significantlydecreased, showing a dose related cells cytotoxic effect.3. Morphology change of SH-SY5Y cells induced by silica nanoparticles After treated with silica nanoparticles for24h, HE staining was used to observethe cell morphology change. In control group, cells form almost the same size, around nucleus in central cell, in12.5,25μg/mL SiO2group, cell number decreased,chromatin acidophilia staining enhancement, in50μg/mL SiO2group cell fell offincreased, with disordered arrangement, visible nucleus fragmentation.4. Effects of silica nanoparticles on SH-SY5Y cell membrane integrityLDH activity assay results showed that compared with control group,3.125,6.25,12.5μg/mL SiO2group showed no significant difference higher LDH activityin cell cultures, while, in25,50μg/mL SiO2group LDH activity increasedsignificantly(P<0.05), silica nanoparticles induced cell membrane damage, increasedmembrane permeability.5. Effects of silica nanoparticles on SH-SY5Y cells intracellular Ca2+FCM results showed that3.125,6.25,12.5,25μg/mL SiO2group intracellularCa2+concentration slightly higher than control group, but no significant differenceswere oberved, only50μg/mL SiO2group had a significant Ca2+concentrationincreased (P <0.05).6. Oxidative damage of SH-SY5Y induced by silica nanoparticlesAfter treated with silica nanoparticles for24h, with the increase of exposuredose, intracellular ROS level in6.25,12.5,25,50μg/mL SiO2group increasedsignificantly compared with control group (P <0.05).7. Effects of silica nanoparticles on SH-SY5Y cells mitochondrial membranepotentialUsing fluorescent probe with JC-1to detect the mitochondrial membranepotential of SH-SY5Y cells, P3door showed red fluorescence intensity, the resultsshowed that with the increase of exposure dose, SH-SY5Y cells with redfluorescence intensity increased, P3door cells gradually increased, compared withcontrol group,6.25,12.5,25,50μg/mL SiO2group cells had a significantmitochondrial membrane potential reduce (P <0.05), in25,50μg/mL SiO2group differences were significant compared with3.125μg/mL SiO2group (P <0.05),Among them,50μg/mL SiO2group had early apoptotic cells, silica nanoparticlescould decrease the mitochondrial membrane potential, induce mitochondrial damage.8. Effects of silica nanoparticles on SH-SY5Y cell cycleAfter treated with silica nanoparticles for24h, cell cycle detection found thatwith the increase of exposure dose, in25and50μg/mL SiO2group cells in G0/G1phase decreased, compared with control and3.125μg/mL SiO2group (P <0.05).cells in G2/M phase increased significantly in50μg/mL SiO2group (P <0.05).silica nanoparticles caused cell G2/M phase retardation, interfered with cellproliferation.9. Effects of silica nanoparticles on SH-SY5Y cells apoptosisFCM to detect cell apoptosis rates, the results showed that after treated withsilica nanoparticles for24h, with the increase of exposed dose, SH-SY5Y cellapoptosis rates increased, compared with control group,6.25,12.5,25,50μg/mLSiO2group cell apoptosis rates increased significantly (P <0.05). AO/EB stainingresults agreed with FCM results, with the increase of exposed dose, the number ofdyed orange apoptotic cells increased gradually.10. Effects of silica nanoparticles on mitochondrial pathways apoptosis relatedproteins expression of SH-SY5Y cellsWestern blot detected SH-SY5Y cells mitochondrial pathways apoptosis relatedproteins, the results showed that among them, the mitochondrial pathways apoptosisrelated proteins Caspase-9content increased with the increase of silica nanoparticlesexposure (P <0.05), compared with control group,12.5,25,50μg/ml SiO2grouphad significant Apaf1content increase (P <0.05),25,50μg/ml SiO2group hadsignificant Cyt C content increase compared with control group (P <0.05).Above all, silica nanoparticles had some toxic effects on SH-SY5Y cell, couldreduce cell vitality, change cell morphology and cell membrane permeability,increase intracellular ROS, cause the change of mitochondrial membrane potential,induce mitochondria damage, increase the intracellular Ca2+, cause cell G2/M phase retardation and change the content of apoptosis related proteins, interfere with cellproliferation. This study aims to provide basis for the safety application of silicananoparticles.