| Objectives In this experiment,a macrophage-fibroblast in vitro model was established,and the nano-silica-stimulated THP-1 derived macrophage supernatant was applied to MRC-5 to detect the change of MRC-5 protein expression level by proteomics method.To explore the effect of nano-silica on the expression of MRC-5 protein,and to provide a basis for exploring the biomarkers of human nano-silica exposure leading to respiratory inflammation and fibrosis.Methods THP-1 and MRC-5 cells were routinely cultured.THP-1 was induced into macrophages before the experiment.Macrophages were divided into control group and five experimental groups.The concentration of dust was(0,6.25,12.5,25,50,100)?g/ml,and the effect was 24 h.Survival rate was measured by MTT,apoptosis rate was measured by Annexin V-FITC,LDH and MDA were detected by enzyme-coupled colorimetry and TBA.Similarly for the MRC-5 group,the corresponding macrophage supernatant was added for 24 h.MTT measured the rate of increase,the sample alkaline hydrolysis method to measure the supernatant Hyp,clarified the nano-silica damage to cells,and evaluated the model construction.The liquid-mass spectrometer had the most toxic effects on MRC-5 and the qualitative and quantitative analysis of the samples in the control group,and the differential proteins were obtained.The difference was confirmed by Western blotting.In the routine experiment,the micron silica group was used as a positive control,and the differential protein detection was only for the nano group.Results Electron microscopy showed that the particle size of the nanoparticles was20-50 nm;the particle size analyzer showed that the nanoparticles in the medium were slightly agglomerated,and the average particle size was 166.8 nm.2 Under the inverted microscope,the morphological changes of the nano-group macrophages occurred.3 MTT assay for macrophage survival: Nanocell survival rate was lower than that of the control group except for dust concentration of 6.25 ?g/ml(P<0.05);nanoparticle concentration was 6.25,12.5,25 ? g/ml.Cell viability was lower than that of the micron group(P<0.05).4 Annexin V-FITC method to detect the apoptosis level of macrophages: the apoptosis rate of each group in the nano-group was higher than that in the control group(P<0.05);the apoptosis rate in the nano-group at the dust concentration of 6.25 and 25?g/ml.Higher in the micron group(P<0.05).5 Enzyme-coupled colorimetric assay for LDH secretion in cell supernatant: The LDH content in the nano-cell supernatant was higher than that in the control group except for the dust concentration of 6.25 ?g/ml(P<0.05);the nano-group had a dust concentration of 25,The LDH activity of the supernatant at 50 and 100 ?g/ml was higher than that of the micron group(P<0.05).6TBA method to detect the level of MDA secretion in cell supernatant: MDA content in the supernatant of the nano-group was increased compared with the control group except for the dust concentration of 6.25 ? g/ml(P<0.05).The MDA activity of the nano-treatment groups was not statistically different from that of the micro-group.significance.7 MTT method to detect the value-added rate of MRC-5: the nano-group had a higher cell proliferation rate than the control group except for the concentration of6.25?g/ml(P<0.05);the nano-group had a higher cell proliferation rate than the micron group at 12.5 ? g/ml.(P<0.05).8 Sample alkaline hydrolysis method to detect the secretion level of Hyp in MRC-5 supernatant: The concentration of Hyp in the nano-separation was 6.25,12.5?g/ml.The content of Hyp in the supernatant was higher than that in the control group(P<0.05);the dust concentration was 100 ? g/ml.The content of Hyp in the nano-group supernatant was higher than that in the micro-group(P<0.05).9 liquid-mass spectrometry was used to detect differential proteins.Western-blot method was used to detect differential proteins.The two groups of samples detected 3174 proteins and 234 differential proteins.It is mainly enriched in the ribosome pathway,involved in regulating apoptosis,growth and differentiation,and producing oxidative stress and other biological processes by exogenous stimulation.P4 HB and HSP90B1 proteins were selected to verify differential proteins.The results showed that the experimental group The expression of the target protein was higher than that of the control group(P<0.05),which verified the reliability of the mass spectrometry results.Conclusions 1 Nano-silica can reduce the survival rate of THP-1-derived macrophages,leading to cell damage and apoptosis,and the intensity of action at the corresponding concentration is greater than that of micron silica.2 Nano-silica induced macrophage production of cytokines can cause MRC-5 proliferation and Hyp secretion,and the intensity of action at the corresponding concentration is greater than that of micron silica.3 Nano-silica induced macrophages to secrete cytokines that cause changes in protein levels in MRC-5,regulating biological processes such as apoptosis,protein synthesis and cell growth.Among them,SerpinB2 protein may be a biomarker for human lung fibroblasts exposed to nano-silica particles to cause inflammation and fibrosis.Figure10;Table10;Reference 115... |
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