Sample collection
Four HIBD infants from neonatal intensive care unit (NICU) of Children’s Hospital of Nanjing Medical University were selected. Inclusion criteria included (1) term infants with acute fetal distress (prolonged resuscitation need, and/or cord pH<7.0, and/or Apgar score at 5min<5); (2) appearing neurological complication; (3) clear brain injury which diagnosed by magnetic resonance imaging (MRI) or computed tomography (CT). Exclusion criteria included serious brain injuries caused by infection, intracranial hemorrhage, genetic metabolic diseases or others. Control CSF samples were obtained from four matched infants without known neurological disease who required diagnostic lumbar punctures for routine sepsis evaluation. Table 1 shows the demographic characteristics of the neonates. All CSF samples were harvested within 24h after birth and centrifuged at 3000rpm for 10min to acquire supernatants which were then stored in liquid nitrogen with protease inhibitor cocktail (Complete mini EDTA-free, Med Chem Express, USA). This study was approved by the ethics committee of Children’s Hospital of Nanjing Medical University and achieved agreements from infants’ parents.
Sample treatment
All CSF samples were grinded under liquid nitrogen conditions, mixed with protein lysate (7M urea, 2M thiourea, 4% SDS, 40mM Tris-HCl, pH8.5), 1mM phenyl methyl sulfonyl fluoride (PMSF), 2mM ethylene diamine tetraacetic acid (EDTA) and 10mM dithiothreitol (DTT). The mixtures were ultra-sounded on ice for 10min and then centrifuged at 12000g, 4°C for 30min to collect liquid supernatant. Equal amounts of proteins were treated by reductive alkylation. Then, equal amounts of proteins were ultrafiltrated using molecular weight cut-off (MWCO) filters (Millipore, USA) of 10kDa according to the manufacturer's recommendation. The flow-through from the filters containing peptide fractions was recycled, desalted, concentrated using C18 solid phase extraction (SPE) (Strata™-X, 33μm, 2g/20mL, Phenomenex), and finally lyophilized.
Labelling
Peptide samples were dissolved in 0.5M Triethylammonium bicarbonate (TEAB) for peptide labeling after desalting. All peptides from samples were labeled with iTRAQ-8 standard kit (AB SCIEX Inc., Framingham, MA, USA). Then, the labeled samples were fractionated using a high-performance liquid chromatography (HPLC) system (Thermo DINOEX Ultimate 3000, USA) with a Durashell C18 (5μm, 100A, 4.6×250mm), and 12 fractions were collected.
SDS-PAGE
The protein samples and protein marker (Fermentas, St. Leon-Rot, Germany) were subjected to 12% sodium docecyl sulfate-polyacrylamide gel (SDS-PAGE), and protein bands were visualized by Coomassie brilliant blue staining.
Liquid chromatography/Mass spectrometry (LC-MS/MS)
Reverse-phase HPLC-ESI-MS/MS was used to analyze the fractionated samples. The data were collected and indentified using the TripleTOF 5600 plus mass spectrometer with the Eksigent Ultra Plus nano-LC 2D HPLC system system (AB SCIEX, USA). The freeze-dried peptide samples dissolving in 2% acetonitrile (ACN)/0.1% formic acid (FA) were loaded to a C18 trap column (5µm, 100μm×20mm, LC packings) and separated using the C18 analytical column (3µm, 75µm×150mm, LC packings) over a 90min gradient at 300nL/min. The two mobile phases of contained A, 2% ACN/98% of 0.1% FA (v/v) in water and B, 98% ACN/2% of 0.1% FA (v/v) in water.
For information-dependent acquisition (IDA), MS1 were scanned in 250ms, and MS/MS of 30 precursor ions were scanned in 50ms. MS1 spectra were collected in the range of 350-1500m/z, and MS/MS spectra were collected in the range of 100-1500m/z. Precursor ions were excluded from reselection for 15s. For MS analysis, ProteinpilotTM database search engine (V4.5, AB SCIEX, USA) was used to protein identification based on the MS/MS spectra data which provides an automatic mass recalibration of the data and obtains more data.
Bioinformatics analysis
The LC-MS/MS data were searched using the Mascot search engine (Matrix science) (http://www.matrixscience.com) against the SwissProt sequence database with the Homo sapiens subset including the following variable modifications: phosphorylation, amidation, deamidation, pyroglutamic acid, oxidation, acetylation, sulfation, oxidized and reduced cysteines. Gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) analysis were used to study potential functions of differentially expressed peptides and their precursor proteins. The protein-protein interaction networks were mapped using STRING database (https://string-db.org/) and UniProt database (http://www.uniprot.org/). The properties of peptides were checked through the website http://www.expasy.org and http://smart.embl-heidelberg.de/.
Cell culture and treatment
PC12 rat pheochromocytoma cells were obtained from American Type Culture Collection (Rockville, MD, USA) and cultured in RPMI 1640 culture medium supplemented with 10% v/v horse serum (HS), 5% v/v fetal bovine serum (FBS) and appropriate antibiotics in a humidified chamber (5% CO2 and 37°C), all of which were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA).
For the induction of oxygen and glucose deprivation (OGD), cells were switched to RPMI 1640 without glucose after washing twice with glucose-free RPMI 1640. TAT-HIBDAP (YGRKKRRQRRR-HSQFIGYPITLFVEKER) and the FITC tagged TAT-HIBDAP (Shanghai Science Peptide Biological Technology Co., Ltd., China) were dissolved in sterile water and added to the glucose-free culture medium. After 1h, cells were placed into an atmosphere of 2% O2, 5% CO2 and 93% N2 at 37°C for 6h. Control cells were maintained in glucose-containing RPMI 1640 and incubated in a normoxic incubator for the same time.
Cell viability assay
Cell viability was measured using cell counting kit-8 (CCK-8) assay (Dojindo Molecular Technologies, Tokyo, Japan) according to the manufacturer’s protocol. Cells were inoculated into a 96-well plate (1×104 cells/hole). Cells in 90μL glucose-free culture medium of each well were added with 10μL CCK-8 solution and incubated for another 1h in hypoxic environment. The absorbance was measured at 450nm using a Microplate Reader (Themo scientific, Vantaa, Finland). All experiments were independently repeated three times.
Annexin V-fluorescein isothiocyanate (FITC) Assay
Cells were labeled by FITC-coupled Annexin V (Annexin V-FITC) (BD Biosciences, NJ, USA) for detection of phosphatidylserine exposure and by propidium iodide (PI) for observation of the loss of membrane integrity. Cells were harvested by trypsinization and washed twice with ice-cold phosphate buffer saline (PBS). Then, cells were resuspended in 1×binding buffer at a concentration of 1×106 cells/ml. Transferred 100μl of the solution (1×105 cells) to a 5ml culture tube and stained with 5μl Annexin V-FITC and 5μl propidium iodide (PI). The suspension was incubated at room temperature in the dark for 15min. Added 400μl of 1×binding buffer to each tube. FACS Calibur Flow Cytometer (BD Biosciences, NJ, USA) was used to distinguish cells and the results were analyzed by FlowJo software (Tree Star Corp, Ashland, OR). All experiments were independently repeated three times.
Transmission electron microscopic examination
PC12 cells were fixed in 2.5% glutaraldehyde and rinsed with 0.1mol/L phosphate-buffered saline (PBS). Samples were placed in 1% osmium acid at 4°C for 4h. After that, they were dehydrated in ethanol, embedded in the embedding agent, and stained with uranyl acetate and lead citrate. Finally, samples were examined under a transmission electron microscope (JEM-1400, JEOL, Japan).
Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA of cells was extracted using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instruction. HiScript® II Q RT SuperMix for qPCR (Vazyme Biotech, Nanjing, China) was used for reverse transcription of mRNA following the manufacturer’s instruction. The RT thermal cycle program was as follows: 50˚C for 15min and 85˚C for 5sec. The qPCR step was performed using a 7900HT Fast Real‑Time PCR system with a TaqMan® MicroRNA Assay kit (Applied Biosystems, CA, USA) as the following conditions: 95°C for 5min, followed by 40 cycles (95°C for 10sec and 60°C for 30sec). The sequences of primers were: NLRP3 F: 5’-TGA AGA GTG TGA TCT GCG GAA AC-3’; R: 5’-GAA AGT CAT GTG GCT GAA GCT GT-3’; NLRP1 F: 5’-GCC CTG GAG ACA AAG AAT CC-3’; R: 5’-AGT GGG CAT CGT CAT GTG T-3’; ASC F: 5’-AGT TGA TGG TTT GCT GGA TGC T-3’ ; R: 5’-GGT CTG TCA CCA AGT AGG GCT G-3’; Caspase-1 F: 5’-AAC CTT GGG CTT GTC TTT-3’; R: 5’-CAG GAG GGA ATA TGT GGG-3’; GAPDH F: 5’-AGA AGG CTG GGG CTC ATT TG-3’; R: 5’-AGG GGC CAT CCA CAG TCT TC-3’. The mRNA levels were calculated using the 2-△△CT method. All experiments were performed in triplicate.
Western blotting
After washed with ice-cold PBS, the cells were ultrasonically homogenized in a RIPA buffer and protease inhibitor cocktail, and then the homogenates were centrifuged at 12000×g for 15min at 4°C. The protein concentration was quantified using a BCA protein assay kit (Pierce, Rockford, IL, USA) and the supernatants of homogenates were boiled at 100°C in a laemmli sample buffer (Abcam, Cambridge, MA, UK) for 5min. Samples were separated on a 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore, MA, USA). Membranes were blocked with 5% (m/v) nonfat dry milk in 0.1% Tween 20 (TBS-T; 2mmol/L Tris-HCl, 50mmol/L NaCl, pH 7.4) for 2h at room temperature and subsequently incubated overnight at 4°C in the blocked buffer with NLRP3 antibody (Cat: 19771-1-AP; Proteintech, Chicago, USA), NLRP1 antibody (Cat: ab3683; Abcam, Cambridge, UK), ACS antibody (Cat: sc-514414; Santa Cruz, CA, USA), Caspase-1 p10 antibody (Cat: sc-514; Santa Cruz, CA, USA), and β-actin antibody (Cat: 8457S; Cell Signaling Technology, MA, USA), respectively. The membranes were washed with 0.1% Tween 20, and then treated with horseradish peroxidase-conjugated anti-mouse IgG (Abcam, Cambridge, UK) or goat anti-rabbit IgG H&L (HRP) (Abcam, Cambridge, UK) for 1h at room temperature. After washing thrice with TBST, proteins were visualized with an electrochemiluminescence detection system and quantified by an image analysis system (Image J, MD, USA).
Statistical analysis
All values are expressed as mean±SEM. The statistical difference was analyzed using the SPSS (version 22.0). GO and pathway analysis were considered to be significantly enriched when Hypergeometric P-value<0.05. Quantitative analysis of our experiments was performed by Student's t-tests or one-way ANOVA. P-value<0.05 was considered statistically significant.