Human brain tissues, including three TBI tissues and three non-contusive tissues (control), were obtained from the Department of Neurosurgery at the First Affiliated Hospital of Nanjing Medical University, which was approved by the Institutional Review Board. Details information of the brain tissues was displayed in Table 1. Brain tissues resected from patients were snap-frozen and stored in liquid nitrogen until assay. The Ethics Committee of Nanjing Medical University approved the use of human brain tissue, and all procedures were conducted in accordance with approved guidelines. The participant's explicit permission was obtained, and the patient provided informed consent.
2. Animals and experimental TBI model
The Laboratory Animal Center of Nanjing Medical University provides adult male C57BL/6J mice (25 ± 2 g). All animals were kept in an SPF condition with regulated temperature (22 ± 2°C), a light and dark cycle of 12:12 hours and received standard laboratory animal food and water. The Institutional Animal Care and Use Committee of Nanjing Medical University approved all research protocols and animal experiments in accordance with the guidelines of the Animal Care and Use Committee (National Institutes of Health Publication No. 85-23, revised 1996). As described previously, eight-week-old mice were subjected surgery to produce a controlled cortical impact (CCI) model. Mice were anesthetized with 4% isoflurane in 70% nitrous oxide and 30% oxygen. and maintained with 1.5% isoflurane. The body temperature was maintained at 37 ± 0.5 °C by a heating blanket. Then, we performed a 4-mm-diameter craniotomy in the left parietal bone (the relative coordinates centre of craniotomy to bregma: 1.5 mm posterior and 2.5 mm lateral). For the sham groups, only the dura mater was exposed. In the TBI groups, the exposed dura mater was struck by impactor at 6.0 ± 0.2 m/s velocity with 1.4 mm depth and 50-ms dwell time. After the injury, we closed the skin incision, and then mice were caged. The mice were then decapitated at various times, and only the cerebral cortex around the lesion was collected and stored in 4% paraformaldehyde or at −80°C for the following study.
3. LC-MS analysis and Proteomic data processing
All the 16 pairs of experimental samples were performed using a QExactive Plus Orbitrap™ mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a nano-electrospray ion source as described previously. Samples were dissolved in water/ formic acid (0.1%, v/v), and peptides were separated by reversed phase liquid chromatography using an EASY-nLC™ 1000 system (Thermo Fisher Scientific). A set-up of pre-column and analytical column was used. The pre-column was a 2 cm EASY-column (1D 100 μm, 5 μm C18) (Thermo Fisher Scientific) while the analytical column was a 10 cm EASY-column (ID 75 μm, 3 μm, C18) (Thermo Fisher Scientific). Peptides were washed with a 90 min linear gradient from 4% to 100% acetonitrile at 250 nL/min. The mass spectrometer was operated in positive ion mode, acquiring a survey mass spectrum with resolving power 70 000 and consecutive high collision dissociation fragmentation spectra of the 10 most abundant ions. The acquired data (.RAW-files) were processed by Maxquant (Version 188.8.131.52) against the Uniprot-Swissprot database using an extracted FASTA file specified for “mouse” taxonomy. The search parameters included: maximum 10 ppm and 0.02Da error tolerance for the survey scan and MS/MS analysis; enzyme specificity was trypsin; maximum 2 missed cleavage sites allowed; cysteine carbamidomethylation was set as static modification; oxidation (M) was set as variable modifications. The protein identification was based on 95% confidence per protein. The acquired data were performed Gene ontology and protein class analysis via the PANTHER (http://pantherdb.org/).
4. BV2 cell culture, transfection and in vitro injury model
The BV2 cell line was purchased from the Chinese Academy of Sciences Cell Bank and was cultured in Dulbecco's modified Eagle's medium (DMEM) with penicillin, treptomycin and 10% foetal bovine serum (FBS) (Gibco). The BV2 cells were cultivated at 37°C with 5% CO2. Generally, the BV2 cells were used for subsequent experiments when the growth density reaches 80-90%.
The siRNA targeting HO-1 was purchased from GenePharma (Shanghai, China), and the sequences of si-RNAs were as follows: sense: 5′-CCAAGUUCAAACAGCUCUAUC-3′, antisense: 5′-UAGAGCUGUUUGAACUUGGUG-3’. At 6 hours after transfection, the medium was changed to DMEM with 10% FBS. Then, 24h later, the BV2 cells were treated with 100 μm glutamate to induce cellular injury for 24 hours according to the study protocol.
5. Primary cortical neuronal culture
Cultivation of primary cortical neurons were obtained from the cerebral cortices of one-day-old C57BL/6J mice. The mice were decollated, and the intact brain was immersed in pre-cooled DMEM/F12 medium immediately. Then the cerebral cortex was dissected and digested with 0.25% trypsin and DNase at 37°C for 20 minutes (with shaking every 5 minutes). The digestion was terminated by the addition of horse serum and filtered through the cell strainer. Then, the filtrate was centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. We resuspended the cell pellet in medium DMEM/F12 medium containing 10% horse serum and 2% penicillin and streptomycin (Gibco). The 6- or 12-well culture dishes contained 6-7×100,000 cells per well. After about 4 hours, the medium was replaced with neurobasal medium containing 2% B27 and 0.5 mM glutamate, and placed in a 37°C, 5% CO2 cell incubator.
6. FITC-Labeled Fetuin-A
Fetuin-A was labeled with fluorescein isothiocyanate (FITC) by using the EZ-Label FITC Protein Labeling kit (Pierce Biotechnology, Rockford, IL, USA) according to the manufacturer’s instructions. At 15 mins following TBI, the FITC-labeled fetuin-A (50 mg/kg) was administered intravenously. The presence of FITC-labeled fetuin-A was examined by a fluorescence microscope.
7. Western blotting
Proteins were extracted from tissues or cells following the previous description. The proteins were separated by 10% or 12% SDS-PAGE gel and then placed to polyvinylidene fluoride (PVDF) membranes (Merck Millipore). Membranes were blocked in 5% non-fat dried milk for 2 hours at ambient temperatures and then incubated overnight at 4°C with antibodies against GAPDH (1:2000, #5174; Cell Signaling Technology), Fetuin-A (1 µg/ml, ab112528; Abcam), Fetuin-A (1:2000, ab187051; Abcam), Iba-1 (1:1000, ab178846; Abcam), CD16 (1:1000, ab223200; Abcam), CD206 (1µg/ml, ab64693; Abcam), Cleaved Caspase-3 (1:1000, #9661; Cell Signaling Technology), Bax (1:1000, ab32503; Abcam), Bcl-2 (1:2000, ab182858; Abcam), p-RIP3 (1:1000, #91702; Cell Signaling Technology), p-MLKL (1:1000, #37333, Cell Signaling Technology), Nrf-2 (1:1000, #12721, Cell Signaling Technology), Histone H3 (1:2000, ab1791; Abcam) and HO-1 (1:1000, #43966; Cell Signaling Technology) followed by incubation with horseradish peroxidase-conjugated secondary antibody (Beyotime, China, A0208, A0216, 1:5000) for 2 h. After washing with PBST, we ascertained the protein bands by using SuperSignal® Maximum Sensitivity Substrate (Thermo Fisher Scientific). And we used ImageJ software (National Institutes of Health) to calculate the achieved bands’ optical density. Samples derive from the same experiment and blots are processed in parallel. The source data file contains uncropped and unprocessed scans of blots.
8. Immunostaining assay
For immunofluorescence assays, the frozen brain sections were permeabilized with 0.1%Triton X-100 (Sigma-Aldrich, St Louis, MO; USA, X100) for 15 min, and blocked with 5% normal goat serum (Millipore; S26-LITER) at 37°C for 1 hours. Then, the sections were incubated with primary antibodies at 4°C throughout the night, washed three times with PBS, and incubated with Alexa Fluor 488- or CyTM3-conjugated secondary antibodies (Jackson, USA, 1:500) for 2 hours at ambient temperature. After additional washed three times with PBS, Nuclei underwent staining processed using Hoechst (C1018, Beyotime, China) at ambient temperature for 10 min. For immunofluorescence staining of cells, different methods were used. The BV2 cells and primary neuronal cells were plated on glass slides which precoated with poly-lysine (PLL). Then, the cells were fixed by 4% paraformaldehyde (PFA) for 1 hours. The rest of the steps were the same as the immunofluorescence assay of brain sections. Finally, A laser scanning confocal microscope (TCS SP5II, Leica, Wetzlar, Germany) was used to observe immunoreactivity, and signal intensities were quantified by ImageJ.
For immunohistochemistry assays, brain sections were incubated overnight with the primary antibodies at 4°C. And the sections underwent incubation with the secondary antibody for 30 min at ambient temperature, and washed with PBS, and incubated with DAB for 15 min at 37°C. The sections were imaged by a light microscope (Leica).
The following primary antibodies were used to perform immunostaining: Fetuin-A (1µg/mL, ab47979; Abcam), Fetuin-A (1:2000, ab187051; Abcam), Iba-1 (1:500, ab178846; Abcam), CD16 (1:500, ab223200; Abcam), CD206 (1µg/ml, ab 64693; Abcam), Ly6g (1:400, #88876; Cell Signaling Technology), p-RIP3 (1:400, #91702; Cell Signaling Technology), p-MLKL (1:1600, #37333, Cell Signaling Technology), Nrf-2 (1:400, #12721, Cell Signaling Technology), TNF-α(1:2000, ab183218; Abcam) and Map2 (1µg/ml, ab183218; Abcam).
9. TUNEL assay
According to the manufacturer's instructions, a TUNEL assay (C1089, Beyotime, China) was used to detect cell death. Briefly, 12-μm brain sections or cells were fixed in 4% PFA. And then they were incubated with 50 μL TUNEL reaction mixture and 0.3% Triton X-100 in the dark (37 °C) for 1 hour. After rinsed three times with PBS, DAPI was used to visualize cell nuclei. Images were obtained using a Nikon Eclipse E600 microscope (Nikon, Melville, NY).
10. Brain water content and cortical lesion volume
The mice brain was removed immediately after decapitation and weighed. Subsequently, the brain was dried at 70°C for 72 hours, and achieve the dry weight. The brain water content was obtained based on water content (%) = [(wet weight − dry weight)/wet weight] × 100%.
All slice thickness was obtained 0.5mm for lesion volume measurement. Lesion volume was assessed from the summation of areas of defect on each slice and multiplied by slice thickness. ImageJ was used to quantitatively analyze the data.
11. ELLSA assay
TNF-α, IL-6, IL-1β, and IL-10 expressions in culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA; R&D Systems Inc) according to the manufacturer's instruction.
12. Cell viability and LDH assay
We ascertained cell viability by Cell Counting Kit-8 (CCK-8, CK04, Dojindo, Tokyo, Japan) assay. In brief, cells was cultured in a 96-well plate, and incubated with the reagent at 37 °C for 2 h. Then, optical density (OD) values were measured at 450 nm by a Thermo Multiskan FC microplate photometer.
Cellular injury-induced cytotoxicity was measured by Cytotoxicity Detection Kit (C0017, Beyotime Biotech, China) in line with the directions of the manufacturer.
13. Transmission Electron Microscopy
The BV2 cells were fixed in PBS (pH 7.4) containing 2.5% glutaraldehyde for at least 1h at room temperature. After this step, cells were post-fixed with 1.5% osmium tetroxide for 2h at 4°C and dehydrated with an ethanol, followed by embedding in epoxy resin. The ultrastructure of the BV2 cells (70 nm ultrathin sections) were observed by transmission electron microscope (Quanta 10, FEI Co.)
14. JC-1 fluorescence assay
The mitochondrial membrane potential was measured using JC-1 (C2003S, Beyotime, China) fluorescence mitochondrial imaging. The BV2 cells were incubated with JC-1 solution for 20 minutes at 37°C. And then, the cells were rinsed twice using JC-1 buffer. Images were obtained using a Nikon Eclipse E600 microscope (Nikon, Melville, NY). The ratio of red to green fluorescence represented the mitochondrial membrane potential.
15. Extraction of cytoplasmic and nuclear protein
After the different treatment described above, Nuclear and Cytoplasmic Protein Extraction Kit (P0027, Beyotime Biotech, China) was used to separate cytoplasmic protein and nuclear protein, and the variation of Nrf-2 expression was detected by Western blot assessment.
16. Measurement of mitoROS levels
After culture of BV2 cells in 6-well dishes with 6×10４cells/well, the mitoROS was examined by MitoSOX molecular probes (Invitrogen, CA) according to the manufacturer's instruction. At the end of treatment, Nikon Eclipse E600 microscope (Nikon, Melville, NY) was used to obtain the image of MitoROS at λ579 nm.
17. Determination of malondialdehyde (MDA), GSH and GSSH level
The MDA, GSH and GSSH level was measured with the Lipid Peroxidation MDA Assay Kit (S0131S, Beyotime Biotech, China) and GSH and GSSG Assay Kit (S0053, Beyotime Biotech, China). After the different treatment described above, the cells were washed with PBS (pH 7.4) and were lysed subsequently. The lysates were centrifugated at 12000 rpm for 10 min at 4 ℃. Then, the supernatant was collected and the absorbance at 532 nm was measured by a microplate reader (Biotech, Winooski, VT, USA). Finally, the datum was normalized by the protein concentration in each sample.
18. Co-immunoprecipitation (Co-IP) assay
Co-IP was performed following the previous description. In brief, the BV2 cells were lysed and total lysates were harvested by weak RIPA lysis buffer (Cell Signaling Technology, Danvers, MA, USA). After cleared with 50% protein A/G agarose for 1 hour, the 500ml of extracted proteins were incubated with primary antibodies of corresponding dilution overnight at 4°C. Then, The immune complexes were pulled down with protein A/G agarose in a shaker at 4°C for 4 h. Microbeads were collected and washed, and then proteins were eluted through boiling in 1 × loading buffer followed by immunoblotting analysis.
19. Statistical analysis
All data were analyzed with GraphPad 8.0 Software and expressed as the means ± standard deviation (SD) at least three independent experiments. Gray levels were detected with ImageJ. A two-tailed unpaired Student’s t test was used to compare the data from two different groups. P < 0.05 was considered as significant difference.