Fetuin-A alleviates neuroinflammation against traumatic brain injury-induced microglial necroptosis by regulating Nrf-2/HO-1 pathway

Patients

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. Detailed information of the brain tissues is 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.

Table 1 Clinical information of human brain specimensAnimals and experimental design

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 h 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).

Six separated experiments were performed as follows.

Experiment 1

To understand the changes of proteins content in brain after CCI. 32 C57BL/6J mice were randomly divided into three groups: sham (n = 16), 6 h (n = 8), 24 h (n = 8) after CCI induction. We collected the cerebral cortex around the lesion at 6 h or 24 h after CCI and then we identify global differences in protein expression between CCI and sham mouse groups by proteomic analysis (Additional file 1: Fig. S1A).

Experiment 2

To determine the endogenous Fetuin-A at each time point after CCI, 30 C57BL/6J mice were randomly divided into five groups: 0 (sham), 6 h, 12 h, 24 h, 72 h (n = 6/group) after CCI induction. The mice were all killed at the scheduled time point and the brain tissue was collected for western blot analysis and immunofluorescence (IF) assays to measure Fetuin-A expression (Additional file 1: Fig. S1B).

Experiment 3

To investigate the intravenous administration of Fetuin-A in the post-CCI brain injury. 48 C57BL/6J mice were randomly assigned into five groups: sham, sham + FITC-Labeled Fetuin-A (25, 50, 75 mg/kg), CCI, CCI + FITC-Labeled Fetuin-A (25, 50, 75 mg/kg) (n = 6/group). The FITC-labeled Fetuin-A was administered by tail vein injection 15 min following CCI. Tissue samples were collected at 6 h or 3 w after CCI, and then we detect the content of Fetuin-A by western blot analysis, immunofluorescence assays and immunohistochemistry (ICH) assays (Additional file 1: Fig. S1C, D). Besides, we test whether intravenous administration of Fetuin-A has biological toxicity via H&E and TUNEL staining (Additional file 1: Fig. S1E).

Experiment 4

To explore the damage degree and repair of blood brain barrier (BBB) after CCI, 30 C57BL/6J mice were randomly assigned into five groups: 0 (sham), 1 d, 3 h, 7 d, 3 w (n = 6/group) after CCI induction. The mice were all killed at the scheduled time point and the brain tissue was collected for western blot analysis and immunofluorescence assays to measure cortex BBB dysfunction (Additional file 1: Fig. S1F).

Experiment 5

To detect the role of Fetuin-A in CCI model and explore the underlying mechanism of Fetuin-A, 70 C57BL/6J mice were randomly assigned into four groups: sham, CCI, CCI + Fetuin-A (50 mg/kg), CCI + sh-Ctrl, CCI + sh-Fetuin-A-1, CCI + sh-Fetuin-A-2, CCI + sh-Fetuin-A-3 (n = 10/group). Tissue samples were collected at 6 h or 3 w after CCI, and then we detect the changes of tissue lesion, cell death, microglia activation and neutrophil infiltration via H&E, TUNEL staining, Brain water content, lesion volume analysis, western blot analysis, immunofluorescence assays and immunohistochemistry assays (Additional file 1: Fig. S1G, H).

Experiment 6

To investigate whether the therapeutic effect of Fetuin-A was dependent on microglia following CCI, 36 C57BL/6J mice were randomly assigned into six groups: sham, sham + vehicle (Veh), sham + PLX5622 (PLX), CCI + Veh, CCI + PLX, CCI + PLX + Fetuin-A (n = 6/group). Tissue samples were collected at 6 h or 3 w after CCI, and we detect the depletion efficiency of microglia via immunohistochemistry assays. Besides, we explored the tissue lesion, neuron death via H&E, TUNEL staining, Brain water content, lesion volume analysis (Additional file 1: Fig. S1I).

CCI model

As described previously, 8-week-old mice were subjected surgery to produce a controlled cortical impact (CCI) model [32]. 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.

Recombinant adenovirus administration

The recombinant adenoviruses sh-Fetuin-A (AD-shFetuin-A) and control (AD-shcontrol) were constructed by GenePharma (Shanghai, China). The three shRNA targeting sequences were as follows: (1) sense: 5′-CCGUGGACUACCUCAAUAATT-3′, antisense: 5′-UUAUUGAGGUAGUCCACGGTT-3’; (2) sense: 5′-GGGAGAAACUCUUCAUUCUTT-3′, antisense: 5′-AGAAUGAAGAGUUUCUCCCTT-3′; (3) sense: 5′-GCCUUCAACACACAGAAUATT-3′, antisense: 5′-UAUUCUGUGUGUUGAAGGCTT-3′. The recombinant adenoviruses were produced and purified according to the manufacturer's instructions. The adenoviruses were subjected to large-scale amplification in AD293A cells and were subsequently collected from supernatant, condensed, and purified. The titers of adenoviruses were determined and calibrated in 293T cells. For tail intravenous injection, C57BL/6J mice were injected with 1 × 1010 viral particles in a total volume 200 μL. Seven days were needed for successful transfection before CCI treatment.

Brain tissue preparation

To prepare brain tissue, mice from each group were randomly selected after experimental TBI. The mice were anesthetized with 10% chloral hydrate (0.4 mL/100 g) injected intraperitoneally. After successful anesthesia, the mice were placed on a wooden board in the supine position. The sternum was cut, the xiphoid was lifted and the chest cavity was cut. Then, the right atrial appendage was cut, and the perfusion needle was inserted from the apex of the left ventricle. A pre-cooled phosphate-buffered saline (PBS) solution was perfused into the lungs and liver, the color of which became grayish-white. Some animals were then reperfused with 4% paraformaldehyde according to the type of experiment. Finally, the mouse was decapitated, the skull was exposed and separated, and the brain was gently removed. Cerebral cortex tissue around the lesion (Fig. 1A) was collected and snap-frozen on dry ice, then stored at − 80 °C for liquid chromatography–mass spectrometry (LC–MS) analysis and western blot assays. For immunostaining assays and hematoxylin and eosin (H&E) staining, the brain tissue was fixed in neutral formalin solution for 24 h, then dehydrated by alcohol gradients, and finally embedded in paraffin.

Fig. 1figure 1

Protein identification and quantification by label-free LC–MS/MS. Quantitative proteomics analysis of the sham mice and TBI mice, and validation of differentially expressed Fetuin-A in different groups. And the animals in the sham group underwent all surgical procedures for TBI induction except the traumatic step. A Location of collected tissues was labeled. Collected cortical tissues were marked by gray frame. B Experimental design for proteomic analysis in the mice brain tissues by label-free LC–MS. C Volcano plot graph of 2499 nonredundant proteins. The − log10 (P-value) was plotted against the log2 (ratio TBI/Sham). The upregulated proteins in TBI tissues were marked with red dots (black arrow: Fetuin-A), and the downregulated proteins in TBI tissues with green dots. Blue plots represented the rest of genes with no significant expression change. D The relative protein level of Fetuin-A in CCI (n = 8 samples) vs. sham (n = 8 samples). E, F Classification of proteins identified through proteomics into their molecular biological processes (BP), cellular components (CC), and molecular functions (MF). The top 10 Gene Ontology (GO) enrichment analysis were listed. Data are presented as the means ± SD; **P < 0.01 vs. sham group

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 [23]. 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 1.5.0.1) against the Uniprot-Swissprot database using an extracted FASTA file specified for “mouse” taxonomy. The search parameters included: maximum 10 ppm and 0.02 Da 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 subject to Gene Ontology and protein class analysis via the PANTHER (http://pantherdb.org/).

Drug administration

Recombinant Mouse Fetuin-A was provided by R&D Systems Inc (USA) and 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 min following CCI, the FITC-labeled Fetuin-A (25, 50, 75 mg/kg) was administered through the tail vein. The presence of FITC-labeled Fetuin-A was examined by a fluorescence microscope.

Microglial cells were treated with Recombinant Mouse Fetuin-A (R&D Systems, USA) at different physiologically relevant concentrations that ranged from 150–900 μg/mL [33].

PLX5622, which is a colony-stimulating factor 1 receptor (CSF1R) inhibitor, was provided by Plexxikon Inc. (Berkeley, CA) and formulated in AIN-76A standard rodent chow by Research Diets Inc. (New Brunswick, NJ) at a concentration of 1200 parts per million. The mice were fed the PLX5622 diet to deplete microglia or AIN-76A chow as vehicle control. After being raised for 2 weeks, mice were subjected to CCI injury for further experiments.

N-Acetyl-l-cysteine (NAC) was purchased from Sigma Chemicals (USA). Microglial cells were treated with NAC (10 mM) 1 h before stimulation with glutamate for 24 h.

ML385, which is Nrf-2 inhibitor, was purchased from Selleck Chem. Microglial cells were treated with different dilution concentrations (0, 2, 4, 6, 8, 10 μmol/L) of ML385 for 72 h.

Extraction of primary neuron and microglia and cell culture

Primary cortical neurons were obtained from the cerebral cortices of 1-day-old C57BL/6J mice. The mice were decollated, and the intact brain was immediately immersed in pre-cooled Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 (DMEM/F12) medium. Then the cerebral cortex was dissected and digested with 0.25% trypsin and DNase at 37 °C for 20 min (with shaking every 5 min). The digestion was terminated by the addition of horse serum and was then 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 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 h, the medium was replaced with neurobasal medium containing 2% B27 and 0.5 mM glutamate, and the dishes were placed in a cell incubator at 37 °C, with 5% CO2.

The preparation of primary microglia was similar to that of neurons. The difference was that DMEM/F12 containing 10% fetal bovine serum, 1 mM sodium pyruvate, 2 mM l-glutamine, 100 mM nonessential amino acids, 50 U/mL penicillin, and 50 mg/mL streptomycin (all from Gibco) was the medium used for the cells. After plating, cells were cultured for 2 days in a 150-cm2 culture flask pretreated with poly-d-lysine (Sigma, USA). Within 2 weeks of seeding, glial cells formed a confluent monolayer. Microglia were separated from astrocytes by shaking the flask and were collected by centrifuging.

Cell transfection and in vitro injury model

The siRNA targeting HO-1 was purchased from GenePharma (Shanghai, China), and the sequences of siRNAs were as follows: sense: (1) sense: 5′-CCACACAGCACUAUGUAAATT-3′, antisense: 5′-UUUACAUAGUGCUGUGUGGTT-3’; (2) sense: 5′-CUCGAAUGAACACUCUGGATT-3′, antisense: 5′-UCCAGAGUGUUCAUUCGAGTT-3′; (3) sense: 5′-CUGCUCAACAUUGAGCUGUTT-3′, antisense: 5′-ACAGCUCAAUGUUGAGCAGTT-3′. At 6 h after transfection, the medium was changed to DMEM with 10% fetal bovine serum. Then, 24 h later, the primary microglia were treated with 100 μm glutamate (Glu) to induce cellular injury for 24 h according to the study protocol.

Western blotting

Proteins were extracted from tissues or cells following the previous description [34]. 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 h 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), Cleaved Caspase-3 (1:1000, #9661; Cell Signaling Technology), Bax (1:1000, ab32503; Abcam), Bcl-2 (1:2000, ab182858; Abcam), RIPK3 (1:1000, #DF10141; Affinity), MLKL (1:1000, DF7412; Affinity), 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 derived from the same experiment and blots are processed in parallel. The source data file contains uncropped and unprocessed scans of blots.

Immunostaining and hematoxylin and eosin (H&E) assay

For immunofluorescence assays, the 8-µm-thick 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 h. 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 h at ambient temperature. After additional washing three times with PBS, nuclei underwent staining process using Hoechst (C1018, Beyotime, China) at ambient temperature for 10 min. For immunofluorescence staining of cells, different methods were used. The primary microglial and neuronal cells were plated on glass slides which precoated with poly-lysine (PLL). Then, the cells were fixed by 4% paraformaldehyde (PFA) for 1 h. 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 each section was imaged randomly by scanning 3–6 fields in each quadrant. Quantitative analysis of signal intensities was performed manually by a blinded investigator using ImageJ. For the count of positive cells, the blinded investigator selected relevant areas and counted them manually. And the number of positive staining cells with a signal-to-noise ratio (S/N) ≥ 10.0 was quantified to distinguish positive fluorescence intensity (The Signal) from spontaneous fluorescence (The Noise) [35].

For immunohistochemistry assays, 8-µm-thick 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), and each section was imaged randomly by scanning 3–6 fields in each quadrant. The average absorbance was measured by ImageJ, and the proportion of positive cells was counted manually.

Hematoxylin and eosin (H&E) staining was performed on prepared 8-mm-thick sections. Prepared 8-mm-thick sections. Slides were imaged under a light microscope (Leica).

The following primary antibodies were used to perform immunostaining: Fetuin-A (1 µg/mL, ab47979; Abcam), Fetuin-A (1:2000, ab187051; Abcam), Iba-1 (1:500, ab178846; Abcam), CD16/32 (1:500, ab223200; Abcam), MPO (1:1000, ab208670; Abcam), p-RIP3 (1:400, #91702; Cell Signaling Technology), p-MLKL (1:1600, #37333, Cell Signaling Technology), Nrf-2 (1:400, #12721, Cell Signaling Technology).

Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (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 h. After rinsing three times with PBS, DAPI was used to visualize cell nuclei. Images were obtained using a Nikon Eclipse E600 microscope (Nikon, Melville, NY).

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 h, and the dry weight determined. The brain water content was obtained based on water content (%) = [(wet weight − dry weight)/wet weight] × 100% [36].

A slice thickness of 0.5 mm was used 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.

Enzyme-linked immunosorbent assay (ELISA) assay

The brain tissues were collected from the mice after CCI 6 h. Briefly, added 5 mg of brain tissue to a tube along with 300 µL of extraction buffer and mix for 2 h at 4 °C with an electric homogenizer. Then, the brain tissues were centrifuged at 13,000 rpm for 20 min at 4 °C. The supernatant was extracted and diluted with sample buffer at a ratio of 1:1 and loaded onto the wells in duplicates. TNF-α, IL-6, IL-1β, and IL-10 expressions in the brain tissues were measured using a specific enzyme-linked immunosorbent assay (ELISA; R&D Systems Inc) according to the manufacturer’s instruction.

The conditioned medium was moved to a centrifuge tube and centrifuged at 1500 rpm for 10 min at 4 °C and samples were loaded onto the wells in duplicates. TNF-α, IL-6, IL-1β, and IL-10 expressions in culture supernatants were measured by a specific ELISA according to the manufacturer’s instruction.

Cell viability and lactate dehydrogenase (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.

Transmission electron microscopy

The microglial cells were fixed in PBS (pH 7.4) containing 2.5% glutaraldehyde for at least 1 h at room temperature. After this step, cells were post-fixed with 1.5% osmium tetroxide for 2 h at 4 °C and dehydrated with ethanol, followed by embedding in epoxy resin. The ultrastructure of the microglial cells (70 nm ultrathin sections) was observed by transmission electron microscope (Quanta 10, FEI Co.)

JC-1 fluorescence assay

The mitochondrial membrane potential was measured using JC-1 (C2003S, Beyotime, China) fluorescence mitochondrial imaging. The microglial cells were incubated with JC-1 solution for 20 min 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.

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.

Measurement of mitoROS levels

After culture of microglial cells in 6-well dishes with 6 × 104 cells/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.

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 centrifuged at 12,000 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.

Co-immunoprecipitation (Co-IP) assay

Co-IP was performed following the previous description [37]. In brief, the microglial cells were lysed and total lysates were harvested by weak RIPA lysis buffer (Cell Signaling Technology, Danvers, MA, USA). After clearing with 50% protein A/G agarose for 1 h, the 500 mL 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.

Statistical analysis

All data were analyzed with GraphPad 8.0 software and expressed as the mean ± standard deviation (SD) of at least three independent experiments. Gray level and fluorescence intensity were detected with ImageJ. An unpaired Student’s t-test or one-way analysis of variance (ANOVA) plus Tukey’s post hoc test was applied to compare the differences between two groups. For the difference of groups at the same time point, the data were analyzed using one-way ANOVA followed by Tukey’s post hoc test. Two-way ANOVA was used to compare the data from all groups. P < 0.05 was considered as a significant difference.

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