C57BL/6J male healthy mice (8-12 weeks old, average weight 18-22g) were accommodated in the IVC animal husbandry system of the Medical Animal Center of the Affiliated Hospital of Southwest Medical University, where the ventilation system and internal hygiene of the cages were kept in good condition, the humidity inside the cages was controlled at 55%-65%. The constant temperature inside the cages was maintained at 24-25°C. The animals were exposed to a 12-hour cycle of light, were protected from light, and rested quietly at night. The animals all have free access to fresh feed and drinking water.
Animal experimental design
Experiment 1. To examine the changes of RIPK1/RIPK3 during early brain injury in subarachnoid hemorrhage, Mice used for protein abundance assay were divided into four groups: Sham (n=5), SAH 24h (n=5), SAH 48h (n=5), SAH 72h (n=5), and the injured lateral hemispheres were harvested after modeling for subsequent validation. Immunofluorescence staining was used to determine the co-localization of MLKL with microglia in the 24h group (N=6), Pyridinium iodide staining to detect cell necrosis and to detect co-localization with microglia (N=6).
Experiment 2. Targeted knockdown of mice microglia Bak1 protein using pAAV-CD68p-Bak1-shRNA adeno-associated virus (shanghai genechem Co., Ltd. CN). Bak1 target sequence is 5'-GCTCTCATCGGAGATGATATT-3', NC negative control sequence is 5′-TTCTCCCCGAACGTGTCACGT-3′. Mice were injected with the adeno-associated virus in the lateral ventricle for 3ul and then monitored for viral expression for three weeks. Different C57BL/6J male mice groups were randomly selected after infection for subsequent experimental validation. WB detection of RIPK1, RIPK3 expression in the ipsilateral cerebral hemisphere 24h after SAH molding, NC group (n=5), Bak1-KD (N=5), ELISA for pro-inflammatory factor concentration (IL-6, IL-1β, TNFα), NC group (n=5), Bak1-KD (n=5). Co-localization of PI and microglia IBA1 were detected by immunofluorescence, NC group (n=3), and Bak1-KD group (n=3).
Subarachnoid hemorrhage model
Induction of SAH model by modified single-clamp puncture method. The primary process is: Anesthesia was induced in experimental mice with 4-5% isoflurane, followed by intraperitoneal injection of 1% pentobarbital sodium to create continuous anesthesia. The mice were placed supine on the operating table with a satisfactory level of anesthesia (no significant pain from skin incision). After skin preparation and disinfection, the skin of the neck was incised along the anterior midline of the neck using a sharp surgical blade (an incision of approximately 1 cm in length was sufficient). Micro forceps bluntly separated the subcutaneous fat, deep fascia, and muscle tissue, preserved the hyoid bone, pulled the hyoid bone to the side, exposed the right common carotid artery, separated the vessels and peripheral nerves, and then freed the right external carotid artery, down and on its serious side, separated the right internal carotid artery. After resistance is felt at the bifurcation of the anterior and middle cerebral arteries, the puncture wire is advanced 2mm further through the vessel and immediately returned. We also observed whether the mice showed typical Cushing's reaction, which was used as an indirect criterion to judge the success of the SAH model. After the puncture is completed, the puncture wire is quickly withdrawn. The blind end of the external carotid artery vessel is ligated, the skin incision is sutured, and the skin is disinfected with dilute iodophor. Mice in the Sham group were performed the same surgical procedure. Still, instead of puncturing the vessel, the puncture line was immediately returned after resistance was felt at the bifurcation of the anterior and middle cerebral arteries. The mice were placed in a rewarming chamber at 25°C and observed and recorded every 15 minutes until they were revived from anesthesia and housed in separate cages by the group.
Cell culture and reagents
The mouse microglial cell-line BV2 were cultivated in Dulbecco’s Modified Eagle’s Medium (DMEM) (#C11995500BT, Gibco) containing 10% fetal bovine serum (FBS) (#04-001-IACS, Biological Industries) and 1% penicillin-streptomycin and maintained under the stable environment with 37°C and 5% CO2. Cell stimulation was induced 10μM oxy-Hb (#JP0200, Jinpin Chemical Technology Co., Ltd. CN). zVAD-FMK was selected (# C1202 Beyotime CN) 30umol/ml, Necrostatin-1 (#SC4359 Beyotime CN) 30μM/ml, the above reagents were added to microglia cultured inappropriate well plates for modeling.
The harvested cells or animal tissues were lysed on ice with RIPA lysis solution for 30 min and then centrifuged at 12000g for 10 min at 4°C. The protein supernatant was added to the loading buffer and denatured at 100°C for 5 min. The samples were added to SDS-PAGE gel for electrophoresis, after which the proteins were transferred to PVDF membrane, 5% skim milk powder was blocked for 1 h, and then overnight at 4°C with the following primary antibody: rabbit polyclonal anti-RIPK1 (1:1000 #17519-1-AP Proteintech CN), rabbit polyclonal anti-RIPK3 (1:1000 #17563-1-AP Proteintech CN), Bak1 Rabbit mAb (1:1000 #12105 CST USA), Phospho-MLKL(Ser345) Rabbit mAb (1:1000 #37333 CST USA ), Rabbit Polyclonal anti-Thrombospondin 1 (1:1000 # 18304-1-AP Proteintech CN), Rabbit Polyclonal anti-Beta Tubulin (1:5000 # 10094-1-AP Proteintech CN). The appropriate secondary antibody (1:5000) was selected and incubated for 1h, X-ray film and Image J software (NIH) were used for detecting and quantifying, respectively.
Propidium iodide staining
Intraperitoneal injection of pyridinium iodide (PI 10mg/kg) 1h before sacrificing mice after SAH 24h, Brain tissue was obtained after cardiac perfusion with ice PBS and immediately embedded by OCT, avoiding light in sections, Brain sections were cut at 10μm intervals near the optic chiasma. The sections were fixed with 4% paraformaldehyde for 10 min, washed with PBS 3 times×10 min, 0.3% TritonX-100 for 10 min, and then blocked with 10% goat serum for 1h, Anti-IBA1 (1:500 #ab178846 Abcam USA) overnight at 4°C, fluorescent secondary antibody Goat anti-Rabbit (H+L 488) (1:200 #ab150077 Abcam USA) incubated for 1h at room temperature, DAPI blocked, fluorescence microscopy photographed(Olympus, Japan) and propidium iodide-positive microglia were quantified in puncture bleeding injury region from 200× in three brain sections per mice.
Oxyhemoglobin stimulated BV2 cells for 24h. 1× PI staining working solution (#C1056 Beyotime) and Hoechst 33342 (#C1027 Beyotime) were added, incubated for 20min at 37°C, avoiding light, and PI-positive BV2 cells were photographed by fluorescence microscopy.
Mice 24h after SAH, ice PBS and 4% paraformaldehyde heart perfusion and immersed mouse brain for 2h, 30% sucrose dehydration overnight at 4°C, OCT embedding, brain tissue section 10μm, 0.01M Sodium citrate antigen repair, 0.3% TritonX-100 10min then 10% goat serum blocked for 1h. Anti-IBA1 (1:500 #ab178846 Abcam USA), anti-MLKL (1:100 #66675-1-Ig proteintech CN) overnight at 4°C, fluorescent secondary antibody Goat anti-Rabbit (H+L) (1:200 #ab150077 Abcam USA) and Goat anti- Mouse (H+L) (1:200 #ab150116 Abcam USA) incubated for 1h at room temperature, DAPI blocked, The MLKL co-localizes with IBA1 in microglia was observed using a fluorescent microscope (Olympus Japan).
Reactive oxygen measurement
BV2 cells after 24h of oxyhemoglobin stimulation, Removal of supernatant, add the working assay solution to the cells according to the Reactive Oxygen Species Assay Kit (#S0033S Beyotime CN) manufacturer’s instruction. Incubate for 20min at 37℃, avoiding light, Wash cells three times with serum-free medium after incubation; Fluorescence situation was examined using a fluorescent microscope (Olympus, Japan).
Bak1-KO cell line construction
Bak1 sgRNA was designed using the online tool (http://crispr-era.stanford.edu/), and the target sequence 5'-TCGGGGTCTTCGTCTTTGCA-3' was cloned into the plasmid lentiGuide-Puro (addgene #52963 USA). The plasmid vector was then transfected with liposomal transfection reagent LipoFiter3.0 (HANBIO, CN) in 293T cells, and supernatants containing viral particles were collected every 24 h for three days after transfection. Infection of BV2 cells after concentrating and purifying the virus. The Cas9 protein expression plasmid lentiCas9-Blast was constructed and infected with BV2 cells, in the same way, followed by puromycin or blasticidin selection. Sequencing to identify completed mutant BV2 cell lines, WB to detect Bak1 protein knockout efficiency.
Bak1 and THBS1 knockdown
THBS1 and Bak1 shRNA were designed using the online tool (https://www.sigmaaldrich.cn/cn/zh). THBS1 target sequence 5’-GCCAGAACTCGGTTACCATCT-3’, Bak1 target sequence 5’-GCTCTCATCGGAGATGATATT-3’, and the scrambled control sequence 5’-TTCTCCGAACGTGTCACGT-3’ were cloned into the lentiviral shRNA expression vector plko.1. the plasmid vector was then transfected with liposomal transfection reagent lipofiter3 (HANBIO, CN) in 293T cells, collecting virus supernatant every 24h. after virus concentration and purification, THBS1 virus-infected Bak1-ko cell line, NC negative control, and Bak1-KD virus-infected BV2 cells, RT-qPCR and WB detected knockdown efficiency.
HiScript III-RT SuperMix synthesized cDNA for qPCR (+gDNA wiper) (#R323, Vazyme Biotech Co., Ltd); reactions were performed on LightCycle®96 (Roche) using ChamQ Universal SYBR qPCR Master Mix (#Q711, Vazyme Biotech Co., Ltd). The result was analyzed using the 2−ΔΔCq, gene expression was normalized to β-actin and presented as a fold change relative to control.
Stimulation of BV2 cells with oxyhemoglobin for 24h, Whole-cell lysates were extracted with IP lysis buffer (NP-40 1%, Tris-HCL 8.0 50mmol/ml, Glycerin 10%, NaCl 150mmol/ml, EDTA 5mmol/ml) supplemented with Protease and phosphatase inhibitors (#P1045, Beyotime). The lysates were sonicated and centrifuged at 12000×g for 10min at 4 °C. The BCA Assay Reagent (#P0012S, Beyotime) was used to determine protein concentration. Take 10% of the total volume of lysate as input. 2mg of whole protein was made for the IgG and IP groups, respectively. Add Rabbit control IgG antibody (1:200 #AC005, ABclonal) anti-RIPK3 (anti-RIPK3 1:200 #17563-1-AP Proteintech CN), 4°C overnight. Antibody-protein mixture is incubated in the appropriate volume of Dynabeads Protein A (#10001D, Invitrogen™) for 30 minutes at room temperature. After washing the beads three times, the samples were denatured at 100°C for 5 min using a 5×SDS loading buffer, and WB detected the pull-down proteins.
Cell viability assays
Cells were seeded at a density of 5× 103 cells/well in 96-well plates. After overnight incubation, fresh medium containing Oxyhemoglobin was added, and the plates were maintained for 0, 12,24h. Add CCK8 reagent (1:100 #HB-CCK-8-10 HANBIO CN) and incubate for 2 hours protected from light, measure the OD450 value, and compare the relative CCK8 values at each time point.
TNFα ELISA kit, IL-6 ELISA kit, IL-1β ELISA kit were obtained from NEOBIOSCIENCE CN (#EMC001b.96, #EMC102a.96, # EMC004.96); subsequent testing follows manufacturer's protocols. The same number of BV2 cells were seeded into the culture well plates, and the cell supernatant was collected for detection after 24h of hemoglobin stimulation. The mice’s brain tissues were obtained from SAH molds for 24h, lysed by RIPA, and BCA measured protein concentrations. Brain tissue lysates with the same protein content were taken for subsequent assays. The samples were added to the test wells, and the standard wells were set up simultaneously. After the operation, according to the kit’s instructions, the OD450 values were measured, and the contents of IL-6, IL-1β, and TNFα were calculated according to the standard curve.
Microglia were seeded in a 6-well plate, and after 12h for complete cell wall adhesion, Oxy-Hb stimulated the cells and reached the scheduled stimulation time. Cellular RNA was extracted using RNAeasy Isolation Reagent (#R701-02, Vazyme Biotech Co., Ltd) according to the manufacturer's instructions. The Universal V6 RNA-seq Library Prep Kit for Illumina (#NR604-02, Vazyme Biotech Co., Ltd) was used for library preparation. Briefly, the samples were normalized at a 500 ng/ml concentration. First, samples were subjected to a tagmentation redaction, indexed, and PCR amplified. Libraries were then purified with VAHTS DNA Clean Beads (#N411-02, Vazyme Biotech Co., Ltd). QC checks for the library preparations included electrophoresis (Agilent Technologies 2100 Bioanalyzer) or library quantification. The Novogene (Beijing CN) sequence was sequenced on an Illumina HiSeq 1500 high-throughput sequencing system.
The raw fastq files were trimmed using trim galore (version 1.18) to remove adaptor sequences and low-quality reads. Then FastQC (version 0.11.9) was used for quality control. The remaining reads were aligned to the GRCm38 mouse genome using HISAT2 (v2.2.0) with default parameters and filtered with samtools (version 1.10, parameters used: samtools view -F 1804 -f 2 -q 30). Gene counts were calculated from the mapped reads using featureCounts (v2.0.1) with the Ensembl gene annotation (version mm10). Subsequently, TPM (Transcripts Per Kilobase of exon model per Million mapped reads) in each gene was calculated for subsequent analysis. Differential expressed genes (DEGs) was evaluated using DESeq2 package in R (version 4.2.0), using adjusted P < 0.05 and |log2FC| > 1 as cutoffs to define the DEGs.
GSEA uses the expression matrix obtained by cluster profile package in R software to carry out GSEA based on biological processes in gene ontology (GO-BP) and based on the Kyoto Encyclopedia of Gene and Genome (KEGG) signal pathway, respectively, to obtain the enrichment results of differentially expressed genes in GO functional annotation and KEGG pathway obtained by differential analysis between NC group and KD group. The gene sequence was arranged according to the descending order of multiple gene expression changes (foldchange, FC) in the Bak1-KD group compared with the NC group. The statistical method was Fisher exact probability test, and error detection rate (false discovery rate, FDR) was used to correct.
GraphPad Prism 8 was used for statistical analysis. We used an unpaired two-tailed t-test to compare two groups, and a one-way ANOVA was applied to compare three or more independent groups. Bar graphs were described as mean ± Standard error of the mean (SEM) of at least three independent experiments. Statistically significant changes relative to a negative control were represented with P < 0.05.