Patients

This study was approved by the Ethics Committee of the Second Hospital of Hebei Medical University (Ethics Approval No: 2024-R558). Thirty patients with stroke hospitalized in the Department of Neurology at the Second Hospital of Hebei Medical University between September 2023 and October 2024 were enrolled. Blood samples from all patients were collected within 48 h of symptom onset to capture the acute phase inflammatory response. Additionally, 25 age and sex-matched healthy controls were recruited from the same organization. At 3-month follow-up, functional outcomes were evaluated using the modified Rankin Scale (mRS); good outcome was defined as an mRS score between 0 and 2.

Blood Sample Processing

Non-coagulated whole blood samples were drawn from the forearm of each participant. One aliquot of the sample was centrifuged at 2000 × g for 20 min to isolate the serum, and TNF-α levels were quantified using a specific ELISA kit (BOSTER, China, Cat. EK0525). The second aliquot was carefully layered on the top of lymphocyte separation medium (Ficoll) after diluting with phosphate-buffered saline (PBS). Peripheral blood mononuclear cell (PBMC) layers were collected by centrifugation at 400 × g for 30 min. Total RNA was extracted from PBMCs using the TRIzol LS reagent (Invitrogen, Cat# 10296028CN), followed by quantitative analysis of Ptbp2 expression.

Animals

Eighty 12-week-old specific pathogen-free (SPF) male C57BL/6N mice (weighing 22–26 g) were obtained from the Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). The animals were housed under SPF conditions in a controlled environment (temperature: 22 ± 3 °C; humidity: 60 ± 5%; 12/12 h light/dark cycle) with ad libitum access to a standard chow and water. Following one-week acclimatization, the mice were randomly allocated to either the control or experimental group. All procedures performed in this study were approved by the Animal Care and Use Committee of the Second Hospital of Hebei Medical University and adhered to the guidelines outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Transient Middle Cerebral Artery Occlusion (tMCAO) Model

The tMCAO model was developed according to a previously established protocol [18]. Briefly, mice were anesthetized while maintaining spontaneous respiration. The right common carotid artery (CCA) and external carotid artery (ECA) were carefully exposed through a midline neck incision. A silicon-coated monofilament (diameter: 0.22 ± 0.01 mm; Yushun Bio, China) was then introduced through the ECA lumen and advanced into the internal carotid artery (ICA) until it reached the origin of the middle cerebral artery (MCA), achieving occlusion. After occlusion for 60 min, the filament was withdrawn to initiate reperfusion. Throughout the procedure, body temperature was maintained at 37 ± 0.5 °C using a heating pad. The sham-operated mice underwent the same surgical procedure, except for filament insertion.

Stereotactic Injection of AAV Vectors

Stereotaxic delivery of the AAV overexpression vector (HBAAV2/9-m-Ptbp2-3xflag-ZsGreen) was performed by injecting into the right lateral ventricle(Zhou, Tao, Yu, Wu, Hui, Xu et al., 2023). Following cranial exposure, the injection site was targeted 1.55 mm right and 1.05 mm posterior to the bregma. A microinjector (Hamilton, Shanghai, China) was implanted at a depth of 3.5 mm, and the viral suspension was infused at a controlled rate over 10 min.

Measurement of Cerebral Infarct Volume

Following extraction, the collected brains were frozen at −20 °C to measure the volume of the infarct. The brain slices were preserved in 4% paraformaldehyde and stained with TTC (2%, 37 °C, 20 min; Sigma T8877). A stereomicroscope (ZEISS Axio Zoom V16) was used to image the sections (Shi, Zou, Jia, Shi, Yang, Liu et al., 2021) and ImageJ software (NIH) was used to measure the infarct area.

Infarct volume was assessed histologically using Nissl staining. After fixation in 10% formaldehyde for 48 h, brain samples were dehydrated, embedded in paraffin, and cut into 3.5-μm sections. Sections were deparaffinized, rehydrated, and incubated with 1% toluidine blue (Solarbio, G1434) according to the manufacturer’s instructions. Following staining, slides were mounted with a neutral resin and examined under the ZEISS Axio Zoom V16 stereomicroscope.

Behavioral Analysis

Neurological functions in mice after tMCAO were assessed using three behavioral tests: the modified Neurological Severity Score (mNSS) [19], rotarod test [20, 21], and the adhesive removal test [22]. Mice were trained for three consecutive days prior to tMCAO surgery and were re-evaluated on days 1, 2, and 3 after tMCAO. All tests were conducted by investigators blinded to the experimental groups.

Quantitative Real-Time PCR

Reverse transcription was performed using the SureScript™ First-Strand cDNA Synthesis Kit (GeneCopoeia). The resulting cDNA was amplified using the real-time PCR system (Agilent, Santa Clara, CA, USA) with BlazeTaq SYBR Green qPCR Mix (GeneCopoeia) as the fluorescent dye. The following primer sequences were used:

Ptbp2:

CD86:

Arg-1:

Immunofluorescence Staining

After anesthetizing the mice, transcardial perfusion was performed sequentially using 0.9% saline and 4% paraformaldehyde. The brains were post-fixed and dehydrated in 30% sucrose for 48 h and sectioned (15 μm thick) on a cryostat (Thermo Scientific, USA). The sections were then processed for staining by permeabilization with 0.5% Triton X-100 (25 min), blocking with 10% donkey serum (1 h, 37 °C), and finally incubation with primary antibodies overnight at 4 °C. The antibodies used included rabbit anti-CD86 (Boster, Cat. BM4121), rabbit anti-Iba-1 (Wako, Cat. 019–19741), rabbit anti-Arg-1 (Proteintech, Cat. 16,001–1-AP), rabbit anti-Occludin (Invitrogen, Cat. 40–4700), rabbit anti-ZO-1 (Proteintech, Cat. 21773–1-AP), and rat anti-CD31 (BD Biosciences, Cat. 550–274). After washing, the sections were incubated with species-appropriate secondary antibodies (Alexa Fluor 488 or 594, Jackson ImmunoResearch, USA) for 1 h, mounted with DAPI Fluoromount-G (Southern Biotech, Cat. 0100–20), and imaged using a laser scanning confocal microscope (ZEISS LSM880, Germany).

Enzyme-Linked Immunosorbent Assay (ELISA)

At 3 days post-tMCAO, blood was obtained via cardiac puncture and serum was isolated by centrifugation (3000 × g, 15 min, RT). Moreover, supernatants were prepared from homogenates of peri-infarct cortical tissue in buffer, which were subsequently sonicated on ice and centrifuged (1500 rpm, 10 min). TNF-α and IL-10 concentrations were determined using the quick ELISA kits (TNF-α, BOSTER EK0527; IL-10, BOSTER EK0417) following the supplier's instructions.

Western Blot

Proteins were isolated from the brain tissues or cultured cells on ice. Equal amounts of proteins were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred onto PVDF membranes (Millipore). Membranes were then blocked with rapid blocking buffer (Boster, Cat. no. AR0041) and incubated with primary antibodies overnight at 4 °C, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (goat anti-rabbit IgG; Abcam, Cat. 423,920) for 1 h. Protein bands were visualized using the imaging system (LI-COR Biosciences). The primary antibodies used were rabbit anti-Ptbp2 (Abcam, Cat. ab154787), rabbit anti-CD86 (Boster, Cat. BM4121), rabbit anti-Arg-1 (Proteintech, Cat. 16,001–1-AP), rabbit anti-Occludin (Invitrogen, Cat. 40–4700), rabbit anti-ZO-1 (Proteintech, Cat. 21,773–1-AP), rabbit anti-TNF-α (Boster, Cat. BA0131), rabbit anti-PFKFB3 (Proteintech, Cat. 13,763–1-AP), rabbit anti-LDHA (Proteintech, Cat. 19,987–1-AP), rabbit anti-HK2 (Proteintech, Cat. no.22029–1-AP), rabbit anti-GLUT1 (Proteintech, Cat. 21,829–1-AP), rabbit anti-NF-κB p65 (Abcam, Cat. ab32356), and rabbit anti-NF-κB p-p65 (Proteintech, Cat. 82,335–1-RR).

Lactate Measurement

Lactate concentration in the brain tissues was determined using the lactate assay kit according to manufacturer’s instructions (Beyotime, Cat. P0393S). Briefly, homogenized tissue samples in the lysis buffer were sonicated on ice and centrifuged at 12,000 rpm for 3 min. The supernatant was incubated with the reaction mixture for 25 min. Finally, the absorbance at 450 nm was measured using a microplate reader, and lactate concentration was determined using a standard curve.

RNA Sequencing (RNA-seq) of Brain Tissue

Transcriptomic analysis was conducted on ischemic brain tissues collected 3 days post-stroke. After RNA extraction and sequencing at Majorbio (Shanghai, China), bioinformatic analyses, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to uncover differential gene expression and pathways linked to microglial polarization. Sequencing depth: the average sequencing depth was 15.08 GB of reads per sample. Normalization: gene read counts were normalized using the DESeq2 package. Differentially expressed genes were identified using the following criteria: |Fold Change|> 2 and adjusted P-value < 0.05.The lists of differentially expressed mRNAs and lncRNAs are provided in Supplementary Files 1, 2, and 3, respectively.

ceRNA Network Construction and Analysis

The ceRNA network was constructed based on RNA-seq data from the ischemic penumbra. We first identified putative ceRNA pairs by selecting RNA molecules that showed a significant positive correlation in expression (Pearson's |r|> 0.9, p < 0.01) and were supported by experimental evidence in the NPInter v5.0 database. CeRNA pairs were further required to share ≥ 2 common miRNAs.Subsequently, miRNA-target interactions for these pairs were identified by querying three experimentally validated databases (miRecords, miRTarBase, and TarBase) using the 'multiMiR' R package to confirm shared miRNA response elements (MREs). The final network was assembled by integrating these co-expression and miRNA interaction data. Hub genes within the network were objectively defined and ranked by their degree centrality, a measure of connection density.The complete list of the detailed LncRNA-mRNA and the detailed miRNA-mRNA interactions have been provided in Supplementary File 4 and Supplementary File 5, respectively.

Cell Culture

BV2 microglial cells (immortalized; RRID: CVCL_0182) were cultured in complete DMEM (supplemented with 10% FBS and 1% penicillin–streptomycin) at 37 °C with 5% CO₂ and passaged every two days. To induce activation, cells were exposed to LPS (2 μg/mL) in fresh DMEM for 24 h.

Establishment of the Oxygen–Glucose Deprivation (OGD) Model

bEnd.3 cells were plated in 384-well plates (Cellvis, Cat. p96-1.59) and maintained in high-glucose complete medium for 48 h. Prior to oxygen–glucose deprivation (OGD), the cells were rinsed with PBS and switched to glucose-free medium. OGD injury was induced by placing the cells in a tri-gas incubator (95% N₂, 5% CO₂) at 37 °C for 4 h. Following OGD, the cells were exposed to conditioned medium collected from differentially treated BV2 cells for an additional 24 h.

Lentiviral Transfection of Ptbp2

BV2 cells were plated in 6-well plates and maintained in DMEM containing 10% FBS and 1% penicillin–streptomycin. After 10 h of culturing, a solution containing the virus and polybrene was added to the medium. To establish stable cell lines, transduced cells were subjected to selection using puromycin (Yeasen, Cat.no.60209ES) for a minimum of seven days until only stably transduced cells survived.

Statistical Analysis

All data are presented as the mean ± standard deviation (SD). The independent samples t-test was used for comparing two groups and one-way analysis of variance (ANOVA), followed by Tukey’s test was used for multi-group comparisons after assessing data distribution and variance equality. The association of blood Ptbp2 levels with TNF-α levels, NIHSS, and mRS was evaluated using Pearson's correlation test. A p-value < 0.05 was considered statistically significant.