The transcriptomic data in this investigation were sourced from the Gene Expression Omnibus (GEO) repository, hosted by the National Center for Biotechnology Information (NCBI; https://www.ncbi.nlm.nih.gov/geo/). The related datasets were retrieved from the GEO database by searching for “Ischemic stroke”, from which the GSE106680, GSE97537, and GSE61616 datasets were screened. GEO2R (https://www.ncbi.nlm.nih.gov/geo/geo2r/) was used to analyze the datasets. Within these three datasets, differentially expressed genes (DEGs) between the IS and Control groups were identified using the following filtering criteria: adjusted p-value (adj. p) 0.05 and absolute logarithmic fold change ($|$logFC$|$) $\ge$1. A volcano plot was created to illustrate the distribution of the DEGs. Common DEGs across the three datasets were identified and presented in a Venn diagram. Additionally, a heat map was created to visualize the expression patterns of common DEGs within the GSE106680, GSE97537, and GSE61616 datasets.

The selected overlapping DEGs were analyzed by Gene ontology (GO) functional enrichment analysis, including the molecular function (MF), biological process (BP), and cellular component (CC) categories. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID, https://david.ncifcrf.gov/summary.jsp). The p value 0.05 was considered statistically significant. The R Package clusterProfiler (R Package 4.00, Yu Laboratories, Los Angeles, CA, USA) was used to further explore the GO and KEGG enrichment results of the DEGs. Enrichment dot plots were used to display the most significantly enriched terms, which exhibited the lowest p values.

The Search Tool for the Retrieval of Interacting Genes (STRING, https://cn.string-db.org/) database was employed to generate protein-protein interaction (PPI) networks based on the identified DEGs, with a confidence score threshold set to 0.4 as an indicator of significance. The topology of the PPI network was visualized using the Cytoscape software, version 3.8.2 (Cytoscape Consortium, Institute for Systems Biology, Seattle, WA, USA). Within Cytoscape, the CytoHubba plugin, version 0.1, was used to calculate the degree of centrality of the protein nodes, facilitating the identification of central hub genes. An enrichment chord diagram was further generated to show the enrichment pathways of the hub genes, and an expression ridgeline map was plotted to display the distribution of hub genes in different sample data using the R package ggplot2. A matrix heat map was created to show the association between the hub genes in the datasets.

Sixty male Sprague-Dawley rats, with ages between 6–8 weeks and body weights ranging from 230–250 g, were procured from SPF Biotechnology Co. (Beijing, China). The animals were housed under controlled conditions with a 12-hour light/dark cycle, and a temperature maintained at 24 $\pm$ 2 °C. The humidity level was kept between 50–60%, and the animals had unrestricted access to food and water. The rats were then randomly assigned to four groups based on body weight: Sham group, Middle cerebral artery occlusion and reperfusion (MCAO/R) model group, MCAO/R+AAV-NC (adeno-associated virus- negative control) group, and MCAO/R+AAV-CLEC7A group.

This study was designed to minimize any potential harm or distress to the animals involved by adhering to the highest standards of animal welfare and ethical conduct. All animal procedures were conducted in strict accordance with the ARRIVE guidelines. The experimental protocol was carefully reviewed and approved by the Ethics Committee of Hainan Medical University (ethics number: HYLL-2020-140).

We used microglia-specific adeno-associated virus (AAV) vectors (Genechem Co., Ltd. Shanghai, China) for targeted gene manipulation in the microglia. The sequences of the AAV vectors are listed in Supplementary Table 1. The viral vectors were delivered via stereotactic surgery. In brief, rats were anesthetized with 4–5% isoflurane (R510-22-10, RWD Life Sciences, Shenzhen, Guangdong, China) sustained with a concentration of 1–2% isoflurane, and fixed to a stereotactic instrument (51600, Stoelting, Mount Vernon, IL, USA).

The MCAO/R ischemic rat model was constructed as previously described [32]. Anesthesia was induced with 4–5% isoflurane and sustained with a concentration–1–2% isoflurane. After exposing the right carotid bifurcation, an 8-0 silicone-coated filament was gently pushed (9.0–10.0 mm) through the common carotid artery to occlude the middle cerebral artery. Removal of the nylon suture allowed the cerebral blood flow to resume after 1 h of temporary blockage. Rats in the sham group underwent surgical exposure of the common carotid arteries without MCAO/R. Neurobehavioral function and infarct volume were assessed 24 h after reperfusion. Neurological function was assessed using the Zea Longa scoring method, as described previously [33]. According to this scale, the animals were scored as follows: 0, no deficits; 1, inability to fully extend the left forepaw; 2, circling behavior to the left; 3, incapacity to support weight on the left side; and 4, lack of spontaneous ambulation coupled with reduced consciousness. Following neurobehavioral assessment, the rats were anesthetized with 4–5% isoflurane and subsequently euthanized via cervical dislocation to collect brain tissues for further experimental analysis.

The brain tissues were sliced into 1-mm-thick sections using a vibration microtome (YD-31S, YIDI Medical Appliance CO., Ltd., Jinhua, Zhejiang, China) and fixed with 4% paraformaldehyde (441244, Sigma, Saint Louis, MO, USA) for 30 min after being submerged in 2% Triphenyl Tetrazolium Chloride (TTC; PM10798, CANSPECCHINA, Shanghai, China ) solution for 30 min at 37 °C. Brain slices were then photographed using a microscope (Leica, Wetzlar, Germany) and images were analyzed using ImageJ software (version 8.0, the National Institutes of Health, Bethesda, MD, USA). A red hue denotes live tissues, whereas a pale color denotes infarcted tissue. The white infarct area/whole slice area $\times$ 100% was used to compute the brain infarct size (%).

The brain water content was measured using the wet/dry weight method [34]. Brain slices (3 mm thick) of were cut and divided into ischemic and non-ischemic hemispheres. The ischemic hemisphere samples were weighed (wet weight), dried in a vacuum oven at 100 °C for 48 h, and then reweighed (dry weight). The brain water content (%) was calculated as [(wet weight – dry weight)/wet weight] $\times$ 100%.

The brain tissue was fixed in 4% paraformaldehyde. Following dehydration and hyalinization, tissues were embedded in paraffin to prepare sections. The sections were subsequently dehydrated using a gradient of 50%–70%–80%–90%–95%-absolute ethanol. After dehydration, the tissues were placed in a 1:1 solution of anhydrous ethanol and xylene for 30 min, and permeated in a pure xylene solution. Subsequently, paraffin sections were stained with hematoxylin (H3136-25G, Sigma, Saint Louis, MO, USA) and alcoholic eosin (E4009, Sigma, Saint Louis, MO, USA). The HE-stained images were examined using an optical microscope (Leica).

Immunofluorescence staining was performed to detect the expression of Iba-1. The paraffin-embedded brain tissue sections were deparaffinized in xylene and rehydrated using a graded ethanol series. Subsequently, ethylenediaminetetraacetic acid (pH 8.0) was used for antigen retrieval, and the sections were washed with phosphate buffered saline (PBS). Subsequently, the sections were incubated in an autofluorescence quencher reagent for 5 min, rinsed with running water for 10 min, and incubated with 5% bovine serum albumin for 30 min to block nonspecific reactions. Next, the sections were incubated with anti-Iba-1 (1:400; ab283319, Abcam, Cambridge, UK) antibody for 15 h at 4 °C. The sections were then washed three times with PBS and incubated with the appropriate secondary antibody (1:200; Abcam) for 90 min. Finally, the sections were covered with a sealer containing 4^′,6-diamidino-2-phenylindole (DAPI), imaged, and analyzed using ImageJ software, version 1.53t (Wayne Rasband, U.S. National Institutes of Health, Bethesda, MD, USA).

Terminal Deoxynucleotidyl Transferase-mediated Nick End Labeling (TUNEL) staining was performed using a commercial in situ cell death detection kit (Roche, Basel, Switzerland). Tissue sections were deparaffinized and rehydrated, followed by a 15-minute digestion with Proteinase K at 10 mmol/L. The treated slides were then incubated in the TUNEL reaction mix for 6 minutes at 37 °C, shielded from light. After staining with DAPI (P36931, Invitrogen, Carlsbad, CA, USA), the cell nuclei were observed under a fluorescence microscope (LWP300-38LFT, CEWEI OPTOELECTRONIC TECHNOLOGY Co., Ltd., Shanghai, China). Nuclei appeared blue under a microscope, whereas green fluorescence indicates the presence of apoptotic cells. The apoptosis index of TUNEL-stained tissues was assessed using the TUNEL index (%), which was calculated as the ratio of TUNEL-positive cells to total cells.

HT22 mouse hippocampal neurons and BV2 mouse microglia cells were obtained from iCell Bioscience, Inc. (Shanghai, China). Both cell lines used in this study were authenticated using Short Tandem Repeat (STR) profiling to confirm species identification prior to commencing the experiments. The authentication confirmed the genetic identity of these cell lines. Upon receipt, the cells were immediately amplified and used for experiments within a short timeframe to ensure stability. All cell lines were tested for mycoplasma contamination prior to use to guarantee the accuracy of the experimental results. The test results were negative, indicating that the cells were free of contamination. Regular testing was conducted throughout the experiments to maintain cleanliness of the cell lines.

The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, New York, NY, USA) containing 10% fetal bovine serum (FBS; A5670701, Gibco) and 1% penicillin-streptomycin. Cells were incubated at 37 °C with 5% CO₂. CLEC7A was knocked down in BV2 cells using three small interfering RNAs (siRNAs; GenePharma Co., Ltd., Shanghai, China) targeting the coding sequence of CLEC7A. Transfection was carried out using the Lipofectamine 3000 liposome transfection kit in accordance with the manufacturer’s instructions (L3000008, Thermo Fisher Scientific, Wilmington, DE, USA). Neuron-microglia co-cultures were seeded in 24-well plates and incubated for 3 days. BV-2 microglia were seeded into the Transwell inserts with a pore size of 0.4 µm at a density that maintained a microglia-to-neuron ratio of 1:2. HT22 and BV2 cells were co-cultured for 2 days before oxygen-glucose deprivation/reperfusion (OGD/R) treatment.

The cells were divided into Control, OGD/R, OGD/R+si-CLEC7A, and OGD/R+si-CLEC7A+nigericin groups. HT22 cells in the OGD/R+si-CLEC7A+Nigericin group were treated with Nigericin (HY-127019, pyroptosis activator,10 µM; MedChemExpress, Shanghai, China) for 1 h. An in vitro model for reperfusion after IS was established, as previously described [33]. In brief, the cells were maintained in DMEM without glucose and transferred to a modular incubation chamber. Subsequently, the cells were exposed to a gas mixture of 95% N₂ and 5% CO₂ at a flow rate of 3 L/min for 15 min. The chamber was subsequently sealed and transferred to an incubator maintained at 37 °C. The OGD/R treatment was performed for 4 h. After OGD/R, the cells were incubated under normal conditions in regular culture medium for 12 h.

Cell viability was assessed using the Cell Counting Kit-8 (CCK-8) (521942, Biosharp, Beijing, China) according to the manufacturer’s protocol. For each treatment group, the cells (1 $\times$ 10⁴ cells/well) were plated in a 96-well plate and cultured for 24 h. The detailed protocol was as follows: the culture medium was replaced with maintenance medium, followed by a 24 h-incubation. After incubation, the culture medium was carefully aspirated and the cells were gently rinsed with Dulbecco’s Phosphate-Buffered Saline (DPBS). Subsequently, the maintenance medium (90 µL) and CCK-8 solution (10 µL) were added to each well. The plates were then incubated for 2 h. Optical density was recorded at a wavelength of 450 nm using a microplate reader (A51119600C, Thermo Fisher Scientific).

RNA was isolated using TRIZOL reagent (Invitrogen). Subsequently, the RNA concentration and absorbance at wavelengths of 260 and 280 nm were precisely measured using a spectrophotometer (OSE-260-03, TIANGEN, Beijing, China) after dilution in RNase-free ultrapure water. cDNA was synthesized using Hiscript II QRT Supermix for the qPCR Reverse Transcription Kit (R222-01, Vazyme, Nanjing, Jiangsu, China). The process of reverse transcription for the synthesis of cDNA templates was carried out using a Polymerase Chain Reaction amplifier (T100, BIO-RAD, Hercules, CA, USA), with the following reaction conditions: 25 °C for 5 minutes; 42 °C for 30 minutes; and 85 °C for 5 seconds. Reverse Transcription-Polymerase Chain Reaction (RT-qPCR) was performed on an ABI7500 quantitative PCR instrument (Applied Biosystems, Foster City, CA, USA). The PCR reaction conditions were set as an initial denaturation step at 95 °C for 30 seconds, followed by 40 cycles consisting of denaturation at 95 °C for 10 seconds, and annealing at 60 °C for 30 seconds. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal control. The Ct values were quantified using the 2^{-Δ⁢Δ⁢Ct} method, and each assay was triplicated. The primer sequences are listed in Supplementary Table 2.

Total protein was fractionated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred onto Polyvinylidene Difluoride (PVDF) membranes (1620177, BIO-RAD). Membranes were blocked with 5% non-fat milk for 2 h. The primary antibodies (Abcam): anti-CLEC7A (1:2000); anti-Caspase-1 (1:2000), anti-GSDMD (1:2000), anti-NLRP3 (1:2000), anti-Iba-1 (1:2000), and anti-$\beta$-actin (1:2000) were diluted in blocking solution and incubated overnight at 4 °C. The membranes were washed to 4–5 times with Tris-Buffered Saline and Tween 20 (TBST, B1009, Applygen, Beijing, China). Subsequently, horseradish peroxidase-labeled goat anti-rabbit Immunoglobulin G (IgG, 1:5000; Abcam) was added and diluted in TBST, followed by incubation for 1 h. $\beta$-actin served as the internal standard for normalization. The PVDF membranes were placed on a Tanon 5200 chemiluminescence imaging system (5200, Tanon, Shanghai, China). An enhanced chemiluminescence substrate was used for color development and images were captured by exposure. The gray values of the protein bands were quantified using ImageJ software. The original figures of Western Blot can be found in the Supplementary Materials.

To detect the concentrations of IL-1$\beta$, IL-18, tumor necrosis factor (TNF)-$\alpha$, and lactate dehydrogenase (LDH) in the supernatant, an Enzyme-linked Immunosorbent Assay (ELISA) kit (Esebio, Shanghai, China) was utilized following the manufacturer’s instructions. The absorbance values for each well were measured at 450 nm, and the sample concentrations were computed based on the equation derived from the standard curve.

Data analysis was performed using GraphPad Prism software (version 9.0, Dotmatics, Boston, MA, USA), and the results were expressed as mean values $\pm$ standard deviation. Comparisons between groups were performed using one- or two-way analysis of variance (ANOVA). Post hoc pairwise comparisons following ANOVA were conducted using Tukey’s multiple comparison test. Comparisons between two groups were performed using a t-test. Statistical significance was set at p 0.05.

From the GSE106680 dataset, we selected three control and three IS samples for analysis. From these, we identified 4558 DEGs, including 2557 upregulated and 2001 downregulated genes. From the GSE97537 dataset, which included 5 control and 7 IS samples, we identified 250 DEGs, of which 240 were upregulated and 10 were downregulated. The GSE61616 dataset comprised five control and five IS samples, from which we screened 2463 DEGs, including 1809 upregulated and 654 downregulated genes. DEGs from both datasets were subjected to clustering analysis to create a volcano plot illustrating notable up- and down-regulated genes between the control and IS samples (Fig. 1A). We selected the top 10 upregulated and downregulated DEGs from each dataset to create a heat map for visualization (Fig. 1B). A Venn diagram showed 140 overlapping DEGs across the three datasets, with 138 upregulated and two downregulated genes, was finally created (Fig. 1C).

Fig. 1.

Differentially expressed genes (DEGs) identification. (A) Volcano plots of DEGs in GSE106680, GSE97537, and GSE61616 datasets; The x-axis represents log2FoldChange, while the y-axis corresponds to –log10. Red dots denote genes with increased expression (up-regulated DEGs), and blue dots denote genes with decreased expression (down-regulated DEGs). (B) Heat map of DEGs in GSE106680, GSE97537, and GSE61616 datasets. (C) Venn diagram of DEGs. The number in each circle represents the number of DEGs in the dataset. The overlapping area between circles indicates the common DEGs found in all three datasets: GSE106680, GSE97537, and GSE61616.

Functional analysis was performed on the 140 common DEGs. The GO enrichment results for these DEGs were organized into categories. For each category, the six GO terms with the most significant enrichment, indicated by the lowest p-values, were identified and illustrated in the GO enrichment dot plot (Fig. 2A). Furthermore, KEGG pathway analysis of the DEGs revealed the 10 most significantly enriched pathways based on the lowest p-values, which are shown in the KEGG pathway enrichment dot plot (Fig. 2B).

Fig. 2.

Enrichment analysis and hub genes analysis. (A) Gene Ontology enrichment dot plot. (B) Kyoto Encyclopedia of Genes and Genome pathway enrichment dot plot. (C) Protein-protein interaction (PPI) network of DEGs. (D) Key modules containing hub genes. (E) Gene expression ridgeline plot of hub genes. The x-axis displays gene expression levels, while the ridge shape illustrates the data distribution within each group. The height of the ridge indicates the number of samples corresponding to that particular gene expression level. (F) The matrix heatmap of GSE61616 dataset. (G) Gene Ontology enrichment chord diagram, consisting of three parts: genes, LogFold Change (representing the fold change of genes for sorting and color-coding gene blocks), and Gene ontology (GO) terms. Different connections between genes indicate their involvement in specific GO terms. NF, nuclear factor; TNF, tumor necrosis factor; AGE-RAGE, advanced glycosylation end-product specific receptor. *** indicates that the p-value is less than 0.001. **** indicates that the p-value is less than 0.0001.

A PPI network was constructed (Fig. 2C), and the PPI network model was visualized using the Cytoscape software, while the Molecular Complex Detection (MCODE) plugin from the CytoHubba plugin suite was used to identify the highest-scoring functional complexes related to the IS within the PPI network. We selected the top-rated Module 1 (24 nodes and 152 edges). Subsequently, we selected hub genes of interest from the top-rated module 1 (Fig. 2D). Fc fragment of Immunoglobulin G (IgG) receptor IIIa (FCGR3A), Fc fragment of Immunoglobulin E (IgE) receptor Ig (FCER1G), Complement component 5a receptor 1 (C5AR1), CLEC7A, Plasminogen activator, urokinase (PLAU), and C-C motif chemokine ligand 6 (CCL6) were screened for subsequent analysis. We finally plotted a ridgeline map using the hub gene expression in the GSE61616 dataset (Fig. 2E). The shape of the ridgelines indicates dispersion within the GSE61616 dataset, with the height of each line representing the number of samples at the corresponding gene expression level, providing an intuitive understanding of both the central tendency and variability in gene expression across the sample population. The matrix heatmap of the expression profile data from GSE61616 revealed the correlation of hub genes across different sample datasets (Fig. 2F). An enrichment chord diagram was further generated to display the relationship between proteins and pathways, as well as to indicate changes in pathway function, which comprised three main components: genes, the fold change in gene expression used for sorting and color-coding of gene blocks, and columns representing GO terms (Fig. 2G).

The brain tissue of rats in the MCAO/R group showed a considerable infarction (34.11 $\pm$ 5.03; N = 6), compared with the Sham group (***p 0.001) (Fig. 3A,B). To determine the extent of brain edema, we measured the brain water content, finding that this metric was substantially increased in the MCAO/R group (81.85 $\pm$ 1.65; N = 6, ***p 0.001), compared with the Sham group (74.71 $\pm$ 2.34) (Fig. 3C). Rats in the MCAO/R group exhibited significantly higher neurological scores (2.67 $\pm$ 0.52; N = 6) compared to the Sham group (0.17 $\pm$ 0.41, ***p 0.001) (Fig. 3D). Representative images (Amplification: 200$\times$, Scale: 100 µm; N = 6) of hematoxylin & eosin (HE) staining revealed that, in the Sham group, the pia mater remained undamaged, with no signs of inflammatory exudates. Neurons in the brain appeared normal, with no evidence of degeneration, necrosis, or inflammatory cell infiltration. In contrast, the model group exhibited a mild presence of lymphocytes in the connective tissue. Extensive necrosis was further found to be more pronounced in the cerebral hemispheres, with the necrotic areas displaying faint staining. The necrotic neuronal nuclei contracted and dissolved, accompanied by neurofibrinolysis (Fig. 3E). To demonstrate microglial activation, we performed immunofluorescence staining for Iba-1, a well-established microglial marker. The results revealed a significant increase in the intensity of Iba-1 immunofluorescence in the MCAO/R group (6.97 $\pm$ 2.33; N = 6) compared to the Sham group (1.00 $\pm$ 0.09, *p 0.05), which revealed an increase in activated microglia (Fig. 3F). Moreover, relative to the Sham group, the MCAO/R group (N = 6) exhibited a marked increase in the relative expression levels of Iba-1 (2.39 $\pm$ 1.20, ***p 0.001), GSDMD (2.64 $\pm$ 0.14, ***p 0.001), Caspase-1 (1.46 $\pm$ 0.05, ***p 0.001), and NLRP3 (2.38 $\pm$ 0.21, ***p 0.001) (Fig. 3G).

Fig. 3.

The expression levels of Iba-1, GSDMD, Caspase-1, and NLRP3 were evaluated in rats after middle cerebral artery occlusion and reperfusion (MCAO/R). (A) Triphenyl Tetrazolium Chloride (TTC) staining. Scale: 5 mm. (B) Infarct volume. (C) Brain water content. (D) Neurological deficit score. (E) Representative images of hematoxylin & eosin (HE) staining (Amplification: 200$\times$, Scale: 100 µm). (F) The expression of Iba-1 was detected by immunofluorescence staining, Scale: 20 µm. (G) Western blot detected the expression of Nod-like receptor protein 3 (NLRP3), caspase-1, gasdermin D (GSDMD), and Iba-1 in rats. *p 0.05, ***p 0.001 vs. Sham group; N = 6. DAPI, 4^′, 6-diamidino-2-phenylindole.

The mRNA expression levels of hub genes (FCGR3A, FCER1G, C5AR1, CLEC7A, PLAU, and CCL6) in the brain tissue (N = 6) were determined by RT-qPCR. The results indicated that the relative mRNA expression levels of FCGR3A (3.32 $\pm$ 1.92, ***p 0.001), FCER1G (3.45 $\pm$ 0.30, ***p 0.001), C5AR1 (3.44 $\pm$ 0.23, ***p 0.001), CLEC7A (3.51 $\pm$ 0.42, ***p 0.001), PLAU (3.51 $\pm$ 0.44, ***p 0.001), and CCL6 (3.81 $\pm$ 0.45, ***p 0.001) were significantly higher in the MCAO/R group than those in the Sham group (Fig. 4).

Fig. 4.

The mRNA expression levels of FCGR3A, FCER1G, C5AR1, CLEC7A, PLAU, and CCL6 in brain tissues were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). ***p 0.001 vs. Sham group; N = 6.

A comprehensive literature review revealed that the roles of FCGR3A, FCER1G, C5AR1, PLAU, and CCL6 in IS and other central nervous system disorders have all been extensively studied [35, 36, 37, 38, 39]. Consequently, CLEC7A was deliberately chosen as the research object for IS in this study. For CLEC7A siRNA transfection, si-CLEC7A-1 (0.28 $\pm$ 0.45, ***p 0.001), si-CLEC7A-2 (0.34 $\pm$ 0.01, ***p 0.001), and si-CLEC7A-3 (0.32 $\pm$ 0.05, ***p 0.001) all caused a significant downregulation of CLEC7A expression in BV2 cells (N = 3), compared to si-NC group (Fig. 5A). Western blotting (WB) was performed to detect the transfection efficiency of CLEC7A protein. The results revealed that the relative expression of CLEC7A protein in BV2 cells was significantly decreased in the si-CLEC7A-1 (0.37 $\pm$ 0.06, ***p 0.001), si-CLEC7A-2 (0.39 $\pm$ 0.06, ***p 0.001), and si-CLEC7A-3 (0.40 $\pm$ 0.02, ***p 0.001) groups, compared to the si-NC group (0.94 $\pm$ 0.01; N = 3) (Fig. 5B). Among these, si-CLEC7A-1 demonstrated the highest transfection efficiency. Therefore, subsequent experiments were performed using the si-CLEC7A-1. In the present study, we used a co-culture system of HT22 and BV2 cells to investigate the role of CLEC7A-silenced microglia in neurons. Compared to the co-culture system of the Control group (N = 3; 98.72 $\pm$ 1.77), the viability of HT22 cells was significantly inhibited in the OGD/R group (49.65 $\pm$ 3.14, ***p 0.001). Compared to the co-culture system of OGD/R+si-NC group (N = 3; 49.25 $\pm$ 2.23), the viability of HT22 cells significantly increased after CLEC7A knockdown in BV2 cells (79.26 $\pm$ 4.26, ***p 0.001) (Fig. 5C). ELISA results (N = 3) demonstrated that the expression levels of IL-1$\beta$ (596.50 $\pm$ 34.51, ***p 0.001), IL-18 (423.57 $\pm$ 8.11, ***p 0.001), TNF-$\alpha$ (432.70 $\pm$ 19.33, ***p 0.001), and LDH (31.77 $\pm$ 4.32, ***p 0.001) were significantly increased in the co-culture system of HT22 cells and BV2 cells of OGD/R group than those in Control group (161.81 $\pm$ 7.17, 150.62 $\pm$ 17.21, 127.26 $\pm$ 11.91, and 12.04 $\pm$ 1.72, respectively) (Fig. 5D,E). The expression levels of IL-1$\beta$ (310.54 $\pm$ 17.26, ***p 0.001), IL-18 (280.92 $\pm$ 23.31, ***p 0.001), TNF-$\alpha$ (229.49 $\pm$ 10.98, ***p 0.001), and LDH (18.53 $\pm$ 2.99, ***p 0.001) in HT22 cells were significantly lower in the co-culture system of OGD/R+si-CLEC7A group than those in OGD/R+si-NC group (610.28 $\pm$ 41.57, 432.81 $\pm$ 30.70, 442.74 $\pm$ 38.88, and 31.45 $\pm$ 3.36, respectively) (Fig. 5D,E). WB revealed that the relative expression of GSDMD (2.55 $\pm$ 0.19, ***p 0.001), Caspase-1 (3.61 $\pm$ 0.20, ***p 0.001), and NLRP3 (2.60 $\pm$ 0.19, ***p 0.001) was substantially increased in HT22 cells (N = 3) of the OGD/R group, compared to the Control group. In the co-culture system of HT22 cells and BV2 cells (N = 3), the expression of GSDMD (1.62 $\pm$ 0.12, ***p 0.001), Caspase-1 (1.97 $\pm$ 0.04, ***p 0.001), and NLRP3 (1.50 $\pm$ 0.04, ***p 0.001) was markedly decreased in HT22 cells of the OGD/R+si-CLEC7A group, compared to OGD/R+si-NC group (2.50 $\pm$ 0.21, 3.46 $\pm$ 0.22, and 2.57 $\pm$ 0.15, respectively) (Fig. 5F).

Fig. 5.

CLEC7A knockdown in BV cells promotes viability and inhibits pyroptosis of HT22 cells after oxygen-glucose deprivation/reperfusion (OGD/R) model. (A) The transfection efficiency was detected by RT-qPCR. (B) The transfection efficiency was detected by western blot. (C) Cell counting kit-8 (CCK-8) was used to detect cell viability of HT22 cells. (D) The level of lactate dehydrogenase (LDH) was detected by enzyme-linked immunosorbent assay (ELISA). (E) The levels of interleukin (IL)-1$\beta$, IL-18, and tumor necrosis factor-$\alpha$ (TNF-$\alpha$) were detected by ELISA. (F) Western blot detected the expression of NLRP3, caspase-1, GSDMD, and Iba-1. ^Δ⁢Δ⁢Δp 0.001 vs. si-NC group; ***p 0.001 vs. Control group; ^#⁢#p 0.01 vs. OGD/R-si-NC; ^#⁢#⁢#p 0.001 vs. OGD/R-si-NC; N = 3. CLEC7A, C-type lectin domain family 7 member A.

TTC staining revealed that CLEC7A knockdown significantly reduced the infarction caused by cerebral ischemia (N = 6, 15.00 $\pm$ 4.01), relative to the MCAO/R+AAV-NC (adeno-associated virus- negative control) group (34.14 $\pm$ 4.76, ***p 0.001) (Fig. 6A,B). Additionally, CLEC7A knockdown significantly reduced the brain water content of MCAO/R treated rats (N = 6, 80.53 $\pm$ 1.32), compared with the MCAO/R+AAV-NC group (86.12 $\pm$ 1.26, ***p 0.001) (Fig. 6C). The assessment of neurological function in rats further indicated that the MCAO/R+AAV-CLEC7A group (N = 6, 0.83 $\pm$ 0.75) had notably reduced neurological scores in comparison to the MCAO/R+AAV-NC group (2.67 $\pm$ 0.52, ***p 0.001) (Fig. 6D). Representative images of HE staining (Amplification: 200$\times$, Scale: 100 µm; N = 6) revealed that the injury of brain tissues in the MCAO/R+AAV-CLEC7A group was alleviated compared to the MCAO/R+AAV-NC group (Fig. 6E). Furthermore, TUNEL-positive neurons were observed in the ischemic areas after 24 h of reperfusion (N = 6, 41.10 $\pm$ 6.32), compared to the Sham group (1.00 $\pm$ 0.50, ***p 0.001). Compared with the MCAO/R+AAV-NC group (N = 6, 41.80 $\pm$ 3.10), the TUNEL-positive neurons were considerably reduced in the MCAO/R+AAV-CLEC7A group (20.03 $\pm$ 4.20, ***p 0.001) (Fig. 6F).

Fig. 6.

Knockdown of CLEC7A alleviates ischemic stroke in MCAO/R model. (A) TTC staining. Scale: 5 mm. (B) Infarct volume. (C) Brain water content. (D) Neurological deficit score. (E) Representative images of HE staining (Amplification: 200$\times$, Scale: 100 µm). (F) Terminal Deoxynucleotidyl Transferase-mediated Nick End Labeling staining, Scale: 100 µm. ***p 0.001 vs. Control group; ^#⁢#⁢#p 0.001 vs. OGD/R-si-NC; N = 6. AAV, adeno-associated virus.

The concentrations of IL-1$\beta$ (354.60 $\pm$ 26.60, ***p 0.001), IL-18 (272.94 $\pm$ 9.16, ***p 0.001), TNF-$\alpha$ (260.77 $\pm$ 20.68, ***p 0.001), and LDH (298.47 $\pm$ 30.21, ***p 0.001) were apparently lower in rats of the MCAO/R+AAV-CLEC7A group compared with those in the MCAO/R+AAV-NC group (632.45 $\pm$ 41.78, 430.04 $\pm$ 18.29, 440.00 $\pm$ 24.33, and 415.33 $\pm$ 15.41, respectively) (Fig. 7A,B). The intensity of Iba-1 immunofluorescence in the MCAO/R+AAV-CLEC7A group (2.00 $\pm$ 0.25; N = 6) was markedly reduced compared to the MCAO/R+AAV-NC group (6.12 $\pm$ 0.41, ***p 0.001), indicating a reduction of activated microglia (Fig. 7C). WB analysis further revealed that the relative expression levels of the microglial marker, Iba-1 (1.63 $\pm$ 0.07, ***p 0.001) and pyroptosis-related proteins, GSDMD (1.82 $\pm$ 0.01, ***p 0.001), Caspase-1 (2.36 $\pm$ 0.10, ***p 0.001), and NLRP3 (2.86 $\pm$0.04, ***p 0.001) in the brain tissue were markedly reduced in MCAO/R+AAV-CLEC7A group, compared with the MCAO/R+AAV-NC group (2.55 $\pm$ 0.12, 2.40 $\pm$ 0.19, 3.09 $\pm$ 0.16, and 3.87 $\pm$ 0.37, respectively) (Fig. 7D).

Fig. 7.

Knockdown of CLEC7A inhibits pyroptosis and microglia activation in MCAO/R model. (A) The levels of IL-1$\beta$, IL-18, and TNF-$\alpha$ were detected by ELISA. (B) The level of LDH was detected by ELISA. (C) The expression of Iba-1 was detected by immunofluorescence staining, Scale: 20 µm. (D) Western blot detected the expression of NLRP3, caspase-1, GSDMD, and Iba-1. ***p 0.001 vs. Control group; ^#⁢#p 0.01 vs. OGD/R-si-NC; ^#⁢#⁢#p 0.001 vs. OGD/R-si-NC; N = 6.

The CCK-8 assay results showed a significant promotion of viability of HT22 cells in the OGD/R+si-CLEC7A group (83.08 $\pm$ 3.56; N = 3), compared to the OGD/R group (47.69 $\pm$ 2.31, ***p 0.001). However, the addition of Nigericin led to a noticeable decrease in cell viability in the OGD/R+si-CLEC7A+Nigericin group (67.67 $\pm$ 2.36; N = 3), compared to the OGD/R+si-CLEC7A group (83.08 $\pm$ 3.56, ***p 0.001) (Fig. 8A). Knockdown of CLEC7A in BV2 cells resulted in significant reductions in the expression levels of LDH (15.94 $\pm$ 1.80, ***p 0.001), TNF-$\alpha$ (224.39 $\pm$ 20.08, ***p 0.001), IL-1$\beta$ (224.39 $\pm$ 20.08, ***p 0.001), and IL-18 (246.61 $\pm$ 22.08, ***p 0.001) in HT22 cells (N = 3), compared to the OGD/R group (29.41 $\pm$ 1.46, 432.33 $\pm$ 19.09, 432.33 $\pm$ 19.09, and 423.23 $\pm$ 8.15, respectively). However, the introduction of a pyroptosis activator triggered a significant increase in the levels of LDH (23.68 $\pm$ 3.32, ***p 0.001), TNF-$\alpha$ (363.12 $\pm$ 21.24, ***p 0.001), IL-1$\beta$ (363.12 $\pm$ 21.24, ***p 0.001), and IL-18 (349.30 $\pm$ 16.68, ***p 0.001), compared to the OGD/R+si-CLEC7A group (Fig. 8B,C). Additionally, we observed a marked decrease in the relative expression of pyroptosis-related proteins: NLRP3 (2.31 $\pm$ 0.06, ***p 0.001), Caspase-1 (1.33 $\pm$ 0.03, ***p 0.001), and GSDMD (1.25 $\pm$ 0.06, ***p 0.001) in the OGD/R+si-CLEC7A group, relative to the OGD/R group (N = 3; 3.40 $\pm$ 0.20, 2.10 $\pm$ 0.04, and 2.18 $\pm$ 0.04, respectively). Conversely, the addition of Nigericin resulted in a considerable increase in the expression of NLRP3 (3.02 $\pm$ 0.09, ***p 0.001), Caspase-1 (1.64 $\pm$ 0.02, ***p 0.001), and GSDMD (1.71 $\pm$ 0.07, ***p 0.001), relative to the OGD/R+si-CLEC7A group (Fig. 8D).

Fig. 8.

CLEC7A knockdown in BV cells promotes viability of HT22 cells after OGD/R by inhibiting pyroptosis. (A) CCK-8 was used to detect cell viability of HT22 cells. (B) The level of LDH was detected by ELISA. (C) The levels of IL-1$\beta$, IL-18, and TNF-$\alpha$ were detected by ELISA. (D) Western blot detected the expression of NLRP3, caspase-1, and GSDMD. ***p 0.001 vs. Control group; ^#p 0.05, ^#⁢#p 0.01, ^#⁢#⁢#p 0.001 vs. OGD/R group; ^&p 0.05, ^&⁣&&p 0.001 vs. OGD/R+si-CLEC7A group; N = 3.