Study on pyroptosis-related genes Casp8, Gsdmd and Trem2 in mice with cerebral infarction

Data acquisition

GSE137482 and GSE93376 were downloaded from the GEO. RNA-SEQ of the brain tissues from 24 mice were included in GSE137482: including 12 mice in the middle cerebral artery occlusion model group (MCAO) and 12 mice in the sham operation group (Sham). There were the brain tissues of six mice in GSE93376: three MCAO models and three sham operation samples. Finally, a total of 30 samples (15 MCAO models and 15 sham operation samples) were used for bioinformatics analysis.

DEGs analysis

The DEGs between the Sham group and the MACO group (mice aged 3 and 18 months) were comparatively analyzed using the DESeq2 software (—logFC—>1, padj <0.05) in GSE137482. Next, Venn diagram analysis was performed on the down- and up-regulated genes from mice aged 3 and 18 months (Fig. 1C and 1D), respectively, and the common DEGs were obtained.

GO and KEGG analysis

ClusterProfiler software is a general enrichment tool for interpreting omics data (Yu et al., 2012; Wu et al., 2021), and we used it to analyze the GO and KEGG enrichment pathways of the DEGs.

GSEA

GSEA was performed on mice aged 3 and 18 months using the GSEA 4.1.0 software.

Identification of Pyroptosis-related Genes

Thirty-five genes in the pyroptosis Gene Ontology Term (GO:0070269) was downloaded from the GO database (Table S1). The Venn plot software was used for the intersection of 35 genes in GO:0070269 and DEGs in GSE137482 to obtain distinctively expressed pyroptosis-related genes. Simultaneously, the PPI network analysis of the pyroptosis-related genes was performed using STRING. Finally, the distinctively expressed pyroptosis-related genes associated with cerebral infarction were confirmed.

Establishment of Mouse MCAO Model

Twenty C57BL/6J mice (23–25 g), aged 8–10 weeks (Nanjing Institute of Biomedical Sciences, Nanjing University), divided into two groups according to the random number table method: the MCAO model group and the sham operation group (n = 6). Finally, a total of 12 samples (6 MCAO models and 6 sham operation samples) were used for experimental verification. The mice were kept in an SPF grade experimental animal room with a 12-hour cycle light system, a humidity of 50 ± 5%, and a temperature of 24 ± 2 °C. Before the experiment, the mice were fed adaptively for one week, and had free eating and drinking during this period. The mouse MCAO model was constructed according to the Longa method (Longa et al., 1989), and the successful establishment of the MCAO model was judged by evaluating the neurological function of the mice. The sham operation group served as the control group, and mice in this group underwent the same surgical procedures, excluding middle cerebral artery occlusion. Neurological examination is divided into 5 levels: 0 points: normal; 1 point: The left front paw cannot be fully extended; 2 points: When walking, rotate the mouse towards the paralyzed side; 3 points: When walking, the mouse body tilts towards the paralyzed side; 4 points: Unable to walk independently and lose consciousness. The mice with a score of 1–4 were included in the MCAO model group, and mice with a score of 0 were enrolled in the sham operation group. After the experiment, the mice were put into a clean container, where carbon dioxide was slowly introduced, reaching a final concentration of 60%. The incremental rise in carbon dioxide concentration induced painless euthanasia of the mice. The animal experiments in this research were approved by the Animal Care and Use Committee (IACUC) of the Laboratory Animals Committee of Zhejiang Province (ZJCLA) (approval number: ZJCLA-IACUC-20050029).

Cell culture and grouping

Mouse microglia BV-2 (CL-0493B; Pricella) were cultured in high-glucose DMEM medium (12491015; Thermo Fisher Scientific, Waltham, MA, USA) containing 10% FBS in a 5% CO₂, 37 °C incubator. The cells were exposed to oxygen-glucose deprivation (OGD) for cell model preparation. Cells in the OGD group were cultured with sugar-free DMEM medium (ThermoFisher, #12491015) in a hypoxic incubator containing a pre-mix of gases (94% N₂, 1% O₂, 5% CO₂) at 37 °C. Control group cells cultured under normal conditions (95% air, 5% CO₂).

Interference with Trem2 expression

BV2 cells were seeded into 12-well plates at a density of 1. 4 ×10⁵/well, and then incubated with jetPRIME^® transfection reagent (Polyplus, #101000001) and siRNA oligonucleotide of Trem2 (Sangon Biotech, Shanghai, China) for 6 h, followed by a change of DMEM culture medium after 24 h. si-NC was constructed as a negative control.

TTC Staining

Twelve mice were euthanized after 3 days, and their brain tissues were obtained after dissection. Cerebral infarction was identified by TTC staining. The brain tissue sections from the MCAO group were prepared and incubated in 1% TTC solution. The slices were turned over to ensure even staining. Finally, organize fixation with 4% paraformaldehyde and photographed for image collection.

qRT-PCR Assay

Total RNA was obtained from the brain tissues of mice using the Trizol kit (Invitrogen, Ca, USA). cDNA was further synthesized and qRT-PCR was performed. The gene expression levels of Casp8, Gsdmd and Trem2 were calculated by the 2^−ΔΔCT method, and the calculation was repeated three times.

Primer sequence: Casp8 Forward primer (F): 5′-TGCCCTCAAGTTCCTGTGCTTGGAC-3′, Reverse primer (R): 5′-GGATGCTAAGAATGTCATCTCC-3′; Gsdmd F: 5′-GTGCCTCCACAACTTCCTGA-3′, R: 5′-GTCTCCACCTCTGCCCGTAG-3′; Trem2 F: 5′-CTACCAGTGTCAGAGTCTCCGA-3′, R: 5′-CCTCGAAACTCGATGACTCCTC-3′; β-actin F: 5′-CTAGGCACCAGGGTGTGAT-3′, R: 5′-TGCCAGATCTTCTCCATGTC-3′.

Western Blot Assay

The brain tissues of mice were collected and lysed with RIPA solution. After electrophoresis, the samples were transferred to PDVF membrane, sealed with 5% skim milk for 1 h, and reacted with primary antibody (NLRP3, 1:1000, Casp8, 1:1000, Gsdmd, 1:1000, Trem2, 1:1000, Cleaved caspase-1, 1:1000, GAPDH, 1:1000, Cell Signaling Technology, USA) at 4 °C overnight. After cleaning, the samples were reacted with goat anti-mice IgG (1:3000; Cell Signaling Technology, Danvers, MA, USA) at room temperature for 1 h.

ELISA

After collecting the cell culture supernatant, the expression levels of IL-6, TNF-α and IL-1β were detected by ELISA kit. For details, see the kit instructions.

Statistical analysis

IBM SPSS Statistics 27 was applied for student’s t-test, and P < 0.05 was considered statistically remarkable.

Screening of DEGs

In this study, the DESeq2 software was used to analyze the DEGs of 3-month-old mice between the MACO group and the Sham group in GSE137482, and 1,844 DEGs were found, including 125 down-regulated and 1,719 up-regulated DEGs (Fig. 1A). We comparatively analyzed the DEGs of 18-month-old mice between the MACO group and the Sham group, and 2,527 DEGs were screened out, including 337 down-regulated and 2,190 up-regulated DEGs (Fig. 1B). Meanwhile, Venn diagram analysis showed that there were total 34 down-regulated and 1,483 up-regulated DEGs by intersection of DEGs from mice aged 3 and 18 months (Fig. 1C and 1D).

GO and KEGG

In order to explore the potential biological functions of DEGs, GO and KEGG analyses were performed on the up- and down-regulated DEGs. The up-regulated DEGs were revealed to be enriched in pathways of Human T-cell Leukemia virus 1 infection, T cell activation, positive regulation of cytokine production, Epstein-Barr virus infection, and ECM-receptor interaction (Fig. 2A and 2B). At the same time, down-regulated DEGs were found to be enriched in positive regulation of camp-mediated signaling, cellular response to interleukin −1, and Apelin signaling (Fig. 2C and 2D).

GSEA

In addition, for further investigate the functions and effects of DEGs, the GSEA 4.1.0 software was used to conduct GSEA on genes. GSEA showed that genes from MCAO mice aged 3 and 18 months were significantly enriched in pyroptosis (Fig. 3A and 3B).

Identification of differentially expressed pyroptosis-related genes

To identify the distinctively expressed pyroptosis-related genes, Venn plot software was used to analyze up-regulated DEGs in GSE137482 and 35 genes in the pyroptosis Gene Ontology Term (GO:0070269), and seven distinctively expressed pyroptosis-related genes were obtained accordingly (Fig. 4A). Meanwhile, a PPI network with 15 edges and seven nodes was obtained (Fig. 4B). In addition, these seven genes (Aim2, Casp8, Gsdmd, Naip2, Naip5, Naip6 and Trem2) were up-regulated in MCAO mice aged 3 and 18 months (Fig. 4C and 4D).

Verification of differentially expressed pyroptosis-related genes in GSE93376

At the same time, we verified the differential expression of seven pyroptosis-related genes (Aim2, Casp8, Gsdmd, Naip2, Naip5, Naip6 and Trem2) based on GSE93376. It was revealed that Casp8, Gsdmd and Trem2 were significantly up-regulated in MCAO mice, while no significant differences were observed for the remaining four genes (Fig. 5).

Differentially expressed pyroptosis-related genes were up-regulated in MCAO mice

In addition, mouse MCAO models were constructed to verify the expression of Casp8, Gsdmd and Trem2. The evaluation of neurologic function and TTC staining results showed that the mouse MCAO model was successfully built (Figs. 6A and 6B). Moreover, the inflammatory factor TNF-α as well as the pyroptosis-related proteins were both increased in the MCAO group (Figs. 6C and 6D). Finally, Western blot and qRT-PCR analysis both showed that the expression levels of Casp8, Gsdmd and Trem2 were significantly higher in the MCAO group than those in the Sham group (Figs. 6E and 6F). Taken together, pyroptosis-related genes Casp8, Gsdmd, and Trem2 exhibited higher expression levels in the brain tissues of mice with cerebral infarction.

Differentially expressed pyroptosis-related genes were up-regulated in BV2 cells

Previous studies have already identified Casp8 and Gsdmd as marker proteins for pyroptosis, so we chose Trem2 for follow-up studies. Trem2 has been shown to be primarily expressed in microglia, which are known to play an important role in ameliorating the progression of cerebral infarction. In vitro, BV2 microglia were subjected to OGD treatment, resulting in increased expression of Trem2 (Figs. 7A and 7B). We found a significant increase in Trem2 in the OGD group, consistent with the results obtained from both previous bioinformatics analyses and animal–level validations. Compared with the Control group, BV2 cells in the OGD group exhibited shortened processes, round or rod-shaped cell morphology, and enlarged cell bodies, indicative of microglial activation (Fig. 7C). Additionally, the BV2-secreted inflammatory factors IL-1β, TNF-α, and IL-6 increased (Fig. 7D), along with a significant increase in pyroptosis-related proteins in the OGD group (Fig. 7E). In both the OGD+si-NC group and the OGD+si-Trem2 group (Fig. 7C), BV2 activation was observed. Compared with the OGD+si-NC group, the OGD+si-Trem2 group demonstrated significantly increased secretion of inflammatory factors IL-1β, TNF-α, and IL-6, as well as pyroptosis-related proteins (Fig. 7D and 7E). In OGD-treated BV2 cells, both the expression of Trem2 and the levels of pyroptosis increased. Upon knockdown of Trem2 expression in OGD-treated BV2 cells, the levels of pyroptosis further increased. Therefore, we conclude that Trem2 is a protective factor that regulates pyroptosis, thus impacting the progression of cerebral infarction.