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PubMed2025

Suppression of OTUD4 protects against myocardial ischemia-reperfusion injury by increasing autophagic flux and inhibiting apoptosis in cardiomyocytes.

Dysregulated autophagic flux plays a critical role in myocardial ischemia-reperfusion injury (MIRI), complicating cardiac reperfusion therapy. In this study, we identified OTUD4 as a potential regulator of autophagic flux in MIRI using CRISPR/Cas9 sgRNA sequencing. However, the underlying mechanism is poorly understood. The purpose of this study is to investigate the effects of OTUD4 on autophagic flux in OGD-R treated AC16 cells (IRI model in vitro) and LAD artery ligation induced myocardial ischemia-reperfusion mice (MIRI model in vivo). In the in vitro IRI cell model, OTUD4 knockdown significantly reversed impaired autophagic flux, increased mitochondrial membrane potential, and decreased LDH activity, ROS production, autophagy and apoptosis. Overexpression of OTUD4 showed the opposite result. In the in vivo MIRI model, OTUD4 knockdown also significantly decreased infarct area, improved cardiac structure and function, reduced serum BNP and LDH levels, attenuated cardiac tissue injury/fibrosis/myocardial hypertrophy, and ultimately exerted myocardial protective effects against ischemia-reperfusion injury. Importantly, OTUD4 knockdown inhibited autophagosome-associated markers (LC3II/LC3I, Beclin1, ATG9), autophagy substrate p62, increased lysosomal activity marker LAMP2, and activated the autophagy pathway (AKT/mTOR), thereby promoting the recovery of impaired autophagic flux in the MIRI model. Moreover, OTUD4 showed strong interaction with UBAC1, and OTUD4 deficiency decreases UBAC1 protein expression by impairing its deubiquitination, thereby regulating autophagy. In short, blocking OTUD4 restored damaged autophagic flux in I/R induced myocardial injury both in vivo and in vitro, inhibited myocardial cell apoptosis, and greatly improved cardiac function in ischemia-reperfusion mice. KEY MESSAGES: OTUD4 was identified as a key negative regulator of autophagy flux in myocardial ischemia-reperfusion injury (MIRI) via genome-wide CRISPR/Cas9 screening. OTUD4 knockdown exerts cardioprotective effects by reducing apoptosis and ROS generation and improving heart function in both in vitro and in vivo models. The interaction between OTUD4 and UBAC1 was confirmed, and OTUD4 maintains UBAC1 stability through deubiquitination, providing new insights into the ubiquitination regulatory mechanism in myocardial injury. Targeting OTUD4 has therapeutic potential for MIRI, as OTUD4 knockdown alleviated MIRI in both in vitro and in vivo models, suggesting the possibility of developing OTUD4 inhibitors for cardiac reperfusion treatment.

Animals↗
PubMed2026

MLL4 protects cardiomyocytes against ischemia-reperfusion injury through STAT3-mediated mitochondrial function.

Myocardial ischemia-reperfusion injury (MIRI) is an inevitable pathophysiological response during the revascularization process following myocardial ischemia. Despite its clinical significance, effective targeted therapies for MIRI remain an unmet medical need. Mixed-lineage leukemia 4 (MLL4), a member of the SET family of histone methyltransferases, exhibits particular methyltransferase action toward histone H3 lysine 4 (H3K4). This study establishes a protective role for MLL4 in MIRI pathogenesis. Utilizing cardiomyocyte-specific Mll4 knockout mice and an in vivo ischemia-reperfusion (I/R) model induced by left anterior descending coronary artery ligation, we observed significant upregulation of MLL4 expression in cardiac tissue following I/R. Genetic ablation of Mll4 in cardiomyocytes markedly exacerbated both acute and chronic phases of MIRI. In vitro, Mll4 knockdown in neonatal rat cardiomyocytes (NRCMs) amplified mitochondrial dysfunction and apoptosis under hypoxia/reoxygenation (H/R) conditions. Integrated analysis of Cleavage Under Targets and Tagmentation sequencing (CUT&Tag-seq) and RNA sequencing (RNA-seq) revealed that Mll4 deficiency induces a pronounced reduction in H3K4 monomethylation (H3K4me1) and histone H3 lysine 27 acetylation (H3K27ac) enrichment at the Stat3 genomic locus. Mechanistically, MLL4 functions as a transcriptional activator of Stat3 by depositing H3K4me1 and H3K27ac, thereby facilitating STAT3 transcription. This regulatory cascade ultimately governs STAT3-dependent mitochondrial homeostasis. Collectively, these findings identify MLL4 as a critical epigenetic regulator of MIRI and suggest its therapeutic targeting may offer a promising strategy for mitigating reperfusion injury.

Animals↗
PubMed2026

Necroptosis in alveolar epithelium orchestrates lung ischemia-reperfusion injury: a multi-omics study.

BACKGROUND: Lung ischemia-reperfusion injury (LIRI) is a leading cause of early morbidity and mortality following lung transplantation and other cardiopulmonary procedures. It is characterized by acute sterile inflammation driven by regulated cell death (RCD). While various RCD modalities, including apoptosis, necroptosis, pyroptosis, and ferroptosis, have been implicated in lung injury, their relative contributions and distinct activation patterns in LIRI remain poorly defined. METHODS: We employed an integrated multi-omics approach combining transcriptomics and proteomics with histological and functional validations in a murine hilar clamping model of LIRI. Key findings were further corroborated using single-cell RNA sequencing (scRNA-seq) data from human lung transplant recipients. The functional role of necroptosis was validated using pharmacological inhibitors (Nec-1, GSK'872) and Mlkl-deficient (Mlkl-/-) mice. RESULTS: LIRI triggered acute, time-dependent lung injury peaking within 24 h of reperfusion. Although transcriptomic profiling suggested broad activation of multiple RCD pathways, proteomic and biochemical analyses revealed a distinct landscape in our experimental setting: markers of apoptosis, pyroptosis, and ferroptosis were either downregulated or showed no significant positive correlation with injury severity and inflammatory peaks. In contrast, the necroptotic pathway emerged as a highly activated modality. Specifically, necroptosis, marked by phosphorylated RIPK1, RIPK3, and MLKL, was localized primarily in alveolar epithelial cells, correlated strongly with cytokine release and histological lung injury, and preceded the inflammatory response. Pharmacological inhibition or genetic ablation of necroptosis significantly attenuated tissue damage and inflammation. This pronounced necroptotic signature appeared distinct from the broad multi-pathway activation observed in lipopolysaccharide (LPS)-induced lung injury. Translational analysis of human scRNA-seq data further confirmed the selective upregulation of necroptosis signatures in alveolar type 2 (AT2) cells following lung transplantation. CONCLUSION: Our multi-omics analysis identifies necroptosis, particularly in alveolar epithelial cells, as a critical driver of sterile inflammation and tissue injury in the early phase of LIRI. Targeting alveolar epithelial necroptosis may represent a precise and promising therapeutic strategy for lung transplantation and ischemia-reperfusion-associated pulmonary disorders.

Animals↗
PubMed2026

Development of Electrocardiography Standards for Evaluating Myocardial Infarction and Ischemia-Reperfusion Injury in Mice.

BACKGROUND: Acute and chronic heart failure secondary to myocardial infarction (MI) and cardiac ischemia-reperfusion injury (IRI) are leading causes of death in ischemic heart disease. A mouse model is indispensable for investigating MI and IRI, and the development of reliable mouse MI and IRI models is essential for advancing research in this field. The clear early diagnostic criteria for confirming successful induction of MI and IRI in mice remain lacking. METHODS: Adult C57BL/6J background mice underwent left anterior descending coronary artery ligation to induce acute MI, or ligation followed by reperfusion to induce IRI. The success of the MI and IRI model establishment was confirmed by 2,3,5-triphenyltetrazolium chloride staining and echocardiography. Electrocardiography was used to monitor the electric activity in the mice. CONCLUSIONS: Electrocardiography demonstrated that ST-segment elevation in ECG lead II and corrected QTc interval prolongation at 30 minutes following left anterior descending ligation as 2 key early indicators of successful MI. Echocardiography analysis revealed that the magnitude of ST-segment elevation strongly correlated with the left anterior descending ligation site, where a more proximal ligation produced a greater ST-segment elevation amplitude and more severe ischemia. In IRI models, ST-segment elevation typically resolved and returned to baseline within 20 minutes of reperfusion. This study developed quantifiable early diagnostic criteria for successful MI and IRI induction based on characteristic ECG changes. These quantifiable ECG parameters provide early diagnostic standards that can significantly streamline and optimize modeling procedures.

Animals↗
PubMed1977

Studies on myocardial reperfusion injury. I. Favorable modification by adjusting reperfusate pH.

This study tests the hypothesis that postischemic myocardial depression can be reduced by providing an initial reperfusate pH which is appropriate for myocardial temperature (i.e., metabolic systems function optimally when pH is kept slightly alkaline to the neutral point, which changes with temperature in concordance with the pK of water). Ten dogs underwent 1 hour of ischemic arrest with topical hypothermia (intramyocardial temperature 16+/-2 degrees C). The initial reperfusate (500 cc of blood from the extracorporeal circuit) was infused (100 cc/minute) into the proximal aorta just before removing the cross-clamp. Reperfusate pH was kept at 7.4 in five dogs (control) and raised to 7.8 with THAM [tris (hydroxymethyl) aminomethane] in five dogs. Measurements 30 minutes after reperfusion showed that raising reperfusate pH to 7.8 resulted in (1) higher subendocardial blood flows (109+/-20 vs 61 cc+/-8 cc/100 gm/minute), (2) redistribution of postischemic blood flow toward the subendocardium (endocardial/epicardial flow 1.25+/-0.1 vs 1.0+/-0.03), (3) higher left ventricular oxygen uptakes (0.046 vs 0.033 cc/100 gm/beat), (4) better postischemic left ventricular compliance (56+/-3% more compliant), and (5) improved left ventricular performance (88+/-7% recovery vs only 57+/-3% recovery at pH 7.4). Postischemic edema (2% water gain) was unchanged by pH modification. We conclude that initial reperfusion with the appropriate pH provides an optimal milieu for restoration of cellular metabolism, counteracts the acidosis of ischemia, and improves postischemic left ventricular blood flow, distribution, oxygen uptake, compliance, and performance.

Animals↗
PubMed2026

Human biopsy-defined ischemia-reperfusion injury-selective reperfusion signature prioritizes reperfusion-timed mitogen-activated protein kinase kinase inhibition after donation after circulatory death liver transplantation.

Early post-liver transplant ischemia-reperfusion injury (IRI) in donation after circulatory death grafts lacks therapies targeted to the immediate postreperfusion window, in part because generic reperfusion transcription obscures IRI-selective amplification. We analyzed paired prereperfusion/postreperfusion liver biopsies from 2 cohorts (GSE151648 and GSE87487) using a difference-in-differences interaction estimand (&#x394;&#x394; = [Post-Pre]IRI+ - [Post-Pre]IRI-) to define an IRI-selective early reperfusion program. Genome-wide &#x394;&#x394; effects were summarized using pathway-responsive genes, and pathway concordance was tested using permutation (B = 5000). The reproducible &#x394;&#x394; footprint highlighted epidermal growth factor receptor-mitogen-activated protein kinase signaling (Spearman &#x3c1; = 0.811; P = .001). Directional &#x394;&#x394; gene sets (interaction P < .05) were submitted to the L1000 characteristic direction signature search engine2; cross-cohort overlap identified 8 shared perturbagens, including 3 mitogen-activated protein kinase kinase (MEK)1/2 inhibitors. In a hepatic ischemia/reperfusion time course (GSE117915), epidermal growth factor receptor and mitogen-activated protein kinase activities increased within 0.5 hours of reperfusion, and transplant single-cell RNA sequencing (GSE189539) localized MEK/extracellular signal-regulated kinase pathway engagement predominantly to parenchymal cells. A representative MEK inhibitor, PD-0325901, reduced hepatocyte oxygen-glucose deprivation/reoxygenation injury and, when administered at reperfusion in a rat donation after circulatory death liver transplantation model (5-20 mg/kg), attenuated histologic and biochemical injury, apoptosis, and redox-inflammatory readouts and improved 7-day survival. Collectively, this biopsy-anchored &#x394;&#x394; interaction-phenotype framework, with cross-cohort concordance as a prespecified robustness gate, nominates reperfusion-timed MEK inhibition as a mechanism- and window-aligned strategy to blunt early post-liver transplant IRI.

difference-in-differences (time &#xd7; IRI interac↗
PubMed2026

Transcription Factor SP1 Drives Myocardial Ischemia/reperfusion Injury By Transcription Activation-mediated GADD45G Upregulation.

Myocardial ischemia-reperfusion injury (MIRI) is an unresolved clinically fatal complication in the management of acute myocardial infarction (AMI). Growth arrest and DNA damage-inducible gene 45 Gamma (GADD45G) plays a vital role in the regulation of MIRI. However, the underlying mechanisms remain unclear. GADD45G and SP1 expression were upregulated in hypoxia/reoxygenation (H/R)-treated H9C2 cells. H/R treatment repressed H9C2 cell viability, and induced apoptosis, oxidative stress, and inflammatory response. Moreover, GADD45G deficiency could relieve H/R-triggered H9C2 cell injury. In mechanism, SP1 was a transcription factor of GADD45G and activated the transcription of GADD45G via binding to its promoter region. Besides, SP1 knockdown alleviated MI/R-induced pathological damage in the myocardial tissue of rats by regulating GADD45G. In conclusion, SP1 could promote H/R-induced cardiomyocyte injury and MI/R-caused rat myocardial tissue pathological injury by increasing GADD45G, providing a promising therapeutic target for MIRI treatment.

Animals↗
PubMed2025

TCF25 serves as a nutrient sensor to orchestrate metabolic adaptation and cell death by enhancing lysosomal acidification under glucose starvation.

Cells adapt to nutrient limitation by activating catabolic and inhibiting anabolic pathways, yet prolonged stress may lead to cell death. How cells orchestrate metabolic adaptation and cell death to nutrient stress is poorly understood. We conduct a genome-wide CRISPR-Cas9 screen to identify regulators in glucose-starvation-induced cell death and find a group of genes in lysosomal pathway is enriched following glucose starvation. We focus on one candidate gene, Transcriptional Factor 25 (TCF25). We find TCF25 enhances lysosomal acidification by targeting V-ATPase, promoting autophagy and ATP generation under glucose starvation. However, prolonged glucose starvation constitutively activates ferritinophagy via TCF25, increasing lysosomal membrane permeability (LMP) and leading to lysosome-dependent cell death (LDCD). Knocking out TCF25 or V-ATPase components prevents cell death. Furthermore, TCF25 deficiency protects mice from hepatic ischemia-reperfusion injury. Our findings identify TCF25 as a crucial nutrient sensor that regulates lysosomal activity, offering potential therapeutic targets for metabolic and ischemic disorders.

Lysosomes↗
PubMed2025

The effect of penile tourniquet and continuous artificial erection on penile erectile tissues: An experimental study.

INTRODUCTION: Penile tourniquet (PT) is known to cause ischemic injury, which worsens with prolonged application. Artificial erection (AE), formed by intracorporal saline injection mostly under PT, has been practiced for decades to evaluate penile curvature, yet its effect on erectile tissues has never been investigated. In this study, we examined a modified approach, continuous artificial erection (CAE), and investigated its effects on erectile tissues. OBJECTIVE: This study aims to investigate the histopathological and immunohistochemical effects of CAE on penile erectile tissues. STUDY DESIGN: Thirty-five rats were randomized into five groups. Four experiment groups received 20 or 40 min of isolated PT (20T and 40T) or PT with CAE (20T&E and 40T&E). CAE was achieved through continuous intracavernosal saline injection. Penectomy was performed three weeks post-procedure in the experiment groups and directly in the control group. Erectile tissue samples were evaluated using light microscopy for histopathological parameters including inflammation, neovascularization and fibrosis, and by immunohistochemistry. Endothelial function was assessed by eNOS and e-selectin staining, while ICAM-1 staining was used to assess chronic inflammation. RESULTS: 40T showed the highest levels of inflammation, fibrosis, and endothelial dysfunction. 20T had significantly less inflammation than 40T, with a non-significant increase in fibrosis and alteration of endothelial markers. 40T&E displayed the second-highest fibrosis rate (adjusted p > 0.05), while 20T&E showed complete absence of fibrosis. Both 40T&E and 20T&E preserved strong eNOS and e-selectin expression, identical to controls. ICAM-1 expression in 20T&E was also consistent with the control group. The most significant difference in erectile tissue damage was noted between 40T and 20T&E. CONCLUSION: This is the first study to evaluate the effects of AE on erectile tissues. Findings of this experimental model support that, CAE does not increase the tissue damage that is already caused by PT, but rather reduces it, likely through the washout of blood elements contributing to reperfusion injury. CAE possibly provides a protective effect on erectile tissues by preserving endothelial function, reducing inflammation and fibrosis, especially under 20 minutes of duration. These findings may support that AE maneuvers such as "artificial erection test" and CAE are potentially safe, while further studies are needed to assess the detailed effects of CAE.

Male↗
PubMed2026

Nrsn1-Smarcc1 Coupling Regulates Neural Stem Cell Differentiation and Chronic-phase Recovery After Ischemic Stroke.

Stroke remains a leading cause of long-term neurological disability worldwide, largely due to irreversible neuronal loss and the limited regenerative capacity of the adult mammalian brain. Neural stem cells (NSCs) in the adult brain possess the potential to generate new neurons after injury, yet the molecular mechanisms regulating their neuronal differentiation following ischemic insult remain incompletely understood. Here, integrating single-cell multi-omics analyses with spatial transcriptomics, we systematically delineated cell type-specific spatiotemporal dynamics in the striatum of a mouse model of ischemia-reperfusion injury. We identified Neurensin 1 (Nrsn1) as a gene markedly upregulated during NSC-derived neuronal differentiation in the recovery phase. Mechanistically, Foxa2 directly activates Nrsn1 transcription, whereas Nrsn1 promotes neuronal differentiation by facilitating the nuclear translocation of the chromatin-remodeling factor Smarcc1 in vitro. In vivo, both endogenous NSCs and transplanted NSCs overexpressing Nrsn1 significantly enhanced neuronal regeneration and improved functional recovery in mice subjected to middle cerebral artery occlusion and reperfusion (MCAO/R). Collectively, these findings identify Nrsn1 as a key regulator of NSC neuronal differentiation and uncover a Nrsn1-Smarcc1 coupling mechanism that promotes neural regeneration after ischemic brain injury, highlighting a potential molecular target for strategies aimed at enhancing post-stroke recovery.

Foxa2↗
PubMed2026

Intravenous Nicorandil in Patients With ST-Segment Elevation Myocardial Infarction Undergoing Primary PCI: The CLEAN Randomized Clinical Trial.

BACKGROUND: Nicorandil, an adenosine triphosphate-sensitive potassium-channel opener with nitrate-like properties, may reduce reperfusion injury and microvascular obstruction in ST-segment elevation myocardial infarction (STEMI), but large-scale randomized evidence on long-term clinical outcomes is inconclusive. OBJECTIVES: The CLEAN trial aimed to assess whether adjunctive intravenous nicorandil improves 12-month clinical outcomes in patients with STEMI undergoing primary percutaneous coronary intervention. METHODS: In this multicenter, randomized, double-blind, placebo-controlled trial conducted at 49 hospitals in China, patients aged 18 to 80 years with STEMI within 12 hours of symptom onset were randomly assigned (1:1) to receive intravenous nicorandil (6 mg bolus before reperfusion followed by 6 mg/h infusion for 48 h) or matching placebo. Oral nicorandil was prohibited during follow-up. The primary outcome was a composite of cardiovascular death, nonfatal myocardial infarction, target vessel revascularization, or unplanned hospitalization for heart failure within 12 months. RESULTS: Between January 2021 and December 2023, 1,503 patients were enrolled and randomly assigned to nicorandil (n = 748) or placebo (n = 755). The primary composite outcome occurred in 98 patients (13.1%) in the nicorandil group (113 events over 717.2 person-years) and 99 (13.1%) in the placebo group (136 events over 710.3 person-years), with no significant difference between groups (rate ratio: 0.869; 95% CI: 0.650-1.162; P = 0.3429). Among secondary outcomes, nominal reductions were observed in cardiovascular death (1.9% vs 3.6%; HR: 0.515; 95% CI: 0.269-0.983) and target-vessel revascularization (1.1% vs 3.0%; HR: 0.322; 95% CI: 0.143-0.727), whereas rates of nonfatal myocardial infarction and unplanned hospitalization for heart failure were similar between groups. Adverse events did not differ between groups. CONCLUSIONS: In patients with STEMI undergoing primary percutaneous coronary intervention, adjunctive intravenous nicorandil did not significantly reduce the 12-month primary composite outcome. These findings do not support routine use of intravenous nicorandil in unselected patients with STEMI. (Clinical Efficacy and sAfety of Intravenous Nicorandil; NCT04665648).

Humans↗
PubMed1979

Nifedipine: a myocardial protective agent.

The effectiveness of the calcium antagonist nifedipine in preserving postischemic myocardial function and structural integrity was experimentally demonstrated in isolated rabbit hearts, in conscious dogs subjected to myocardial infarction, in open chest anesthetized dogs with normothermic regional ischemia induced for 1 to 2 hours and in dogs undergoing hypothermic global ischemia for 2 hours followed by 2 hours of reperfusion. Nifedipine had a beneficial effect on postischemic myocardial stiffness and mitochondrial calcium accumulation, which were correlated. Administration of nifedipine at the onset of myocardial infarction increased blood flow to ischemic zones of myocardial infarction and resulted in less loss of creatine kinase. It reduced by two- to three-fold the volume of the ischemia-reperfusion injury induced by left anterior descending coronary arterial occlusion and release and preserved indexes of hemodynamic function. Nifedipine was found effective in protecting myocardial performance and structure after 2 hours of global ischemia during hypothermic cardiopulmonary bypass. It is suggested that this agent may be useful as an adjunct to cold cardioplegia in man for enhanced myocardial protection during cardiac surgery.

Animals↗
PubMed1977

No-reflow phenomenon and acute myocardial ischemia. The need for further investigation.

It has been shown that localized myocardial ischemia is accompanied by microvascular changes which produce capillary obstruction when blood flow is restored. This so-called no-reflow phenomenon has been noted in the brain, kidney, dermis and, more recently, in the myocardium. Ultrastructural studies have pointed out the role of myocardial and endothelial cells swelling. It seems likely that such damage of the vascular bed may constitute the first irreversible change during ischemia and result in failure to obtain successful reperfusion of involved myocardium. It can be suggested on the basis of recent clinical and experimental observations, that this phenomenon may play a role in subendocardial necrosis associated with cardiac surgery, and in myocytolytic necrosis. In both of these conditions, the most salient features are the microcirculatory defect and the reperfusion injury following transient ischemia. Further investigations are needed to determine the basic alterations induced by no-reflow phenomenon and the value of various prophylactic and therapeutic measures.

Animals↗
PubMed2026

Spatiotemporal transcriptomic analysis during cold ischemic injury to the murine kidney reveals compartment-specific changes.

BACKGROUND: Kidney transplantation is the preferred treatment strategy for end-stage kidney disease. Deceased donor kidneys usually undergo cold storage until kidney transplantation, leading to cold ischemia injury that may contribute to poor graft outcomes. However, the molecular characterization of potential mechanisms of cold ischemia injury remains incomplete. RESULTS: To bridge this knowledge gap, we leverage 10x&#x2009;Visium spatial transcriptomic technology to perform full transcriptome profiling of murine kidneys subject to varying durations of cold ischemia typical in a deceased donor kidney transplant setting. We develop a computational workflow to identify and compare spatiotemporal transcriptomic changes that accompany the injury pathophysiology in a tissue compartment-specific manner. We identify proportional enrichment of oxidative phosphorylation (OXPHOS) genes with increasing duration of cold ischemia injury within the oxygen-lean inner medulla region, suggestive of atypical metabolic presentation. This is distinct in cold ischemia injury tissue compared to warm ischemia-reperfusion kidney injury tissue. Spatiotemporal trends are validated by qPCR and immunofluorescence in a larger cohort of mice. CONCLUSIONS: Altogether, our spatiotemporal transcriptomic analysis identifies coordinated molecular changes within metabolic pathways such as OXPHOS deep within the cold ischemic kidney, highlighting the need for increased attention to the inner medulla and potential opportunities for new insights beyond those available from superficial biopsy-focused tissue examination.

Animals↗
PubMed2025

Bioinformatics identification and validation of pyroptosis-related gene for ischemic stroke.

BACKGROUND: Ischemic stroke (IS) is one of the common and frequent diseases with extremely high lethality and disability in the world, and there is no effective treatment at present. This study aimed to screen hub genes involved in cerebral ischemia/reperfusion injury (CIRI) and pyroptosis, and explore promising intervention targets. METHODS: CIRI-related genes (GSE202659 and GSE131193) and pyroptosis-related genes (PRGs) in mice were obtained from the Gene Expression Omnibus (GEO) and GeneCards database. We screened for LASSO regression to construct a prognostic model of GSE131193 and PRGs and examined by GSE137482. The functional enrichment analysis of Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Set Enrichment Analysis (GSEA) and Gene Set Variation Analysis (GSVA) were performed on pyroptosis-related differentially expressed genes (PRDEGs) of GSE202659.The key modules for CIRI and pyroptosis were identified by Weight Gene Co-expression Network Analysis (WGCNA). Subsequently, Protein-protein Interaction (PPI) network and the Cytoscape was constructed to screen out hub genes. Used the starBase to predict miRNA interacting with hub genes and constructed mRNA-miRNA-lncRNA interaction networks. CIRI-related Molecular Subtypes were constructed for hub genes. The relationship between immune cells and hub genes was verified via CIBERSORT. Finally, we selected C57BL/6 mice to construct models to confirm hub genes by enzyme linked immunosorbent assay (ELISA), reverse transcription-polymerase chain reaction (RT-PCR), western blot, and Immunofluorescence. RESULTS: A total of 272 PRGs and 35 PRDEGs were screened. An eight-gene risk prediction models were established (AUC&#x2009;=&#x2009;0.868). GO, KEGG, GSEA and GSVA analyses revealed that PRDEGs were mainly involved in positive regulation of cytokine production, and NOD-like receptor signaling pathway. And then, seven hub genes (Irf1, Icam1, Tlr2, Tnf, Cebpb, Il1rn, and Casp8) were identified by PPI. Icam1, Tnf, Cebpb, Il1rn, and Casp8 had high expression profiles in Cluster2 by hierarchical clustering. The immune infiltration analysis results showed that among the hub genes, Cebpb, Il1rn, and Casp8, showed a significant positive correlation with the degree of NK.Actived, and Icam1 showed a significant negative correlation with B.Cells.Memory. The results of animal experiments significantly demonstrated an upregulation of Irf1, Icam1, Tlr2, Cebpb, and Il1rn. CONCLUSION: Our finding indicated that Irf1, Icam1, Tlr2, Cebpb, and Il1rn are hub genes associated with pyroptosis, and these genes are all associated with different immune cells, so as to provide new targets for the prevention and treatment of IS from the perspective of pyroptosis.

Pyroptosis↗