Intracranial aneurysm (IA) is a severe cerebrovascular disease, and effective gene therapy and drug interventions for its treatment are still lacking. Oxidative stress (OS) is closely associated with the IA, but the key regulatory genes involved are still unclear. Through multiomics analysis and experimental validation, we identified two diagnostic markers for IA associated with OS. In this study, we first analyzed the IA dataset GSE75436 and conducted a joint analysis of oxidative stress-related genes (ORGs). Differential analysis, functional enrichment analysis, immune infiltration, WGCNA, PPI, LASSO, and other methods were used to identify IA diagnostic markers related to OS. Next, the functions of TLR4 and ALOX5 expression in IA and their potential targeted therapeutic drugs were analyzed. We also performed single-cell sequencing of patient IA and control (superficial temporal artery, STA) tissues. 23,342 cells were captured from 2 IA and 3 STA samples obtained from our center. Cell clustering and annotation were conducted using R software to observe the distribution of TLR4 and ALOX5 expression in IAs. Finally, the expression of TLR4 and ALOX5 were validated in IA patients and in an elastase-induced mouse IA model using experiments such as WB and immunofluorescence. Through bioinformatics analysis, we identified 16 key ORGs associated with IA pathogenesis. Further screening revealed that ALOX5 and TLR4 were highly expressed to activate a series of inflammatory responses and reduce the production of myocytes. Methotrexate (MTX) may be a potential targeted drug. Single-cell analysis revealed a notable increase in immune cells in the IA group, with ALOX5 and TLR4 primarily localized to monocytes/macrophages. Validation through patient samples and mouse models confirmed high expression of ALOX5 and TLR4 in IAs. Bioinformatics analysis indicated that ALOX5 and TLR4 are the most significant ORGs associated with the pathogenesis of IA. Single-cell sequencing and experiments revealed that the high expression of ALOX5 and TLR4 are closely related to IA. These two genes are promising new targets for IA therapy.

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Background: Intracranial aneurysm (IA) is the most common cerebrovascular disease, and subarachnoid hemorrhage caused by its rupture can seriously impede nerve function. Pyroptosis is an inflammatory mode of cell death whose underlying mechanisms involving the occurrence and rupture of IAs remain unclear. In this study, using bioinformatics analysis, we identified the potential pyroptosis-related genes (PRGs) and performed their inflammatory response mechanisms in IAs. Methods: The mRNA expression matrix of the IA tissue was obtained from the Gene Expression Omnibus database, and 51 PRGs were obtained from previous articles collected from PubMed. The differentially expressed PRGs (DEPRGs) were performed using R software. Subsequently, we performed enrichment analysis, constructed a protein–protein interaction network, performed weighted gene coexpression network analysis (WGCNA) and external validation using another dataset, and identified a correlation between hub genes and immune cell infiltration. Finally, the expression and tissue distribution of these hub genes in IA tissues were detected using Western blotting and immunohistochemical (IHC) staining. Results: In total, 12 DEPRGs associated with IA were identified in our analysis, which included 11 up-regulated and one down-regulated genes. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses revealed that the DEPRGs were mostly enriched in the NOD-like receptor signaling pathway, interleukin-1 beta production, and the inflammasome complex. Three hub genes, NLRP3, IL1B and IL18, were identified using Cytoscape software and the WGCNA correlation module, and external validation revealed statistically significant differences between the expression of these hub genes in the ruptured and unruptured aneurysm groups (p < 0.05). Furthermore, all AUC values were > 0.75. Immune cell infiltration analysis suggested that the hub genes are related to CD8 T cell, macrophages and mast cells. Finally, IHC staining revealed that the protein levels of these hub genes were higher in ruptured and unruptured IA tissues than in normal tissues (p < 0.05). Conclusion: The results of bioinformatics analysis showed that pyroptosis is closely related to the formation and rupture of IA, and identified three potential hub genes involved in the pyroptosis and infiltration ofcells. Our findings may improve the understanding of the mechanisms underlying pyroptosis in IA.

Background: Vascular smooth muscle cell (VSMC) dysfunction is one of the crucial pathologic processes in the development of intracranial aneurysm (IA). Secreted protein acidic and rich in cysteine (SPARC), a multifunctional glycoprotein, is overexpressed in many tumor, but its underlying mechanism in vascular disease has not been elucidated. The aim of this study is to evaluate the potential function of SPARC in IA generation and regulation of mitochondrial function in VSMC.Methods: Human brain vascular smooth muscle cells were treated with recombinant SPARC to detect apoptosis-related markers. The downstream targets affecting mitochondrial dysfunction after SPARC treatment were explored by transcriptome sequencing and bioinformatics analysis, and verified using by immunohistochemistry and western blot. Further in vitro experiments verified the role of downstream targets in regulating VSMC mitochondrial function.Results: Secreted protein acidic and rich in cysteine (SPARC) expression was associated with the risk of IA rupture. SPARC induces mitochondrial pathway apoptosis in human brain VSMC. We screened 40 differentially expressed genes related to mitochondrial function after SPARC treatment. Hexokinase 2 (HK2) was identified as a downstream target of mitochondrial pathway apoptosis in VSMC induced by SPARC. In addition, immunohistochemical results confirmed that the difference between SPARC and HK2 expression is located mainly in the smooth muscle layer of IA. Overexpression of HK2 reversed the SPARC-induced increase in apoptosis and mitochondrial damage in VSMC.Conclusion: Secreted protein acidic and rich in cysteine (SPARC) regulated mitochondrial function in VSMC and induced apoptosis through HK2, which plays an important role in the formation and rupture of IA. Targeting SPARC may be a novel strategy to delay the development of intracranial aneurysms.