Background: The pathophysiological progression during the hyperacute phase of acute ischemic stroke (AIS) critically determines clinical outcomes. Identification of phase-specific biomarkers and elucidation of their temporal regulatory mechanisms are pivotal for optimizing therapeutic interventions.

Methods: Disease signature genes and their mechanisms of action were screened based on the Gene Expression Omnibus database. This involved the use of differentially expressed gene screening, weighted gene co-expression network analysis, Mfuzz analysis, Gene Ontology, Kyoto Encyclopedia of Genes and Genomes enrichment analysis, support vector machines, random forest algorithms, and gene set enrichment analysis. The expression of disease-characteristic genes and their related mechanisms were further validated in both in vivo and in vitro models.

Results: Six hyperacute-phase signature genes (Pip5k1c, Nlgn2, Fzd2, Cd86, Agpat1, and Degs2) were identified in the hyperacute phase of AIS. In light of the gene effect mechanism, the regulation of the neuroinflammatory response and apoptosis by the TLR2/TLR4/NF-κB pathway was monitored in the hyperacute phase of AIS at three times: 3, 6, and 12 h. The results indicated a progressively intensified neuroinflammatory response and the fluctuating growth of early apoptosis changes.

Conclusion: This study systematically identifies hyperacute-phase-specific biomarkers in AIS and delineates their temporal regulatory logic. The time-course dynamics of neuronal apoptosis and inflammatory regulation in the hyperacute phase of AIS were monitored. The observed biphasic apoptotic pattern provides mechanistic insights for developing chronologically targeted therapies, such as timed inhibition of TLR4/CD86 during 0–3 h to block inflammatory initiation, or administration of Agpat1 agonists at 3–6 h to stabilize mitochondrial function. These findings help alleviate the current ‘molecular blind spot’ in early stroke diagnosis and intervention.