Exploring the molecular mechanism of danshensu targeting PANoptosis therapy for stroke based on multi omics and experimental verification.

BACKGROUND: One of the main factors aggravating nerve damage following a stroke is the interplay between inflammatory response and cell death. The significance of PANoptosis, a mixed kind of cell death that includes necroptosis, pyroptosis, and apoptosis, in the pathophysiological process of stroke remains unknown. AIM: The objective of this work is to systematically identify the core genes of PANoptosis associated with stroke by multi-omics analysis and to investigate the intervention mechanism of Danshensu, the active ingredient of salvianolic acids. METHODS: We combine the GSE16561 and GSE22255 transcriptome datasets for differential expression analysis, WGCNA, and immune infiltration evaluation; we examine cell heterogeneity and communication networks using the GSE174574 single-cell data; we employ network pharmacology and molecular docking to predict Danshensu's potential targets; finally, we use the BV-2 microglial cell OGD/R model for experimental validation with ZBP1 siRNA knockdown as a functional control. RESULTS: A total of 10 PANoptosis core genes (ZBP1, CASP8, TNF, SIRT1, MAPK3, MAPK1, TLR4, NLRP3, MLKL, HMGB1) were identified, mainly enriched in necroptosis, TNF signaling, and cytoplasmic DNA sensing pathways. Immune infiltration showed significant infiltration of neutrophils and M0 macrophages in stroke patients, and single-cell data confirmed that macrophages/microglia are the hub of TNF and MIF signaling networks. Network pharmacology screening identified seven potential targets of Danshensu (TLR4, NLRP3, SIRT1, MAPK1, MAPK3, TNF, CASP8). Molecular docking predicted that Danshensu has binding energies to TNF, MAPK1, and NLRP3 of less than -5 kcal/mol. In vitro experiments confirmed that Danshensu significantly inhibits OGD/R-induced upregulation of ZBP1 and its downstream effectors CASP8, NLRP3, and MLKL at the protein level, reverses SIRT1 downregulation and TLR4/ERK phosphorylation, and exhibits effects similar to ZBP1 siRNA knockdown. CONCLUSION: Our findings suggest that Danshensu may control macrophage inflammatory responses and programmed cell death-related signaling by regulating the PANoptosis-related signaling network, thereby modulating the immunological milieu following a stroke. This study provides novel molecular targets and a theoretical foundation for stroke treatment, although further validation with activation-state markers (e.g., p-MLKL, cleaved CASP8) is warranted.