<p>Perioperative neurocognitive disorders (PND) are common in elderly patients, with emerging evidence linking their pathogenesis to abnormal cell death. PANoptosis is a newly identified inflammatory form of programmed cell death, but its role in PND remains unknown. Here, we employed animal behavioral tests, single-cell sequencing (scRNA-seq), methylated RNA immunoprecipitation-sequencing (meRIP-seq), RNA sequencing (RNA-seq), immunofluorescence, and TUNEL staining to investigate the role and underlying mechanisms of PANoptosis in PND. Y-maze and open-field experiments confirmed cognitive and locomotor deficits in a PND mouse model. scRNA-seq revealed altered cellular composition and neuronal heterogeneity in PND brain tissues, with elevated PANoptosis-related gene expression and scores in PND neurons. In vivo and in vitro assays confirmed increased expression of PANoptosis markers (ASC, AIM2, Pyrin) and neuronal apoptosis in PND. Based on the differentially expression gene (DEG) analysis conducted using scRNA-seq, we identified NSUN6 as a key gene that was significantly downregulated in neurons of the PND group, and overexpression of NSUN6 suppressed isoflurane-induced neuronal PANoptosis. RNA-seq and meRIP-seq further revealed that NSUN6 promoted m5C modifications enriched in CDSs and 3′ UTRs, with pathway analysis implicating PANoptosis-associated signaling. OAS2 was identified as a direct substrate of NSUN6-mediated m5C modification, with NSUN6 overexpression enhancing both its m5C enrichment and expression. Collectively, our findings suggest that NSUN6 may be involved in OAS2-associated m5C modification and neuronal PANoptosis, supporting a potential role for the NSUN6/OAS2 axis in the pathogenesis of PND.</p>