To investigate the role of dynamin-related protein 1 (Drp1), a mitochondrial fission protein, on neuronal PANoptosis in oxygen-glucose deprivation (OGD) and reoxygenation (OGD/R) models.

SH-SY5Y cells were cultured in vitro, and OGD and OGD/R models were established. The optimal concentration of the Drp1 inhibitor Mdivi-1 was determined by CCK-8 assay. Cells were divided into control group, OGD group, OGD/R group, OGD+Mdivi-1 group, OGD/R+Mdivi-1 group, OGD/R-2 h+Mdivi-1 group, and OGD/R-4 h+Mdivi-1 group. Mdivi-1 (10 μmol/L) was administered at the beginning of OGD and OGD/R models, and at 2 h and 4 h after reoxygenation of OGD/R, respectively. Reactive oxygen species (ROS) were detected using DCFH-DA and Mito-SOX methods. Mitochondrial membrane potential was measured by JC-1 assay. Western blotting was used to detect the expression of mitochondrial fission-related proteins [phosphorylated dynamin-related protein 1 (p-Drp1), mitochondrial fission protein 1 (Fis1), and mitochondrial fission factor (Mff)] and PANoptosis-related proteins [apoptosis: B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), cleaved cysteinyl aspartate specific proteinase 3 (cleaved caspase-3); pyroptosis: NOD-like receptor protein 3 (NLRP3), N-terminal Gasdermin D (GSDMD-N); necroptosis: receptor-interacting protein kinase 3 (RIPK3), phosphorylated mixed lineage kinase domain-like protein (p-MLKL)]. When comparing two groups of data, the independent samples t-test was used if the data were normally distributed and had homogeneity of variance; otherwise, nonparametric test (Mann-Whitney U) was applied. Differences among more than two groups were compared using one-way ANOVA, with LSD-t or Dunnett's T3 test for pairwise comparisons.

Compared with the control group, both OGD/R and OGD injuries significantly induced excessive mitochondrial fission, triggered mitochondrial dysfunction, and concurrently activated apoptotic, pyroptotic, and necroptotic pathways. The relative expression levels of mitochondrial fission and PANoptosis-related proteins induced by OGD (GSDMD-N: 2.70±0.38 vs 2.12±0.26, t=3.761, P=0.044; p-MLKL: 2.83±0.72 vs 1.76±0.20, t=5.049, P=0.009; p-Drp1/Drp1: 4.54±0.94 vs 2.53±0.47, t=3.821, P=0.024; Mff: 1.80±0.14 vs 1.37±0.10, t=5.181, P=0.011) were significantly higher than those in OGD/R. 10 μmol/L Mdivi-1 increased cell viabilities in OGD [(66.79±6.54)% vs (48.60±4.66)%, t=4.527, P=0.025], OGD/R [(76.68±6.55)% vs (59.96±5.18)%, t=3.008, P=0.030], OGD/R-2 h [(86.17±8.95)% vs (59.33±6.90)%, t=4.747, P=0.016], and OGD/R-4 h [(93.91±7.28)% vs (59.33±6.90)%, t=6.892, P=0.001] groups, with statistically significant differences. Intervention with 10 μmol/L Mdivi-1 effectively inhibited excessive mitochondrial fission in both OGD/R and OGD models, and significantly downregulated the expression of key proteins in the pathway.

In OGD and OGD/R models, Drp1-mediated excessive mitochondrial fission is a common critical mechanism inducing neuronal PANoptosis. Inhibiting Drp1 significantly improves mitochondrial function and suppresses the occurrence of neuronal PANoptosis.