Effects of zinc ions on RIPK3-mediated necroptosis and caspase-8-dependent apoptosis following cerebral ischemia-reperfusion in rats

doi:10.3969/j.issn.1006-7795.2026.04.017

Abstract

Abstract: Objective To establish a rat middle cerebral artery occlusion (MCAO) model and investigate the protein expression and cellular localization of receptor-interacting serine/threonine-protein kinase 3 (RIPK3) and cleaved caspase-8, as well as the expression of B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) in the ischemic brain tissue 6 h and 24 h following reperfusion. Additionally, zinc chelator N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) was administered to investigate the effects of zinc ions on the expression of RIPK3, cleaved caspase-8 and Bcl-2/Bax at 6 h and 24 h after reperfusion, providing novel theoretical insights into the mechanisms of zinc ion-mediated cerebral ischemia-reperfusion injury.Methods Adult male Sprague-Dawley rats were randomly allocated to Sham, MCAO, and TPEN + MCAO groups. MCAO model was established by intraluminal filament technique, with 90 min of occlusion followed by reperfusion for 6 h and 24 h. The ischemic brain tissue was separated and Western blotting was used to quantify the protein expression of RIPK3, cleaved caspase-8, Bcl-2 and Bax in the ischemic hemisphere. The co-localization of RIPK3 and cleaved caspase-8 with neuronal marker NeuN was observed through immunofluorescence staining.Results ① Compared with the Sham group, the expression of RIPK3 was increased significantly in the ischemic brain tissues of MCAO group rats at 6 h and 24 h after reperfusion (P<0.05), and RIPK3 was co-localized with the neuronal marker NeuN. Compared with the MCAO group, TPEN treatment markedly decreased the expression of RIPK3 at 6 h (P<0.05) and 24 h after reperfusion. ② Compared with the Sham group, the expression of cleaved caspase-8 in the ischemic brain tissues of MCAO group rats was increased at 6 h and 24 h after reperfusion and co-localized with NeuN. Compared with the MCAO group, TPEN treatment markedly downregulated the expression of cleaved caspase-8 at 6 h (P<0.05) and 24 h after reperfusion. ③ Compared with the Sham group, the ratio of Bcl-2/Bax was decreased significantly in the ischemic brain tissues of MCAO group rats at 6 h and 24 h after reperfusion (P<0.05). Compared with the MCAO group, the Bcl-2/Bax ratio was increased significantly in the TPEN + MCAO group at 6 h and 24 h after reperfusion (P<0.05).Conclusion This study demonstrates that zinc upregulates the expression of RIPK3 and cleaved caspase-8 and downregulates the Bcl-2/Bax ratio within the ischemic brain tissue following cerebral ischemia-reperfusion in rats, indicating that zinc ions may exacerbate cerebral ischemia-reperfusion injury by promoting RIPK3-mediated necroptosis and cleaved caspase-8-dependent apoptosis. This study provides novel insights into the role of zinc ions in the mechanisms underlying cerebral ischemia-reperfusion injury.

Key words: cerebral ischemia-reperfusion, zinc, necroptosis, receptor interacting serine/threonine kinase 3, cleaved caspase-8, B-cell lymphoma 2, Bcl-2-associated X protein

CLC Number:

Zhu Yuequan, Liu He, Zhou Yingnan, Li Yakun, Zhao Haiping, Zhao Yongmei. Effects of zinc ions on RIPK3-mediated necroptosis and caspase-8-dependent apoptosis following cerebral ischemia-reperfusion in rats[J]. Journal of Capital Medical University, 2026, 47(4): 766-774.

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