锌离子调控大鼠脑缺血再灌注后RIPK3介导的坏死性凋亡与caspase-8依赖凋亡的作用

doi:10.3969/j.issn.1006-7795.2026.04.017

摘要/Abstract

摘要： 目的建立大鼠大脑中动脉闭塞（middle cerebral artery occlusion，MCAO）模型，检测再灌注后缺血侧脑组织受体相互作用丝氨酸/苏氨酸蛋白激酶3（receptor interacting serine/threonine kinase 3，RIPK3）和裂解型半胱天冬酶-8（cleaved caspase-8）的表达及细胞定位，以及B细胞淋巴瘤2蛋白（B-cell lymphoma 2，Bcl-2）和Bcl-2相关X蛋白（Bcl-2-associated X protein，Bax）的表达水平，并给予锌离子螯合剂N,N,N′,N′-四(2-吡啶甲基)乙二胺［N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine，TPEN］，探讨锌离子对再灌注后不同时间（6 h和24 h）缺血侧脑组织RIPK3、cleaved caspase-8和Bcl-2/Bax表达的影响。方法将健康雄性SD大鼠随机数字表法分为 Sham、MCAO和TPEN + MCAO 3组。采用线栓法建立MCAO模型，再灌注6 h和24 h后分离缺血侧脑组织，Western blotting法检测RIPK3与cleaved caspase-8、Bcl-2和Bax的蛋白表达，免疫荧光染色观察RIPK3和cleaved caspase-8与神经元标记物NeuN的共定位表达。结果 ①与Sham组相比，MCAO组大鼠再灌注6 h和24 h后，缺血侧脑组织RIPK3表达显著升高（P<0.05），并与NeuN共定位表达；与MCAO组相比，锌离子螯合剂TPEN干预降低RIPK3在再灌注后6 h（P<0.05）和24 h的表达。②与Sham组相比，MCAO组大鼠再灌注6 h和24 h后，缺血侧脑组织cleaved caspase-8表达升高，并与NeuN共定位表达；与MCAO组相比，TPEN干预降低cleaved caspase-8在再灌注后6 h（P<0.05）和24 h的表达。③与Sham组相比，MCAO组大鼠再灌注6 h和24 h后缺血侧脑组织Bcl-2/Bax比值均显著降低（P< 0.05）；与MCAO组相比，TPEN干预升高再灌注后6 h和24 h的Bcl-2/Bax比值（P< 0.05）。结论大鼠脑缺血再灌注后，锌离子上调缺血侧脑组织神经元中RIPK3、cleaved caspase-8的表达、下调Bcl-2/Bax比值，说明锌离子可能通过促进RIPK3介导的坏死性凋亡与caspase-8依赖的细胞凋亡，加剧脑缺血再灌注损伤。本研究为锌离子参与缺血性脑卒中再灌注损伤的机制提供了新的理论依据。

关键词: 脑缺血再灌注, 锌离子, 坏死性凋亡, 受体相互作用丝氨酸/苏氨酸蛋白激酶3, 裂解型半胱天冬酶-8, B细胞淋巴瘤2蛋白, Bcl-2相关X蛋白

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

中图分类号:

朱玥荃, 刘鹤, 周英楠, 李亚堃, 赵海苹, 赵咏梅. 锌离子调控大鼠脑缺血再灌注后RIPK3介导的坏死性凋亡与caspase-8依赖凋亡的作用[J]. 首都医科大学学报, 2026, 47(4): 766-774.

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.

参考文献

［1］Tu W J, Zhao Z P, Yin P, et al. Estimated burden of stroke in China in 2020［J］. JAMA Netw Open, 2023, 6(3): e231455.
［2］Tuo Q Z, Zhang S T, Lei P. Mechanisms of neuronal cell death in ischemic stroke and their therapeutic implications［J］. Med Res Rev, 2021, 42(1): 259－305.
［3］Hilkens N A, Casolla B, Leung T W, et al. Stroke［J］ Lancet, 2024, 403(10446): 2820-2836.
［4］Uzdensky A B. Regulation of apoptosis in the ischemic penumbra in the first day post-stroke［J］. Neural Regen Res, 2020, 15(2): 253－254.
［5］Morgan M J, Kim Y S. Roles of RIPK3 in necroptosis, cell signaling, and disease［J］. Exp Mol Med, 2022, 54(10): 1695－1704.
［6］Li L, Song J J, Zhang M X, et al. Oridonin ameliorates caspase-9-mediated brain neuronal apoptosis in mouse with ischemic stroke by inhibiting RIPK3-mediated mitophagy［J］. Acta Pharmacol Sin, 2023, 44(4): 726－740.
［7］Sun X, Yang Y P, Meng X N, et al. PANoptosis: mechanisms, biology, and role in disease［J］. Immunol Rev, 2023, 321(1): 246－262.
［8］Mandal P, Berger S B, Pillay S, et al. RIP3 induces apoptosis Independent of pronecrotic kinase activity［J］. Mol Cell, 2014, 56(4): 481－495.
［9］Tummers B, Green D R. Mechanisms of TNF-independent RIPK3-mediated cell death［J］. Biochem J, 2022, 479(19): 2049－2062.
［10］Oltvai Z N, Milliman C L, Korsmeyer S J. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death［J］. Cell, 1993, 74(4): 609－619.
［11］Aboutaleb N, Shamsaei N, Khaksari M, et al. Pre-ischemic exercise reduces apoptosis in hippocampal CA3 cells after cerebral ischemia by modulation of the Bax/Bcl-2 proteins ratio and prevention of caspase-3 activation［J］. J Physiol Sci, 2015, 65(5): 435－443.
［12］Zhou Y, She R N, Mei Z G, et al. Crosstalk between ferroptosis and necroptosis in cerebral ischemia/reperfusion injury and Naotaifang formula exerts neuroprotective effect via HSP90-GCN2-ATF4 pathway［J］. Phytomedicine, 2024, 130: 155399.
［13］Chen L L, Shen Q Q, Liu Y J, et al. Homeostasis and metabolism of iron and other metal ions in neurodegenerative diseases［J］. Signal Transduct Target Ther, 2025, 10(1): 31.
［14］Liu H, Zhu Y Q, Geng X K, et al. Zinc dyshomeostasis, mitochondrial dysfunction, and their pathological cycle in ischemic stroke pathophysiology: mechanisms and potential therapeutic targets［J］. Redox Biol, 2026, 94: 104208.
［15］Zhao Y M, Yan F, Yin J, et al. Synergistic interaction between zinc and reactive oxygen species amplifies ischemic brain injury in rats［J］. Stroke, 2018, 49(9): 2200－2210.
［16］Bai M Y, Cui Y, Sang Z L, et al. Zinc ions regulate mitochondrial quality control in neurons under oxidative stress and reduce PANoptosis in spinal cord injury models via the Lgals3-Bax pathway［J］. Free Radic Biol Med, 2024, 221: 169－180.
［17］Guo J, Meng S S, Zhang J, et al. Zn2+ regulates mitochondrial DNA efflux to inhibit AIM2-mediated ZBP1-PANoptosome pathway and alleviate septic myocardial injury［J］. Chem Biol Interact, 2025, 417: 111525.
［18］Ajibare A J, Odetayo A F, Akintoye O O, et al. Zinc ameliorates acrylamide-induced oxidative stress and apoptosis in testicular cells via Nrf2/HO-1/NfkB and Bax/Bcl2 signaling pathway［J］. Redox Rep, 2024, 29(1): 2341537.
［19］Moriwaki K, Balaji S, McQuade T, et al. The necroptosis adaptor RIPK3 promotes injury-induced cytokine expression and tissue repair［J］. Immunity,2014, 41(4): 567－578.
［20］Wang Q S, Yang F, Duo K, et al. The role of necroptosis in cerebral ischemic stroke［J］. Mol Neurobiol, 2024, 61(7): 3882-3898.
［21］Deng X X, Li S S, Sun F Y. Necrostatin-1 prevents necroptosis in brains after ischemic stroke via inhibition of RIPK1-Mediated RIPK3/MLKL signaling［J］. Aging Dis, 2019, 10(4): 807-817.
［22］Jiang Q, Ding Y C, Li F W, et al. Modulation of NLRP3 inflammasome-related-inflammation via RIPK1/RIPK3-DRP1 or HIF-1α signaling by phenothiazine in hypothermic and normothermic neuroprotection after acute ischemic stroke［J］. Redox Biol, 2024, 73: 103169.
［23］Liu A H, Han X, Yang Q Q, et al. Zinc ions trigger PANoptosis-like cell death in magnetic hyperthermia therapy of magnesium based implant for hepatocellular carcinoma［J］. Apoptosis, 2025, 30(7/8): 1826-1838.
［24］Zhang Y Y, Su S S, Zhao S B, et al. RIP1 autophosphorylation is promoted by mitochondrial ROS and is essential for RIP3 recruitment into necrosome［J］. Nat Commun, 2017, 8: 14329.
［25］Newton K, Wickliffe K E, Maltzman A, et al. Caspase cleavage of RIPK3 after Asp333 is dispensable for mouse embryogenesis［J］. Cell Death Differ, 2024, 31(2): 254-262.
［26］Rodhe J, Burguillos M A, de Pablos R M, et al. Spatio-temporal activation of caspase-8 in myeloid cells upon ischemic stroke［J］. Acta Neuropathol Commun, 2016, 4(1): 92.
［27］Liang H, Matei N, McBride D W, et al. TGR5 activation attenuates neuroinflammation via Pellino3 inhibition of caspase-8/NLRP3 after middle cerebral artery occlusion in rats［J］. J Neuroinflammation, 2021, 18(1): 40.
［28］Nishi K, Iwaihara Y, Tsunoda T, et al. ROS-induced cleavage of NHLRC2 by caspase-8 leads to apoptotic cell death in the HCT116 human colon cancer cell line［J］. Cell Death Dis, 2017, 8(12): 3218.
［29］Yan J K, Yan W H, Cai W. Fish oil-derived lipid emulsion induces RIP1-dependent and caspase 8-licensed necroptosis in IEC-6 cells through overproduction of reactive oxygen species［J］. Lipids Health Dis, 2018, 17(1): 148.
［30］Liu G Y, Wang T, Wang T Q, et al. Effects of apoptosis-related proteins caspase-3, Bax and Bcl-2 on cerebral ischemia rats［J］. Biomed Rep, 2013, 1(6): 861-867.
［31］Zhao Y M, Pan R, Li S, et al. Chelating intracellularly accumulated zinc decreased ischemic brain injury through reducing neuronal apoptotic death［J］. Stroke, 2014, 45(4): 1139-1147.
［32］Peng T M, Li S, Liu L L, et al. Artemisinin attenuated ischemic stroke induced cell apoptosis through activation of ERK1/2/CREB/BCL-2 signaling pathway in vitro and in vivo［J］. Int J Biol Sci, 2022, 18(11): 4578-4594.
［33］Feng P, Li T L, Guan Z X, et al. The involvement of Bax in zinc-induced mitochondrial apoptogenesis in malignant prostate cells［J］. Mol Cancer, 2008, 7: 25.
［34］Kim B M, Choi Y J, Han Y, et al. N, N-dimethyl phytosphingosine induces caspase-8-dependent cytochrome c release and apoptosis through ROS generation in human leukemia cells［J］. Toxicol Appl Pharmacol, 2009, 239(1): 87-97.

[1]	朱玥荃, 刘鹤, 白尚颖, 赵海苹, 赵咏梅. 螯合锌离子对大鼠脑缺血再灌注后焦亡通路NLRP3/cleaved caspase-1及GSDMD表达的影响[J]. 首都医科大学学报, 2026, 47(2): 290-298.
[2]	刘鹤, 朱玥荃, 周英楠, 白尚颖, 赵海苹, 赵咏梅. 脑缺血大鼠再灌注后组蛋白去乙酰化酶2与O-GlcNAc转移酶表达变化及锌离子的调控作用[J]. 首都医科大学学报, 2026, 47(2): 299-307.
[3]	李亚堃, 李蔚, 杨雪琦, 师文娟, 朱玥荃, 赵咏梅. 活性氧自由基清除剂EUK-134对脑缺血再灌注损伤大鼠线粒体相关蛋白PGC-1α和VDAC1的影响[J]. 首都医科大学学报, 2024, 45(6): 1016-1022.