Abstract: Objective  To investigate the effects of the zinc ion chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) on the expression levels of key pyroptosis pathway proteins following cerebral ischemia-reperfusion in rats, including  nucleotide-binding domain leucine-rich repeat containing protein 3 (NLRP3), cleaved caspase-1, and the key execution protein gasdermin D (GSDMD). This study designed to evaluate the impact of zinc ions on the cellular pyroptosis pathway, providing a novel theoretical basis for the development of zinc-targeted therapeutic strategies for ischemic-reperfusion injury. Methods  Healthy male rats were randomly divided into three groups: Sham, middle cerebral artery occlusion (MCAO), and TPEN+MCAO. The MCAO model was established using the modified suture method, with 90 min of ischemia followed by reperfusion upon suture removal. The TPEN+MCAO group received an intraperitoneal injection of TPEN at 30 min before ischemia, while the Sham and MCAO groups received an equal volume of solvent. The ischemic hemisphere tissue was isolated at 6 h and 24 h after reperfusion. Immunofluorescence staining was performed to observe the co-localization of NLRP3 and the neuronal marker NeuN. Western blotting was used to detect the protein expression levels of NLRP3, cleaved caspase-1, and GSDMD. Results  ①NLRP3 was co-localized with the neuronal marker NeuN in the ischemic brain tissue of MCAO rats at 6 h and 24 h after reperfusion.  ②Compared to Sham group, the protein expression of NLRP3 increased to 1.8-fold at 6 h and 2.2-fold at 24 h after reperfusion in the ischemic tissue of MCAO group (P<0.01). TPEN treatment significantly reduced the protein expression of NLRP3 in the ischemic tissue at 24 h after reperfusion (P<0.05).  ③Compared to Sham group, the expression of cleaved caspase-1 increased at 24 h after reperfusion in ischemic tissue of MCAO group rats (P<0.01), and this increase was attenuated by TPEN treatment (P<0.01). ④Compared to Sham group, the protein expression of GSDMD increased to 3.5-fold and 2.0-fold at 6 h and 24 h after reperfusion, respectively, in the ischemic tissue of MCAO group rats (P<0.001). TPEN treatment significantly reduced the protein expression of GSDMD in the ischemic tissue at 24 h after reperfusion (P<0.001). Conclusion  The present study  reveals that neuronal pyroptosis and inflammatory response were significantly increased in the ischemic tissue following cerebral ischemia-reperfusion injury in rats. Chelating zinc  with TPEN was able to alleviate neuronal pyroptosis and inflammatory response,.It provides new scientific evidence for targeting zinc ions as a therapeutic strategy for cerebral ischemic-reperfusion injury.

Key words: cerebral ischemia-reperfusion, pyroptosis, zinc, nucleotide-binding domain leucine-rich repeat containing protein 3 (NLRP3), cleaved caspase-1, gasdermin D (GSDMD), neuron