IMM-H004 attenuates neutrophil pyroptosis following ischemic stroke via a CKLF1 dependent mechanism.

Abstract

BACKGROUND: Neutrophil pyroptosis is considered one of the key pathophysiological mechanisms in the progression of cerebral ischemia-reperfusion (I/R), However, the specific molecular mechanism underlying their regulation remains incompletely understood, and the development of targeted drugs for this mechanism is relatively scarce. METHODS: In this study, a mouse model of cerebral I/R injury was established by middle cerebral artery occlusion and reperfusion (MCAO/R), and the effects of IMM-H004 on the pyroptosis level of peripheral blood neutrophils, cerebral blood flow perfusion status, cerebral infarction volume and neurobehavioral function were systematically investigated. An in vitro model of neutrophil pyroptosis induced by chemokine-like factor 1 peptide C27 (CKLF1-C27) was constructed, and combined with Western blot, immunofluorescence staining, flow cytometry and other molecular and cell biology methods, the intrinsic molecular mechanism of CKLF1-induced neutrophil pyroptosis and the target of IMM-H004's therapeutic effect were deeply explored. RESULTS: The levels of CKLF1 in the serum of MCAO/R model showed a significant elevation with inflammatory cytokines, including TNF-α, IL-18 and IL-1β. Further experiments confirmed that CKLF1 could activate gasdermin E (GSDME) pathway and induce neutrophil pyroptosis in a dose-dependent manner. Inhibition of CKLF1 by IMM-H004 could significantly reduce the occurrence of neutrophil pyroptosis by inhibiting the excessive activation of GSDME, thereby achieving a mitigating effect on cerebral I/R injury. Moreover, loss of CKLF1 significantly reduced the volume of cerebral infarction in MCAO/R mice, effectively improving the perfusion level in the ischemic area, and significantly alleviating the neurobehavioral deficits in the model animals. CONCLUSION: IMM - H004 exerts a protective effect on cerebral I/R injury by targeting and inhibiting the CKLF1-GSDME signaling pathway-mediated pyroptosis of neutrophils.