REG1B attenuates post-cardiac arrest-induced brain injury identified by integrated proteomic and metabolomic profiling and experimental validation

Abstract

Post-cardiac arrest brain injury is a major determinant of mortality and neurological disability after post-cardiac arrest syndrome. However, effective targeted therapies for post-cardiac arrest brain injury and reliable circulating biomarkers for early neurological prognosis are lacking. This study aimed to identify circulating molecules associated with neurological outcomes after cardiac arrest and to investigate their potential neuroprotective mechanisms. Serum samples collected 24 hours after the return of spontaneous circulation were subjected to integrated proteomic and untargeted metabolomic analyses at Renmin Hospital of Wuhan University, and REG1B was identified as a candidate protein associated with favorable neurological outcomes after cardiac arrest (|log ₂ FC|>1.4; P < 0.05). Moreover, metabolomic profiling revealed distinct metabolic reprogramming between the favorable and unfavorable outcome groups. Additionally, a cardiac arrest/cardiopulmonary resuscitation mouse model was developed. Given that human REG1B and mouse Reg1 are orthologous genes with conserved protein sequences, recombinant Reg1 protein was administered exogenously, followed by comprehensive assessments of neurological deficits and pathological changes. To elucidate the mechanism underlying these effects, we conducted a series of experiments in HT22 cells, and Reg1 gain- and loss-of-function assays, Extl3 knockdown, and protein interaction assays were performed to investigate the underlying mechanisms of oxygen–glucose deprivation/reperfusion-induced neuronal injury. In vivo experiments indicated that recombinant Reg1 administration post-cardiac arrest could attenuate neuropathological damage, enhance neurological function scores, and diminish endoplasmic reticulum stress, along with its associated apoptosis. In vitro validation confirmed that Reg1 overexpression markedly enhanced the morphology and neurite outgrowth of HT22 cells while effectively mitigating endoplasmic reticulum stress and related apoptosis. Conversely, loss of Reg1 function exacerbated neuronal injury. Extl3 knockdown further enhanced the neuroprotective effects of Reg1 and attenuated endoplasmic reticulum stress, suggesting that Extl3 may negatively regulate Reg1-dependent neuronal protection. Protein interaction assays further supported a physical interaction between Reg1 and Extl3. Collectively, our findings revealed that REG1B is associated with favorable neurological outcomes and is linked to the homeostatic adaptation of the endoplasmic reticulum following post-cardiac arrest syndrome. Reg1 may exert neuroprotective effects by promoting neuronal repair and suppressing endoplasmic reticulum stress-associated apoptosis, whereas Extl3 may act as a negative regulator of Reg1-mediated neuroprotection. REG1B may therefore represent a candidate circulating biomarker for neurological prognosis, and modulation of REG1B-associated signaling may provide a potential therapeutic strategy for post-cardiac arrest brain injury.