Abstract

First discovery of the role of Circ_0000376 in neurons following ischaemic injury • Elucidation of the mechanism whereby Circ_0000376 regulates CCND1 via the sponge mmu-let-7b-5p • Demonstration that the Circ_0000376/mmu-let-7b-5p/CCND1 signalling axis mitigates neuronal apoptosis and metabolic dysfunction following ischaemic injury Background Post cardiac arrest brain injury (PCABI) results from global ischemia–reperfusion. Circular RNAs (circRNAs) are stable regulators of post transcriptional networks that may modulate neuronal survival and metabolism after ischemic injury. We investigated the role and mechanism of mmu_circ_0000376 (circ_0000376) in PCABI. Methods Through integrated bioinformatics analysis of circular RNA/microRNA/messenger RNA datasets via the GEO platform, a regulatory network involving circ_0000376/mmu-let-7b-5p/CCND1 was identified. We validated its circular structure, stability, and cytoplasmic localisation in HT22 neuronal cells. Through in vitro and in vivo experiments modulating circ_0000376 expression, we investigated cellular functions by assessing cell viability, apoptosis (Annexin V/PI staining, TUNEL, caspase/Bcl-2 family proteins), and mitochondrial/energy parameters (ATP, glucose consumption, lactate, G6PD activity). Results Circ_0000376 is a stable, cytoplasmic circRNA. Overexpression of Circ_0000376 reduced ischemia-reperfusion injury induced ROS, preserved ATP and glycolytic flux, increased G6PD activity, decreased apoptosis, and restored CCND1 and Bcl 2 while lowering Bax and cleaved caspases. Bioinformatic and reporter assays showed circ_0000376 sponges mmu-let-7b-5p, relieving repression of CCND1. mmu-let-7b-5p overexpression or CCND1 knockdown reversed circ_0000376 mediated protection. AAV mediated circ_0000376 overexpression in mice decreased hippocampal apoptosis and restored CCND1 in vivo. Conclusions Circ_0000376/mmu-let-7b-5p/CCND1 axis mitigates neuronal apoptosis and metabolic dysfunction after ischemic injury. Targe