Liposomes-Loaded miR-9-5p Alleviated Hypoxia-Ischemia-Induced Mitochondrial Oxidative Stress by Targeting ZBTB20 to Inhibiting Nrf2/Keap1 Interaction in Neonatal Mice

<b><i>Aims:</i></b> Hypoxia ischemia (HI) is a leading cause of cerebral palsy and long-term neurological sequelae in infants. Given that mitochondrial dysfunction in neurons contributes to HI brain damage, this study aimed to investigate the regulatory role of miR-9-5p in mitochondrial function following HI injury. <b><i>Results:</i></b> Overexpression of miR-9-5p in HI mice or H₂O₂-exposed PC12 cells suppressed neuronal injury, associated with increased mitochondrial copy number, normalizing mitochondrial membrane potential, improved nuclear factor-erythroid factor 2-related factor 2 (Nrf2) activation, and downregulation of Keap1. This was mediated, in part, through the ability of this miR-9-5p to bind and regulate the transcriptional activity of zinc finger and BTB domain-containing protein 20 (ZBTB20). Further study suggested that the knockdown of ZBTB20 exerts neuroprotection by inhibiting Nrf2/Keap1 interaction to promote the translocation of Nrf2 from the cytoplasm to the nucleus and the consequent expression of antioxidant proteins. Notably, the protective effects of miR-9-5p overexpression against HI-induced mitochondrial damage were reversed by the Nrf2 inhibitor ML385. Finally, the utilization of liposomes for the delivery of miR-9-5p (miR-9-5p@Lip) presents a promising therapeutic strategy for the treatment of HI injury. <b><i>Innovation:</i></b> miR-9-5p is a potential therapeutic agent for ischemic stroke through its modulation of the ZBTB20/Nrf2/Keap1 signaling pathway, influencing mitochondrial function and antioxidant response. Furthermore, the use of liposomal delivery for miR-9-5p offers a promising therapeutic strategy for HI injury. <b><i>Conclusion:</i></b> Overexpression of miR-9-5p protects against cerebral HI injury by modulating mitochondrial function through the ZBTB20/Nrf2/Keap1 signaling pathway. <i>Antioxid. Redox Signal.</i> 42, 512-528. [Figure: see text].