With unknown etiology and limited treatment options, neonatal hypoxic‐ischemic brain damage (HIBD) remains a major cause of mortality in newborns. Ferroptosis, a recently discovered type of cell death triggered by lipid peroxidation, is closely associated with HIBD. High‐mobility group box 1 (HMGB1), a molecule associated with inflammation damage, can induce neuronal death in HIBD. However, it remains unknown whether HMGB1 contributes to neuronal ferroptosis in patients with HIBD. Herein, glycyrrhizin (GL), an HMGB1 inhibitor, was used to investigate the relationship between ferroptosis and HMGB1. RAS‐selective lethal 3(RSL3), a ferroptosis agonist, was administered to further confirm the changes in the signaling pathway between HMGB1 and ferroptosis. Western blot analysis revealed that GL markedly suppressed the expression of HMGB1 and increased the level of GPX4 in the context of HIBD. We observed changes in neuronal ultrastructure via transmission electron microscopy to further confirm the occurrence of ferroptosis. Real‐time PCR indicated that GL inhibited the expression of ferroptosis‐related genes and inflammatory factors. Immunofluorescence and immunohistochemistry staining confirmed GL inhibition of neuronal damage and ferroptosis in HIBD associated with GPX4 and ROS. GL not only inhibited ferroptosis induced by RSL3 and oxygen‐glucose deprivation in vitro but also inhibited ferroptosis induced by HIBD in vivo. More importantly, GL may improve oxidative stress imbalance and mitochondrial damage, alleviate the downstream production of inflammatory factors, and ultimately reduce ferroptosis and damage to cortical neurons following HIBD via the HMGB1/GPX4 pathway. In conclusion, we showed for the first time that GL could suppress the occurrence of neuronal ferroptosis and reduce neuronal loss in HIBD via the HMGB1/GPX4 pathway. These findings highlight the potential of HMGB1 signaling antagonists to treat neuronal damage by suppressing ferroptosis, provide new and unique insights into GL as a neuroprotective agent, and suggest new prevention and treatment strategies for HIBD.

Neonatal hypoxic-ischemic (HI) encephalopathy is a severe disease for which there is currently no curative treatment. Recent evidence suggests that high-mobility group box 1 (HMGB1) protein can promote neuroinflammation after stroke in adult rodents, but its role in perinatal hypoxic-ischemic brain damage (HIBD) remains largely uninvestigated. In the present work, the potential role of HMGB1 in the pathogenesis of HIBD was explored. A HIBD model was established in postpartum day 7 rat pups. HMGB1 expression, the cellular distribution of HMGB1, and microglial activation were all evaluated. Glycyrrhizin (GL), an inhibitor of HMGB1, was used to investigate whether the inhibition of HMGB1 modulated microglial M1/M2 polarization or attenuated brain damage after HI. HAPI microglial cells and primary neurons were cultured in vitro and an oxygen-glucose deprivation model was established to evaluate the effects of different microglial-conditioned media on neurons using GL and recombinant HMGB1. Results showed that the expression of HMGB1 was increased in both the ipsilateral cortex and peripheral blood 72 h after HI. Immunofluorescence analyses showed that HMGB1 in the cortex was primarily expressed in neurons. This increase in cortical HMGB1 expression 72 h after HI was characterized by increased co-expression with microglia, rather than neurons or astrocytes. The expression of both M1 and M2 microglia was upregulated 72 h after HI. The administration of GL significantly suppressed M1 microglial polarization and promoted M2 microglial polarization. Meanwhile, GL pretreatment significantly alleviated brain edema and cerebral infarction. In vitro experimentation showed that HMGB1-induced M1-conditioned media aggravated neuronal damage, but this effect was neutralized by GL. These findings suggest that HMGB1 may result in an imbalance of M1/M2 microglial polarization in the cortex and thus cause neuronal injury. Pharmacological blockade of HMGB1 signaling may attenuate this imbalanced polarization of microglia and thus could be used as a therapeutic strategy against brain injury in HIBD.

Introduction The objective of the Chinese Neonatal Network (CHNN) is to provide a platform for collaborative research, outcomes evaluation and quality improvement for preterm infants with gestational age less than 32 weeks in China. The CHNN is the first national neonatal network and has the largest geographically representative cohort from neonatal intensive care units (NICUs) in China. Methods and analysis Individual-level data from participating NICUs will be collected using a unique database developed by the CHNN on an ongoing basis from January 2019. Data will be prospectively collected from all infants <32 weeks gestation or <1500 g birth weight at 58 participating NICUs. Infant outcomes and inter-institutional variations in outcomes will be examined and used to inform quality improvement measures aimed at improving outcomes. Information about NICU environmental and human resource factors and processes of neonatal care will also be collected and analysed for association with outcomes. Clinical studies, including randomised controlled trials will be conducted using the CHNN data platform. Ethics and dissemination This study was approved by the ethics review board of Children’s Hospital of Fudan University, which was recognised by all participating hospitals. Waiver of consent were granted at all sites. Only non-identifiable patient level data will be transmitted and only aggregate data will be reported in CHNN reports and publications.