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[Formononetin downregulates P53/SAT1/ACSL4 pathway-mediated ferroptosis to improve hypoxic-ischemic brain injury in
Tao Guo1,2, Bolin Chen1, Xiao Yang2
1Department of Human Anatomy & Histology and Embryology, School of Basic Medical Sciences, Kunming Medical University, Kunming 650500, China.
Objectives:
To investigate the neuroprotective effects of formononetin (FMN) against hypoxic-ischemic brain damage (HIBD) in neonatal mice and the underlying mechanism.
Methods:
Twenty-four neonatal C57BL/6J mice were randomly divided (n=6) into sham-operated group, HIBD model group, HIBD+FMN-L (50 mg/kg) group, and HIBD+FMN-H (100 mg/kg) group. Mouse models of HIBD were established by left common carotid artery ligation followed by hypoxia (92% N₂, 8% O₂) for 40 min. FMN at the two doses was administered by intraperitoneal injection, and 3 days later, brain tissues from the cortical ischemic penumbra were collected for assessing expressions of ferroptosis-related proteins (P53, SAT1, and ACSL4) using Western blotting and immunofluorescence staining and for detecting the levels of Fe²⁺, superoxide, malondialdehyde (MDA), and glutathione (GSH). In cultured HT22 neurons with oxygen-glucose deprivation (OGD), the effects of 100 μmol/L FMN, 10 μmol/L Nutlin-3 (a P53 agonist), or their combination on expressions of ferroptosis proteins, intracellular Fe²⁺, reactive oxygen species (ROS), lipid peroxidation, GSH, mitochondrial membrane potential, and cell viability were evaluated.
Results:
In the neonatal mouse models of HIBD, FMN treatment significantly suppressed the protein expression of P53, SAT1, and ACSL4, reduced Fe²⁺, ROS, and MDA levels and increased GSH content in the cortical ischemic penumbra. In HT22 neurons with OGD, FMN obviously alleviated OGD-induced ferroptosis as shown by lowered expressions of the key ferroptosis proteins, reduced Fe²⁺ accumulation and lipid peroxidation, and significant increases of GSH levels, mitochondrial membrane potential, and cell viability. Mechanistic experiments showed that activation of P53 signaling by Nutlin-3 markedly reversed the protective effects of FMN.
Conclusions:
FMN produces neuro-protective effects against HIBD in neonatal mice by mitigating neuronal ferroptosis, primarily through downregulation of the P53/SAT1/ACSL4 signaling pathway.
