Genistein mitigates oxidative stress and inflammation by regulating Nrf2/HO-1 and NF-κB signaling pathways in hypoxic-ischemic brain damage in neonatal mice

Abstract

Background: Oxidative stress and neuroinflammation play crucial roles in the progression of neonatal hypoxic-ischemic brain damage (HIBD). Genistein, a natural phytoestrogen, has been found to protect against ischemic brain injury. However, its effects and potential mechanisms in HIBD have not yet been explored.Methods: A neonatal mouse model of hypoxia-ischemia (HI) and a cell model of oxygen-glucose deprivation/reperfusion (OGD/R) were employed. In the in vivo study, genistein (10 mg/kg; ip) was administered in mice once daily for 3 consecutive days before the operation and once immediately after HI.The effects of genistein treatment on acute brain damage and long-term responses were evaluated. Neuronal injury and apoptosis were estimated using hematoxylin and eosin (H&E) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, respectively. The expression of apoptosis-related proteins were also measured by Western blot analysis. Dihydroethidium (DHE) staining and glutathione (GSH) and malondialdehyde (MDA) production were determined to assess the extent of oxidative stress. The messenger RNA (mRNA) levels of proinflammatory cytokines were detected using real-time quantitative polymerase chain reaction (RT-qPCR) to evaluate the extent of neuroinflammation. In the in vitro study, cell counting kit-8 (CCK-8) and lactate dehydrogenase (LDH) assays, as well as propidium iodide (PI) staining, were performed to analyse the neuroprotective effects of genistein on primary cortical neurons. Western blot assays were used to detect the levels of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), phosphorylated inhibitor kappa B-α (p-IκB-α) and phosphorylated nuclear factor-kappa B (p-NF-κB) both in vivo and in vitro.Results: Our results showed that genistein treatment effectively reduced cerebral infarction, attenuated neuronal injury and apoptosis, and contributed to the long-term recovery of neurological outcomes and brain atrophy in neonatal HIBD mice. Moreover, genistein ameliorated HIBD-induced oxidative stress and neuroinflammation. Meanwhile, genistein significantly increased cell viability, reversed neuronal injury and decreased cell apoptosis after OGD/R injury. Finally, the activation of the Nrf2/HO-1 pathway and inhibition of the NF-κB pathway by genistein were verified in the brain tissues of neonatal mice subjected to HIBD and in primary cortical neurons exposed to OGD/R. Conclusions: Genistein exerted neuroprotective effects on HIBD by attenuating oxidative stress and neuroinflammation through the Nrf2/HO-1 and NF-κB signalling pathways.

Echinocystic acid alleviated hypoxic-ischemic brain damage in neonatal mice by activating the PI3K/Akt/Nrf2 signaling pathway

“…The pups were sacrificed 24 h after HIBD, and TUNEL staining was performed as previously described ( Li Y. et al, 2022 ). The TUNEL-positive cells in six randomly selected fields in the ipsilateral parietal cortex and the hippocampal CA1 region were photographed by using a Nikon DS-U3 camera with a ×40 objective.…”

“…The whole brains of mice were quickly collected and stored at −80°C at 24 h after HIBD, and DHE staining was performed as previously described ( Li Y. et al, 2022 ). The images were collected in six randomly selected fields in the ipsilateral parietal cortex and the hippocampal CA1 area with a fluorescence Nikon C2 confocal microscope (Tokyo, Japan) with a ×20 objective.…”

“…Then, these proteins work against ROS and confer endogenous protection against neonatal HIE ( Zeng et al, 2020 ). Our previous study demonstrated that genistein provides neuroprotective effects against hypoxic-ischemic brain damage (HIBD) in neonatal mice via the Nrf2/HO-1 axis ( Li Y. et al, 2022 ). It has also been shown that Nrf2 can be regulated by the PI3K/Akt signaling pathway, which exerts neuroprotective effects in neonatal HIE ( Luo et al, 2021 ).…”

Beneficial effects of natural flavonoids on neuroinflammation

“…Genistein ( Figure 3 ) distributed in soy is an isoflavone and has anti-neuroinflammatory effects. Genistein ameliorates hypoxic-ischemic brain damage-induced neuroinflammation with reducing TNF-α, IL-1β, and IL-6 secretion by the up-regulation of Nrf2, HO-1 and IκBα, and the inactivation of IκBα and NF-κB ( 130 ). Moreover, genistein improves cognitive disorders, and reduces MCP-1 release, and elevates the levels of IL-10, IGF-1, BDNF, and CREB in the hippocampus of hypoxia-induced mice ( 131 ).…”

Therapeutic Potential and Mechanisms of Novel Simple O-Substituted Isoflavones against Cerebral Ischemia Reperfusion

“…Genistein reduced oxidative stress and neuroinflammation state. Meanwhile, genistein increased the ratio of Bcl-2/Bax, the HO-1 expression, and the nuclear translocation of Nrf-2, but inhibited cleaved caspase-3 expression and the phosphorylation of NF-κB and IκB [ 89 ]. In the 4-VO ischemia model (permanent bilateral vertebral artery occlusion and 15-min bilateral carotid artery occlusion), signal transducer and activator of transcription 3 (STAT3) phosphorylation and STAT3 DNA binding activity increased with the reperfusion time until 72 h after reperfusion.…”