Puerarin attenuates intracerebral hemorrhage-induced early brain injury possibly by PI3K/Akt signal activation-mediated suppression of NF-κB pathway.

Intracerebral hemorrhage (ICH) can induce intensively oxidative stress, neuroinflammation, and brain cell apoptosis. However, currently, there is no highly effective treatment available. Puerarin (PUE) possesses excellent neuroprotective effects by suppressing the NF-κB pathway and activating the PI3K/Akt signal, but its role and related mechanisms in ICH-induced early brain injury (EBI) remain unclear. In this study, we intended to observe the effects of PUE and molecular mechanisms on ICH-induced EBI. ICH was induced in rats by collagenase IV injection. PUE was intraperitoneally administrated alone or with simultaneously intracerebroventricular injection of LY294002 (a specific inhibitor of the PI3K/Akt signal). Neurological deficiency, histological impairment, brain edema, hematoma volume, blood-brain barrier destruction, and brain cell apoptosis were evaluated. Western blot, immunohistochemistry staining, reactive oxygen species (ROS) measurement, and enzyme-linked immunosorbent assay were performed. PUE administration at 50 mg/kg and 100 mg/kg could significantly reduce ICH-induced neurological deficits and EBI. Moreover, PUE could notably restrain ICH-induced upregulation of the NF-κB pathway, pro-inflammatory cytokines, ROS level, and apoptotic pathway and activate the PI3K/Akt signal. However, LY294002 delivery could efficaciously weaken these neuroprotective effects of PUE. Overall, PUE could attenuate ICH-induced behavioral defects and EBI possibly by PI3K/Akt signal stimulation-mediated inhibition of the NF-κB pathway, and this made PUE a potential candidate as a promising therapeutic option for ICH-induced EBI.

Inhibition of NOX4/ROS Suppresses Neuronal and Blood-Brain Barrier Injury by Attenuating Oxidative Stress After Intracerebral Hemorrhage.

Intracerebral hemorrhage (ICH) is a common and severe neurological disorder that can effectively induce oxidative stress responses. NADPH oxidase 4 (NOX4) is a member of the NOX family of oxidases. It is expressed in the brain normally and involved in cell signal transduction and the removal of harmful substances. In some pathological conditions, it mediates inflammation and the aging of cells. However, few studies have focused on whether NOX4 is involved in brain injury caused by ICH. Therefore, this study aimed to clarify the role of NOX4 in the pathological process that occurs after ICH and the potential mechanism underlying its role. A rat model of ICH was established by the injection of collagenase type IV, and the expression of NOX4 was then determined. Further, siRNA-mediated protein expression knockdown technology was used for NOX4 knockdown, and western immunoblotting, immunohistochemistry, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), and other molecular biological techniques were performed to assess the effects of NOX4 knockdown. Neurobiological scoring, brain water content determination, and other brain injury detection methods were also performed to assess the role of NOX4 following ICH. We found that the expression of NOX4 increased in the brains of rats after ICH, and that it was mainly expressed in neurons, astrocytes, vascular endothelial cells and microglia. Following NOX4 knockdown, the level of oxidative stress in the brain decreased considerably, the neurobehavioral scores improved, the levels of neuronal apoptosis reduced markedly, and the impairment of blood-brain barrier function was significantly ameliorated in rats with ICH. In conclusion, this study suggests that NOX4 expression is upregulated after ICH, which may cause an imbalance in the oxidative stress of relevant cells in the brain, leading to subsequent apoptosis of neurons and damage to the blood-brain barrier due to secondary brain injury following ICH.

Isoliquiritigenin alleviates early brain injury after experimental intracerebral hemorrhage via suppressing ROS- and/or NF-κB-mediated NLRP3 inflammasome activation by promoting Nrf2 antioxidant pathway.

BACKGROUND: Intracerebral hemorrhage (ICH) induces potently oxidative stress responses and inflammatory processes. Isoliquiritigenin (ILG) is a flavonoid with a chalcone structure and can activate nuclear factor erythroid-2 related factor 2 (Nrf2)-mediated antioxidant system, negatively regulate nuclear factor-κB (NF-κB) and nod-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome pathways, but its role and potential molecular mechanisms in the pathology following ICH remain unclear. The present study aimed to explore the effects of ILG after ICH and underlying mechanisms. METHODS: ICH model was induced by collagenase IV (0.2 U in 1 µl sterile normal saline) in male Sprague-Dawley rats weighing 280-320 g. Different doses of ILG (10, 20, or 40 mg/kg) was administrated intraperitoneally at 30 min, 12 h, 24 h, and 48 h after modeling, respectively. Rats were intracerebroventricularly administrated with control scramble small interfering RNA (siRNA) or Nrf2 siRNA at 24 h before ICH induction, and after 24 h, ICH model was established with or without ILG (20 mg/kg) treatment. All rats were dedicated at 24 or 72 h after ICH. Neurological deficits, histological damages, brain water content (BWC), blood-brain barrier (BBB) disruption, and neuronal degeneration were evaluated; quantitative real-time RT-PCR (qRT-PCR), immunohistochemistry/immunofluorescence, western blot, and enzyme-linked immunosorbent assay (ELISA) were carried out; catalase, superoxide dismutase activities and reactive oxygen species (ROS), and glutathione/oxidized glutathione contents were measured. RESULTS: ILG (20 and 40 mg/kg) markedly alleviated neurological deficits, histological damages, BBB disruption, brain edema, and neuronal degeneration, but there was no significant difference between two dosages. ILG (20 mg/kg) significantly suppressed the NF-κB and NLRP3 inflammasome pathways and activated Nrf2-mediated antioxidant system. Gene silencing of Nrf2 aggravated the neurological deficits, brain edema, and neuronal degeneration and increased the protein levels of NF-κB p65, NLRP3 inflammasome components, and IL-1ß. ILG delivery significantly attenuated the effects of Nrf2 siRNA interference mentioned above. CONCLUSIONS: Intraperitoneal administration of ILG after ICH reduced early brain impairments and neurological deficits, and the mechanisms were involved in the regulation of ROS and/or NF-κB on the activation of NLRP3 inflammasome pathway by the triggering of Nrf2 activity and Nrf2-induced antioxidant system. In addition, our experimental results may make ILG a potential candidate for a novel therapeutical strategy for ICH.