Upregulation Of Peroxiredoxin6and Protein Disulfide Isomerase Expression By4-Hydroxybenzyl Alcohol In Cerebral Ischemic Injury

Background:Pharmacologic stimulation of prosurvival proteins may be an effective strategy to reduce cell damage after ischemia.4-hydroxybenzyl alcohol (4-HBA), one of the phenolic constituents found in many herbal medicinal plants, exhibits beneficial effects in neurological disorders. The mechanisms include antioxidant, ant-anxiety, sedative, and analgesia. More recently,4-HBA protects against stroke. However, the entire picture of the underlying signaling mechanisms has not yet been fully elucidated.Objective:We used an in vitro ischemic model of oxygen-glucose deprivation followed by recovery (OGD/R) and an in vivo ischemic model of middle cerebral artery occlusion (MCAO) to investigate the related neuroprotective mechanisms.Methods:1. Primary cortical neuronal cultures were prepared from Sprague-Dawley rats born within12h as described previously with B27/ neurobasal. Oxygen-glucose deprivation (OGD) was conducted at5-7days in vitro.4-HBA (0.05,0.1,0.5,1mM) was continuously applied from30min before OGD until the end of recovery. The viability of cells was measured by MTT assay. To determine assessment of cell death, cellular cytotoxicity was detected by measuring levels of lactate dehydrogenase (LDH) using a commercially available kit.2. Western blot analyses and immunocytochemistry were used to analyze the protein levels of Prdx6,PDI and Nrf2activation.3. Using antibodies specific to the phosphorylated and active forms of Akt, p38, ERK, and JNK, we analyzed the effects of4-HBA on the survival pathway. To further clarify the signaling pathways involved in4-HBA-mediated neuroprotective effect in neurons exposed to OGD/R insult, inhibition studies of the PI3K/AKT pathway and three MAPK cascades were examined.4. To determine the upstream signaling pathway involved in4-HBA-mediated Nrf2activation and the induction of Prdx6and PDI, we examined the activation of PI3K/AKT in an in vitro model of ischemia.5. Pre-treatment with4-HBA(25,50and100mg/kg) on the brain injury after MCAO, neurologic deficit scores.Infarct volume analysis and morphological changes were performed. Oxidative stress was evaluated superoxide dismutase (SOD) activity and malondialdehyde (MDA) level.6. To reconfirmed the role of PI3K/AKT pathway in4-HBA-induced Nrf2activation and Prdx6and PDI expression in vivo. Rat cortical tissues from MCA regions were harvested and analyzed by Western blotting and immunohistochemistry.Results:1.4-HBA prior to OGD insult can reduce cell death and elevate cell viability. The concentration maximal cytoprotection is0.1mM.2. Pretreatment with4-HBA displayed a significant reduction in cerebral infarction, neurological deficits and histological injury at24h after ischemic stroke. Furthermore, the antioxidant activity of4-HBA was indicated by changes in representative endogenous antioxidant markers (SOD, MDA).The dose of50mg/kg of4-HBA was better protective than the other dose on the brain injury after MCAO.3. We used in vitro and in vivo studies to suggest that4-HBA mediate neuroprotection and the related mechanisms. Pretreatment with4-HBA, we saw a significant increase in transcription of ARE-containing genes such as Prdx6and PDI through Nrf2activation. We further found administration of LY294002blocked the increase in Prdx6and PDI levels in neurons stimulated by4-HBA, although LY294002did not affect vehicle-treated cells. Likewise, the4-HBA-induced elevation and nuclear accumulation of Nrf2was suppressed in the presence of LY294002.Conclusion:4-HBA administration has neuroprotective effects against cerebral ischemic injury both in vitro and in vivo. This is related to the upregulation of Prdx6and PDI expression via Nrf2in a PI3K/AKT-dependent manner. These findings provide further insight into the mechanisms through which4-HBA exerts its neuroprotective effect after ischemic brain damage.4-HBA therefore may serve as a useful agent in stroke treatment.