Ferulic acid, a component of the plants Angelica sinensis (Oliv.) Diels and Ligusticum chuanxiong Hort, exerts a neuroprotective effect by regulating various signaling pathways. This study showed that ferulic acid treatment prevents the injury-induced increase of collapsin response mediator protein 2 (CRMP-2) in focal cerebral ischemia. Glycogen synthase kinase-3β (GSK-3β) regulates CRMP-2 function through phosphorylation of CRMP-2. Moreover, the pro-apoptotic activity of GSK-3β is inactivated by phosphorylation by Akt. This study investigated whether ferulic acid modulates the expression of CRMP-2 and its upstream targets, Akt and GSK-3β, in focal cerebral ischemia. Male rats were treated immediately with ferulic acid (100 mg/kg, i.v.) or vehicle after middle cerebral artery occlusion (MCAO), and then cerebral cortices were collected 24 hr after MCAO. MCAO resulted in decreased levels of phospho-Akt and phospho-GSK-3β, while ferulic acid treatment prevented the decrease in the levels of these proteins. Moreover, phospho-CRMP-2 and CRMP-2 levels increased during MCAO, whereas ferulic acid attenuated these injury-induced increases. These results demonstrate that ferulic acid regulates the Akt/GSK-3β/CRMP-2 signaling pathway in focal cerebral ischemic injury, thereby protecting against brain injury.

Ferulic acid is known to act as a protective agent in cerebral ischemia through its anti-oxidant activity. γ-Enolase is a neuron-specific enolase that also exerts a neuroprotective effect. Here, we investigated whether ferulic acid regulates the expression level of γ-enolase in middle cerebral artery occlusion (MCAO)-induced brain injury and glutamate exposure-induced neuronal cell death. Adult male rats were treated with either vehicle or ferulic acid (100 mg/kg, i.v.) after MCAO and cerebral cortex tissues were collected 24 h after MCAO. Using a proteomics approach, we found that γ-enolase expression was decreased in MCAO-injured animals treated with vehicle alone, whereas ferulic acid treatment attenuated this decrease. Reverse-transcription PCR and Western blot analyses confirmed that ferulic acid treatment prevented MCAO injury-induced decrease in γ-enolase. Furthermore, in hippocampal-derived cell lines, glutamate exposure also decreased γ-enolase expression and ferulic acid treatment attenuated this glutamate-induced decrease in γ-enolase. These findings suggest that ferulic acid mediates a neuroprotective effect by attenuating injury-induced decreases of γ-enolase expression in neuronal cells.

Calbindin is a calcium binding protein that controls intracellular calcium levels and has a neuroprotective function against apoptotic stimuli. We investigated the expression of calbindin in ischemic brain injury. Focal cerebral ischemia was induced in male rats by middle cerebral artery occlusion (MCAO) and cerebral cortices were collected 24 h after MCAO. Cerebral ischemia significantly increased infarct volume. RT-PCR and Western blot analyses showed that MCAO injury induced a decrease of calbindin expression. Moreover, immunohistochemical staining showed that the number of calbindin-positive cells decreased in ischemic regions of MCAO-operated animals. In cultured hippocampal-derived cell lines, glutamate exposure increased intracellular Ca2+ concentrations and decreased calbindin expression. Taken together, both in vivo and in vitro results demonstrated decreases of calbindin after neuronal cell injury. These results suggest that decreases of calbindin in ischemic brain injury contribute to neuronal cell death.

Glutamate induces neuronal damage by generating oxidative stress and neurotoxicities. The neurological damage caused by glutamate is more severe during brain development in newborns than in adults. Resveratrol is naturally present in a variety of fruits and medicinal plants and exerts a neuroprotective effect against brain damage. The goal of this study was to evaluate the neuroprotective effects of resveratrol and to identify changed proteins in response to resveratrol treatment during glutamate-induced neonatal cortical damage. Sprague-Dawley rat pups (7 days old) were randomly divided into vehicle, resveratrol, glutamate, and glutamate and resveratrol groups. The animals were intraperitoneally injected with glutamate (10 mg/kg) and/or resveratrol (20 mg/kg) and their brain tissue was collected 4 hr after drug administration. Glutamate exposure caused severe histopathological changes, while resveratrol attenuated this damage. We identified regulated proteins by resveratrol in glutamate-induced cortical damaged tissue using two-dimensional gel electrophoresis and mass spectrometry. Among identified proteins, we focused on eukaryotic initiation factor 4A2, γ-enolase, protein phosphatase 2A subunit B, and isocitrate dehydrogenase. These proteins decreased in the glutamate-treated group, whereas the combination treatment of glutamate and resveratrol attenuated these protein reductions. These proteins are anti-oxidant proteins and antiapoptotic proteins. These results suggest that glutamate induces brain cortical damage in newborns; resveratrol exerts a neuroprotective effect by controlling expression of various proteins with antioxidant and anti-apoptotic functions.