Jin-Hee Sung

Estradiol attenuates the focal cerebral ischemic injury through mTOR/p70S6 kinase signaling pathway.

We previously showed that estradiol prevents neuronal cell death through the activation of Akt and its downstream targets Bad and FKHR. This study investigated whether estradiol modulates the survival pathway through other downstream targets of Akt, including mammalian target of rapamycin (mTOR) and p70S6 kinase. It is known that mTOR is a downstream target of Akt and a central regulator of protein synthesis, cell growth, and cell cycle progression. Adult female rats were ovariectomied and treated with estradiol prior to middle cerebral artery occlusion (MCAO). Brains were collected 24h after MCAO and infarct volumes were analyzed. We confirmed that estradiol significantly reduces infarct volume and decreases the number of positive cells for TUNEL staining in the cerebral cortex. Brain injury-induced a decrease in phospho-mTOR and phospho-p70S6 kinase. Estradiol prevented the injury-induced decrease in Akt activation and phosphorylation of mTOR and p70S6 kinases, and the subsequent decrease in S6 phosphorylation. Our findings suggest that estradiol plays a potent protective role against brain injury by preventing the injury-induced decrease of mTOR and p70S6 kinase phosphorylation.

Identification of proteins differentially expressed by melatonin treatment in cerebral ischemic injury – a proteomics approach

Abstract:  We previously reported that melatonin protects neuronal cells against ischemic brain damage. In this study, we identified proteins that were differentially expressed by melatonin treatment during ischemic brain injury. Rats were subjected to cerebral ischemia by middle cerebral artery occlusion (MCAO). Adult male rats were treated with melatonin (5 mg/kg) or vehicle prior to MCAO and brains were collected at 24 hr after MCAO. Proteins derived from the cerebral cortex were analyzed using two‐dimensional gel electrophoresis. Protein spots with a greater than 2.5‐fold change in intensity were identified by mass spectrometry. Among these proteins, γ‐enolase, stathmin, thioredoxin, peroxiredoxin‐6, hippocalcin, protein phosphatase 2A, adenosylhomocysteinase, ubiquitin carboxy‐terminal hydrolase L1, and NAD‐specific isocitrate dehydrogenase subunit α were significantly decreased in the vehicle‐treated group in comparison to the melatonin‐treated group. The identified proteins consist of cell differentiation and stabilization proteins, as well as an antioxidant enzyme. In contrast, dehydroprimidinase‐related protein 2 (DRP‐2), a target of protein oxidation in neurodegeneration, was significantly increased in vehicle‐treated animals, while melatonin prevented the injury‐induced increase of DRP‐2. Thus, the results of this study suggest that melatonin prevents cell death resulting from ischemic brain injury and that its neuroprotective effects are mediated by both the up‐ and down‐regulation of various proteins.

Gingko biloba Extract (EGb 761) prevents ischemic brain injury by activation of the Akt signaling pathway.

EGb 761 is a standardized extract of Gingko biloba that exerts protective effects against ischemic brain injury. This study investigated whether EGb 761 modulates the neuroprotective effects through Akt and its downstream targets, Bad and FKHR. Adult male rats were treated with EGb 761 (100 mg/kg) or vehicle prior to middle cerebral artery occlusion (MCAO). Brains were collected 24 hours after MCAO and infarct volumes were analyzed. EGb 761 significantly reduced infarct volume. Potential activation was mearsured by phosphorylation of Akt at Ser(473), Bad at Ser(136), and FKHR at Ser(256) using Western blot analysis. EGb 761 prevented the injury-induced decrease of pAkt and its down stream targets, pBad and pFKHR. Furthermore, EGb 761 prevented the injury-induced increase of cleaved caspase-3 levels. In conclusion, this study suggests that EGb 761 prevents cell death due to brain injury and that EGb 761 protection is affected by preventing the injury-induce decrease of Akt phosphorylation.

Identification of proteins regulated by estradiol in focal cerebral ischemic injury--a proteomics approach.

Estradiol protects neuronal cells against permanent and focal ischemic brain damage. We identified the proteins that are expressed following estradiol administration during cerebral ischemia in an animal model. Adult female rats were ovariectomized and treated with oil or estradiol prior to middle cerebral artery occlusion (MCAO) to induce cerebral ischemia, and brains were collected 24h after MCAO. Protein analysis was performed on the cerebral cortex using two-dimensional gel electrophoresis. Protein spots with difference in intensity between oil- and estradiol-treated groups were identified by mass spectrometry. Among these proteins, levels of protein phosphatase 2A (PP2A) and astrocytic phosphoprotein PEA-15 were significantly decreased in the oil-treated group in comparison to the estradiol-treated group. Moreover, Western blot analysis demonstrated that estradiol treatment prevents injury-induced decrease of PP2A and PEA-15 levels during both MCAO-induced injury and glutamate exposure in HT22 cells. In contrast, levels of the 60kDa heat shock protein (Hsp 60) were significantly increased in oil-treated animals, while estradiol prevented the injury-induced increase of Hsp 60. The results of this study provide an evidence that estradiol protects neuronal cells against ischemic brain injury through the up- and down-modulation of specific proteins.

Ferulic acid regulates the AKT/GSK-3β/CRMP-2 signaling pathway in a middle cerebral artery occlusion animal model.

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 attenuates the cerebral ischemic injury-induced decrease in peroxiredoxin-2 and thioredoxin expression

Ferulic acid, a phenolic phytochemical compound found in various plants, has a neuroprotective effect through its anti-oxidant and anti-inflammation functions. Peroxiredoxin-2 and thioredoxin play a potent neuroprotective function against oxidative stress. We investigated whether ferulic acid regulates peroxiredoxin-2 and thioredoxin levels in cerebral ischemia. Sprague-Dawley rats (male, 210-230g) were treated with vehicle or ferulic acid (100mg/kg) after middle cerebral artery occlusion (MCAO), and cerebral cortex tissues were collected 24h after MCAO. Decreases in peroxiredoxin-2 and thioredoxin levels were elucidated in MCAO-operated animals using a proteomics approach. We found that ferulic acid treatment prevented the MCAO-induced decrease in the expression of peroxiredoxin-2 and thioredoxin. RT-PCR and Western blot analyses confirmed that ferulic acid treatment attenuated the MCAO-induced decrease in peroxiredoxin-2 and thioredoxin levels. Moreover, immunoprecipitation analysis showed that the interaction between thioredoxin and apoptosis signal-regulating kinase 1 (ASK1) decreased during MCAO, whereas ferulic acid prevented the MCAO-induced decrease in this interaction. Our findings suggest that ferulic acid plays a neuroprotective role by attenuating injury-induced decreases in peroxiredoxin-2 and thioredoxin levels in neuronal cell injury.

Ferulic acid attenuates the focal cerebral ischemic injury-induced decrease in parvalbumin expression

Ferulic acid exerts a neuroprotective effect through its anti-oxidant and anti-inflammation properties. Parvalbumin has calcium buffering capacity and protects neuronal cells from cytotoxic Ca(2+) overload. This study investigated whether ferulic acid regulates parvalbumin expression in cerebral ischemia and glutamate toxicity-induced neuronal cell death. Male Sprague-Dawley rats were immediately treated with vehicle or ferulic acid (100 mg/kg, i.v.) after middle cerebral artery occlusion (MCAO), and cerebral cortex tissues were collected 24 h after MCAO. A proteomics approach elucidated the decrease of parvalbumin in MCAO-operated animals, and ferulic acid treatment attenuated the injury-induced decrease in parvalbumin expression. Moreover, RT-PCR and Western blot analyses clearly showed that ferulic acid treatment prevents the injury-induced decrease in parvalbumin levels. The number of parvalbumin-positive cells also decreased in MCAO-operated animals, and ferulic acid attenuated this injury-induced decrease in parvalbumin-positive cells. In cultured hippocampal cells, glutamate toxicity significantly increased the intracellular Ca(2+) concentration, whereas this increase in Ca(2+) levels was inhibited by ferulic acid treatment. In addition, ferulic acid treatment attenuated the glutamate exposure-induced decrease in parvalbumin levels. These results suggest that ferulic acid exerts a neuroprotective effect by attenuating the injury-induced decrease of parvalbumin and modulating intracellular Ca(2+) levels.

Ginkgo biloba extract (EGb 761) prevents the ischemic brain injury-induced decrease in parvalbumin expression.

Ginkgo biloba extract (EGb 761) exerts a neuroprotective effect against ischemic brain injury through an anti-apoptotic mechanism. Parvalbumin is a calcium buffering protein that plays an important role in modulating intracellular calcium concentration and regulating apoptotic cell death. The aim of this study was to investigate whether EGb 761 affects parvalbumin expression in cerebral ischemic injury. Adult male Sprague-Dawley rats were treated with vehicle or EGb 761 (100 mg/kg) prior to middle cerebral artery occlusion (MCAO) and cerebral cortex tissues were collected 24 h after MCAO. A proteomic approach revealed a reduction in parvalbumin expression in the vehicle-treated animals, whereas EGb 761 pretreatment attenuates the ischemic injury-induced decrease in parvalbumin expression. RT-PCR and Western blot analyses clearly confirmed the fact that EGb 761 prevents the injury-induced decrease in parvalbumin. Moreover, the results of immunohistochemical staining showed that the number of parvalbumin-positive cells was lower in vehicle-treated animals than in sham-operated animals, and EGb 761 averted this decrease. Thus, these results suggest that the maintenance of parvalbumin expression is associated with the neuroprotective function of EGb 761 against neuronal damage induced by ischemia.

Proteomic identification of proteins differentially expressed by nicotinamide in focal cerebral ischemic injury.

Nicotinamide exerts a potent neuroprotective effect against ischemia-induced brain injury. We identified proteins that were differentially expressed by nicotinamide treatment in ischemic brain injury. Focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO). Adult male Sprague-Dawley rats were treated with vehicle or nicotinamide (500 mg/kg) 2 h after the onset of MCAO. Brains were collected 24 h after MCAO and cerebral cortex regions were isolated. Protein spots with different intensities between vehicle- and nicotinamide-treated groups were detected using two-dimensional gel electrophoresis and identified by mass spectrometry. Among these proteins, γ-enolase, protein phosphatase 2A (PP2A) subunit B, and peroxiredoxin-2 (Prx-2) were significantly decreased in the vehicle-treated group compared to the nicotinamide-treated group. These identified proteins mediate cell differentiation and stabilization, and play a role as antioxidant enzymes. In contrast, 60 kDa heat shock protein (Hsp 60) was significantly increased in vehicle-treated animals, while nicotinamide prevented the injury-induced increase of this protein. These results suggest that nicotinamide mediates neuroprotective effects by up- and down-regulation of various specific proteins.

Identification of proteins regulated by curcumin in cerebral ischemia

BACKGROUND Curcumin is known to have a neuroprotective effect against cerebral ischemia. The objective of this study was to identify various proteins that are differentially expressed by curcumin treatment in focal cerebral ischemia using a proteomic approach. METHODS Adult male rats were treated with vehicle or curcumin 1 h after middle cerebral artery occlusion. Brain tissues were collected 24 h after the onset of middle cerebral artery occlusion, and cerebral cortices proteins were identified by two-dimensional gel electrophoresis and mass spectrometry. RESULTS We detected several proteins with altered expression levels between vehicle- and curcumin-treated animals. Among these proteins, ubiquitin carboxy-terminal hydrolase L1, isocitrate dehydrogenase, adenosylhomocysteinase, and eukaryotic initiation factor 4A were decreased in the vehicle-treated animal, and curcumin treatment attenuated the injury-induced decreases of these proteins. Conversely, pyridoxal phosphate phosphatase was increased in the vehicle-treated animal, and curcumin treatment prevented decreases in this protein. The identified altered proteins are associated with cellular metabolism and differentiation. CONCLUSIONS The results of this study suggest that curcumin exerts a neuroprotective effect by regulating the expression of various proteins in focal cerebral ischemia.