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Epigallocatechin gallate (EGCG) is one of polyphenol that is abundant in green tea. It has anti-oxidative activity and exerts neuroprotective effects in ischemic brain damage. Ischemic conditions induce oxidative stress and result in cell death. Thioredoxin is a small redox protein that plays an important role in the regulation of oxidation and reduction. This study was designed to investigate the regulation of thioredoxin by EGCG in ischemic brain damage. Middle cerebral artery occlusion (MCAO) was performed to induce focal cerebral ischemia in male Sprague-Dawley rats. The EGCG (50 mg/kg) or was administered before MCAO surgical operation. Neurological behavior test, reactive oxygen species (ROS), and lipid peroxidation (LPO) measurement were performed 24 h after MCAO. The cerebral cortex was isolated for further experiments. EGCG alleviated MCAO-induced neurological deficits and increases in ROS and LPO levels. EGCG also ameliorated the decrease in thioredoxin expression by MCAO. This finding was confirmed using various techniques such as Western blot analysis, reverse transcription PCR, and immunofluorescence staining. Results of immunoprecipitation showed that MCAO decreases the interaction between apoptosis signal-regulating kinase 1 (ASK1) and thioredoxin, while EGCG treatment attenuates this decrease. EGCG also attenuated decrease of cell viability and thioredoxin expression in glutamate-exposed neuron in a dose-dependent manner. It alleviated the increase of caspase-3 by glutamate exposure. However, this effect of EGCG on caspase-3 change was weakened in thioredoxin siRNA-transfected neurons. These findings suggest that EGCG exerts a neuroprotective effect by regulating thioredoxin expression and modulating ASK1 and thioredoxin binding in ischemic brain damage.

Stroke is caused by disruption of blood supply and results in permanent disabilities as well as death. Chlorogenic acid is a phenolic compound found in various fruits and coffee and exerts antioxidant, anti-inflammatory, and anti-apoptotic effects.The purpose of this study was to investigate whether chlorogenic acid regulates the PI3K-Akt-Bad signaling pathway in middle cerebral artery occlusion (MCAO)-induced damage.Chlorogenic acid (30 mg/kg) or vehicle was administered peritoneally to adult male rats 2 h after MCAO surgery, and animals were sacrificed 24 h after MCAO surgery. Neurobehavioral tests were performed, and brain tissues were isolated. The cerebral cortex was collected for Western blot and immunoprecipitation analyses.MCAO damage caused severe neurobehavioral disorders and chlorogenic acid improved the neurological disorders. Chlorogenic acid alleviated the MCAO-induced histopathological changes and decreased the number of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells. Furthermore, MCAO-induced damage reduced the expression of phospho-PDK1, phospho-Akt, and phospho-Bad, which was alleviated with administration of chlorogenic acid. The interaction between phospho-Bad and 14-3-3 levels was reduced in MCAO animals, which was attenuated by chlorogenic acid treatment. In addition, chlorogenic acid alleviated the increase of cytochrome c and caspase-3 expression caused by MCAO damage.The results of the present study showed that chlorogenic acid activates phospho-Akt and phospho-Bad and promotes the interaction between phospho-Bad and 14-3-3 during MCAO damage. In conclusion, chlorogenic acid exerts neuroprotective effects by activating the Akt-Bad signaling pathway and maintaining the interaction between phospho-Bad and 14-3-3 in ischemic stroke model.

Ischemic stroke is a severe neurodegenerative disease with a high mortality rate. Retinoic acid is a representative metabolite of vitamin A. It has many beneficial effects including anti-inflammatory, anti-apoptotic, and neuroprotective effects. The purpose of this study is to identify specific proteins that are regulated by retinoic acid in ischemic stroke. Middle cerebral artery occlusion (MCAO) was performed to induce focal cerebral ischemia. Retinoic acid (5 mg/kg) or vehicle was injected intraperitoneally into male rats for four days prior to MCAO operation. Neurobehavioral tests were performed 24 hr after MCAO and the cerebral cortex was collected for proteomic study. Retinoic acid alleviates neurobehavioral deficits and histopathological changes caused by MCAO. Furthermore, we identified various proteins that were altered by retinoic acid in MCAO damage. Among these identified proteins, adenosylhomocysteinase, isocitrate dehydrogenase [NAD

Ischemic stroke is a fatal disease that has long-term disability. It induces excessive oxidative stress generation and cellular metabolic disorders, result in tissue damage. Epigallocatechin gallate (EGCG) is a naturally derived flavonoid with strong antioxidant property. We previously reported the neuroprotective effect of EGCG in ischemic stroke. The defensive mechanisms of stroke are very diverse and complex. This study investigated specific proteins that are regulated by EGCG treatment in the ischemic brain damage. Middle cerebral artery occlusion (MCAO) was performed to induce focal cerebral ischemia. EGCG (50 mg/kg) or vehicle was intraperitoneally administered just prior to MCAO. MCAO induced severe neurological deficits and disorders. EGCG treatment alleviated these neurological disorder and damage. Cerebral cortex was used for this study. Two-dimensional gel electrophoresis and mass spectrometry were performed to detect the proteins altered by EGCG. We identified various proteins that were changed between vehicle- and EGCG-treated animals. Among these proteins, isocitrate dehydrogenase, dynamin-like protein 1, and γ-enolase were decreased in vehicle-treated animals, while EGCG treatment prevented these decreases. However, pyridoxal-5’-phosphate phosphatase and 60 kDa heat shock protein were increased in vehicle-treated animals with MCAO injury. EGCG treatment attenuated these increases. The changes in these proteins were confirmed by Western blot and reverse transcription-PCR analyses. These proteins were associated with cellular metabolism and neuronal regeneration. Thus, these findings can suggest that EGCG performs a defensive mechanism in ischemic damage by regulating specific proteins related to energy metabolism and neuronal protection.

Ischemic stroke is the most common type of stroke and is caused by vascular closure. Chlorogenic acid is a polyphenolic compound that is present in various plants. It is used as a traditional oriental medicine because of its anti-oxidant and anti-inflammatory properties. We investigated whether chlorogenic acid mediates neuroprotective effects by regulating pro-inflammatory proteins. Focal cerebral ischemia was induced through middle cerebral artery occlusion (MCAO) surgery in adult rats. Chlorogenic acid (30 mg/kg) or vehicle was injected into the abdominal cavity 2 h after MCAO. Rats were sacrificed 24 h after MCAO surgery and brain tissues were isolated immediately. MCAO caused histopathological changes in the ischemic cerebral cortex, and chlorogenic acid attenuated these changes. Chlorogenic acid reduced MCAO-induced reactive oxygen species generation and oxidative stress increase in the cerebral cortex. Furthermore, cerebral ischemia increased the expression of ionized calcium-binding adapter molecule-1 (Iba-1) and glial fibrillary acidic protein (GFAP), which are microglia and astrocyte activation markers, respectively. However, chlorogenic acid prevented MCAO-induced these increases. MCAO damage also increased the expression of nuclear factor-κB (NF-κB), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α). Chlorogenic acid treatment attenuated these increases caused by MCAO. These proteins are representative pro-inflammatory markers. This study confirmed that chlorogenic acid exerts an anti-oxidative effect and elucidated anti-inflammatory effect through regulating NF-κB, IL-1β, and TNF-α on cerebral ischemia. Thus, we can suggest that chlorogenic acid has neuroprotective effects by reducing oxidative stress and controlling pro-inflammatory proteins against cerebral ischemic damage.

Glutamate is the main excitatory neurotransmitter. Excessive glutamate causes excitatory toxicity and increases intracellular calcium, leading to neuronal death. Parvalbumin is a calcium-binding protein that regulates calcium homeostasis. Quercetin is a polyphenol found in plant and has neuroprotective effects against neurodegenerative diseases.We investigated whether quercetin regulates apoptosis by modulating parvalbumin expression in glutamate induced neuronal damage.Glutamate was treated in hippocampal-derived cell line, and quercetin or vehicle was treated 1 h before glutamate exposure. Cells were collected for experimental procedure 24 h after glutamate treatment and intracellular calcium concentration and parvalbumin expression were examined. Parvalbumin small interfering RNA (siRNA) transfection was performed to detect the relation between parvalbumin and apoptosis.Glutamate reduced cell viability and increased intracellular calcium concentration, while quercetin preserved calcium concentration and neuronal damage. Moreover, glutamate reduced parvalbumin expression and quercetin alleviated this reduction. Glutamate increased caspase-3 expression, and quercetin attenuated this increase in both parvalbumin siRNA transfected and non-transfected cells. The alleviative effect of quercetin was statistically significant in non-transfected cells. Moreover, glutamate decreased bcl-2 and increased bax expressions, while quercetin alleviated these changes. The alleviative effect of quercetin in bcl-2 family protein expression was more remarkable in non-transfected cells.These results demonstrate that parvalbumin contributes to the maintainace of intracellular calcium concentration and the prevention of apoptosis, and quercetin modulates parvalbumin expression in glutamate-exposed cells. Thus, these findings suggest that quercetin performs neuroprotective function against glutamate toxicity by regulating parvalbumin expression.

Quercetin is a bioactive flavonoid which abundantly exists in vegetables and fruits. Quercetin exerts a neuroprotective effect against cerebral ischemia. Thioredoxin acts as antioxidant by regulating redox signaling. This study investigated whether quercetin regulates thioredoxin expression in focal cerebral ischemia and glutamate-induced neuronal cell death. Male Sprague Dawley rats (210–230 g) were intraperitoneally injected with vehicle or quercetin (10 mg/kg) 1 h prior to middle cerebral artery occlusion (MCAO). Cerebral cortex was collected 24 h after MCAO. MCAO led to neurological movement deficits, brain edema, and serious histopathological damages in cerebral cortex, and quercetin alleviated these damages following MCAO. We observed the change of thioredoxin expression in MCAO animals with quercetin using proteomic approach, reverse-transcription PCR, and Western blot analyses. Thioredoxin expression decreased in vehicle-treated MCAO animals, while quercetin attenuated this decrease. Moreover, quercetin treatment alleviated the decrease in the number of thioredoxin-positive cells in cerebral cortex of MCAO animals. Furthermore, immunoprecipitation analysis demonstrated that interaction of apoptosis signal-regulating kinase 1 (ASK1) and thioredoxin was decreased in MCAO animals with vehicle, while quercetin prevented MCAO-induced decrease in these binding. In addition, quercetin also alleviated the reduction of cell viability and the decrease in thioredoxin expression in glutamate-treated hippocampal cell line and primary cultures of cortical neurons. However in thioredoxin-silenced cortical neuron, anti-apoptotic effect of quercetin was decreased. Thus, changes of thioredoxin expression by quercetin may contribute to the neuroprotective effect of quercetin in focal cerebral ischemia. Our findings suggest that quercetin mediates its neuroprotective function by regulation of thioredoxin expression and maintenance of interaction between ASK1 and thioredoxin.

Cerebral ischemia is a neurological disorder that leads to cognitive decline and high mortality. Retinoic acid is a metabolite of vitamin A that has anti-inflammatory and anti-apoptotic effects. This study investigated whether retinoic acid prevents neuronal cell damage on focal cerebral ischemia through modulating apoptosis signaling pathway. Middle cerebral artery occlusion (MCAO) was performed to induce focal cerebral ischemia in adult male rats. Retinoic acid (5 mg/kg) or vehicle was injected intraperitoneally for 4 days prior to MCAO. Neurological behavior deficit tests were performed 24 h after MCAO. Brain edema and infarct volume were measured, and TUNEL histochemistry was carried out. We also investigated the changes in apoptosis-related proteins including bcl-2 family proteins and caspases. MCAO injury induced severe neurological behavior deficits and brain edema. It also increased infarct volume, histopathological damages, and the number of TUNEL-positive cells in cerebral cortex. However, retinoic acid pretreatment attenuated MCAO-induced neurological behavior deficits, brain edema, and infarction. It also alleviated histopathological lesion and decreased the number of TUNEL-positive cells. Bcl-2 and bax proteins are representative bcl-2 family proteins. MCAO injury induced a decrease in bcl-2 expression and an increase in bax expression, and retinoic acid pretreatment alleviated these changes. MCAO injury caused a decrease in bcl-2/bax expression ratio in cerebral cortex, while retinoic acid restored this decrease by MCAO. Moreover, our result showed increases in caspase-9, caspase-3, PARP protein levels in MCAO-operated animals. Retinoic acid pretreatment prevented these increases. We identified the changes in cleaved forms of these proteins, similar to the changes in full-length protein. Activation of caspases and PARP proteins are considered to be representative apoptosis indicators. This study showed that retinoic acid regulates bcl-2 family proteins and caspase proteins in focal cerebral ischemia. Thus, our findings demonstrate that retinoic acid exhibits a neuroprotective effect against ischemic damage by modulating apoptosis signaling pathway.

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 anti-apoptotic proteins. These results suggest that glutamate induces brain cortical damage in newborns; resveratrol exerts a neuroprotective effect by controlling expression of various proteins with anti-oxidant and anti-apoptotic functions.

Cerebral ischemia is a neurological disorder with high mortality. Quercetin is a flavonoid compound that is abundant in vegetables and fruits. It exerts anti-inflammatory and anti-apoptotic effects. This study investigated the neuroprotective effects of quercetin in focal cerebral ischemia. Male Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO) to induce focal cerebral ischemia. Quercetin or vehicle was injected 30 min before the onset of ischemia. A neurological function test, brain edema measurement, and 2,3,5-triphenyltetrazolium chloride staining were performed to elucidate the neuroprotective effects of quercetin. Western blot analysis was performed to observe caspase-3 and poly ADP-ribose polymerase (PARP) protein expression. MCAO leads to severe neuronal deficits and increases brain edema and infarct volume. However, quercetin administration attenuated the MCAO-induced neuronal deficits and neuronal degeneration. We observed increases in caspase-3 and PARP protein levels in MCAO-operated animals injected with vehicle, whereas quercetin administration attenuated these increases in MCAO injury. This study reveals the neuroprotective effect of quercetin in an MCAO-induced animal model and demonstrates the regulation of caspase-3 and PARP expression by quercetin treatment. These results suggest that quercetin exerts a neuroprotective effect through preventing the MCAO-induced activation of apoptotic pathways affecting caspase-3 and PARP expression.

Cerebral ischemia leads to neuronal cell death, causes neurological disorder and permanent disability. Chlorogenic acid has antioxidant, anti-inflammatory, and anti-apoptotic properties. This study investigated the neuroprotective effects of chlorogenic acid against cerebral ischemia. Focal cerebral ischemia was induced in male adult rats via middle cerebral artery occlusion (MCAO). Chlorogenic acid (30 mg/kg) or vehicle was injected in the intraperitoneal cavity 2 h after MCAO operation. Neurological behavior tests were performed 24 h after MCAO, brain edema and infarction were measured. Oxidative stress was assessed by investigating the levels of reactive oxygen species (ROS) and lipid peroxidation (LPO) levels. MCAO damage leaded to severe neurobehavioral deficits, increased ROS and LPO levels, and induced brain edema and infarction. MCAO damage caused histopathological damages and increased the number of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells in the cerebral cortex. However, chlorogenic acid treatment improved neurological behavioral deficits caused by MCAO and attenuated the increase in ROS and LPO levels. It also alleviated MCAO-induced brain edema, infarction, and histopathological lesion. Chlorogenic acid treatment attenuated the increase in the number of TUNEL-positive cells in the cerebral cortex of MCAO animals. We also investigated caspase proteins expression to elucidate the neuroprotective mechanism of chlorogenic acid. Caspase-3, caspase-7, and poly ADP-ribose polymerase expression levels were increased in the MCAO damaged cortex, while chlorogenic acid mitigated these increases. These results showed that MCAO injury leads to severe neurological damages and chlorogenic acid exerts neuroprotective effects by regulating oxidative stress and caspase proteins expressions. Thus, our findings suggest that chlorogenic acid acts as a potent neuroprotective agent by modulating the apoptotic-related proteins.

Calcium acts as a second messenger that mediates physiologic functions, such as metabolism, cell proliferation, and apoptosis. Hippocalcin is a neuronal calcium sensor protein that regulates intracellular calcium concentration. Moreover, it prevents neuronal cell death from oxidative stress. Quercetin has excellent antioxidant properties and preventative effects. We studied modulation of hippocalcin expression by quercetin treatment in cerebral ischemic injury and glutamate-induced neuronal cell damage. Focal cerebral ischemia was induced by permanent middle cerebral artery occlusion (pMCAO). Male Sprague-Dawley rats were injected with vehicle or quercetin (10 mg/kg) 1 h prior to pMCAO, and cerebral cortical tissues were isolated 24 h after pMCAO. Quercetin improved pMCAO-induced neuronal movement deficit and infarction. pMCAO induced a decrease in hippocalcin expression in the cerebral cortex. However, quercetin treatment attenuated this pMCAO-induced decrease. In cultured hippocampal cells, glutamate excitotoxicity dramatically increased the intracellular calcium concentration, whereas quercetin alleviated intracellular calcium overload. Moreover, Western blot and immunocytochemical studies showed reduction of hippocalcin expression in glutamate-exposed cells. Quercetin prevented this glutamate-induced decrease. Furthermore, caspase-3 expression in hippocalcin siRNA transfection conditions is higher than caspase-3 expression in un-transfection conditions. Quercetin treatment attenuated the increase of caspase-3. Taken together, these results suggest that quercetin exerts a preventative effect through attenuation of intracellular calcium overload and restoration of down-regulated hippocalcin expression during ischemic injury.

Diabetes is a metabolic health disorder and an important risk factor for stroke. Phosphoprotein enriched in astrocytes 15 (PEA-15) is a multifunctional protein modulating cell proliferation, survival, apoptosis and glucose metabolism. This study investigated whether diabetes modulates the expression of PEA-15 and two phosphorylated forms (Ser 104 and Ser 116) in middle cerebral artery occlusion (MCAO)-induced brain injury. Male Sprague-Dawley rats were administrated with streptozotocin (40 mg/kg) and were underwent right middle cerebral artery occlusion (MCAO) 4 weeks after streptozotocin injection. Brain tissues were collected 24 hr after MCAO and stained using triphenyltetrazolium chloride. Western blot analysis was performed to elucidate the expression of PEA-15 and two phosphorylated forms (Ser 104 and Ser 116) in right cerebral cortex. Infarct volume during MCAO injury was severely increased in diabetic animals compared to non-diabetic animals. We identified the decrease in PEA-15 in animals that underwent MCAO using proteomic approach. PEA-15 expression during MCAO was strongly decreased in diabetic animals compared to non-diabetic animals. Western blots analysis confirmed that diabetes exacerbated the decrease in PEA-15 expression after MCAO. Moreover, decrease in expression of phospho-PEA-15 (Ser 104 and Ser 116) was greater in diabetic than in non-diabetic animals. These results suggested that a diabetic condition may aggravate brain damage through decreasing expression of PEA-15 and phospho-PEA-15 (Ser 104 and Ser 116) in ischemic brain injury.

Resveratrol has a neuroprotective effect against cerebral ischemia. The objective of this study was to identify proteins that are differentially expressed in the cerebral cortex of vehicle- and resveratrol-treated animals during ischemic injury. Focal cerebral ischemia was induced as middle cerebral artery occlusion (MCAO) in male rats. Rats were treated with vehicle or resveratrol before MCAO, and cerebral cortex was collected 24 hr after MCAO. Cerebral cortex proteins were identified by two-dimensional gel electrophoresis and mass spectrometry. Several proteins were identified as differentially expressed between vehicle- and resveratrol-treated animals. Among these proteins, expression of peroxiredoxin-5, isocitrate dehydrogenase [NAD+], apolipoprotein A-I and ubiquitin carboxy terminal hydrolase L1 was decreased in the vehicle-treated group, whereas resveratrol attenuated the injury-induced decrease in expression of these proteins. However, expression of collapsing response mediator protein 2 was increased in the vehicle-treated group, whereas resveratrol prevented the injury-induced increase in the expression of this protein. These findings suggest that resveratrol modulates the expression of various proteins that associated with oxidative stress and energy metabolism in focal cerebral ischemia.

Testicular torsion is a urological emergency that leads to serious testicular damage and male infertility. We performed this study to identify specific proteins that are differentially expressed in response to testicular torsion and detorsion-induced ischemia-reperfusion (I-R) injury. Adult male rats were divided into two groups: a sham-operated group and a testicular I-R group. Testicular torsion was induced by rotating the left testis 720° in a clockwise direction for 1 hr, and then, detorsion was performed for 24 hr. After this testicular tissues were collected, protein analysis was performed using two-dimensional gel electrophoresis and Western blot analyses. Testicular I-R injury resulted in serious histopathologic damage to the germinal cells in the seminiferous tubules and increased the number of TUNEL-positive cells in testicular tissue. Specific protein spots with a greater than 2.5-fold change in intensity between the sham-operated and testicular I-R groups were identified by mass spectrometry. Among these proteins, levels of peroxiredoxin 6, thioredoxin, heterogeneous nuclear ribonucleoproteins, ubiquitin carboxyl terminal hydrolase isozyme L5 and zinc finger AN1-type domain 3 were decreased in the testicular I-R group compared to the sham-operated group. Moreover, Western blot analysis clearly showed the decrease of these proteins in the testicular I-R group. These proteins have spermatogenesis and anti-oxidative functions. These findings suggest that testicular I-R results in cell death due to altered expression of several proteins with spermatogenesis and anti-oxidation functions.

Diabetes is a severe metabolic disorder and a major risk factor for stroke. Stroke severity is worse in patients with diabetes compared to the non-diabetic population. The 14-3-3 proteins are a family of conserved acidic proteins that are ubiquitously expressed in cells and tissues. These proteins are involved in many cellular processes including metabolic pathways, signal transduction, protein trafficking, protein synthesis, and cell cycle control. This study investigated 14-3-3 proteins expression in the cerebral cortex of animals with diabetes, cerebral ischemic injury and a combination of both diabetes and cerebral ischemic injury. Diabetes was induced by intraperitoneal injection of streptozotocin (40mg/kg) in adult male rats. After 4 weeks of treatment, middle cerebral artery occlusion (MCAO) was performed for the induction of focal cerebral ischemia and cerebral cortex tissue was collected 24h after MCAO. We confirmed that diabetes increases infarct volume following MCAO compared to non-diabetic animals. In diabetic animals with MCAO injury, reduction of 14-3-3 β/α, 14-3-3 ζ/δ, 14-3-3 γ, and 14-3-3 e isoforms was detected. The expression of these proteins was significantly decreased in diabetic animals with MCAO injury compared to diabetic-only and MCAO-only animals. Moreover, Western blot analysis ascertained the decreased expression of 14-3-3 family proteins in diabetic animals with MCAO injury, including β/α, ζ/δ, γ, e, τ, and η isoforms. These results show the changes of 14-3-3 proteins expression in streptozotocin-induced diabetic animals with MCAO injury. Thus, these findings suggest that decreases in 14-3-3 proteins might be involved in the regulation of 14-3-3 proteins under the presence of diabetes following MCAO.

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.

Parvalbumin is a calcium-binding albumin protein that is involved in neuronal maturation, differentiation, axonal transport, and neurotransmitter release. Parvalbumin protects neuron from cell death through reduction of intracellular Ca²⁺ concentrations. In this study, we investigated parvalbumin expression after neuronal cell injury. Middle cerebral artery occlusions (MCAO) were surgically performed in a rat model to induce focal cerebral ischemic injury. Adult male rats were used and brain tissues were collected 24 h after MCAO. MCAO increases infarct damages and apoptotic cell death in cerebral cortex. A proteomic approach revealed a decrease of parvalbumin expression in MCAO-operated animals. RT-PCR and Western blot analyses showed that MCAO induces a reduction in parvalbumin transcript and protein levels, respectively. The numbers of parvalbumin-positive cells were also decreased in the cerebral cortices of MCAO-operated animals. Moreover, glutamate exposure significantly increased intracellular Ca²⁺ concentrations and induced a reduction of parvalbumin expression in a hippocampal-derived cell line. These results suggest that the reduction in parvalbumin levels after ischemic brain injury can modulate neuronal cell death.

Ferulic acid protects neuronal cells against focal cerebral ischemic injury through its anti-oxidative and anti-inflammatory effects. Phosphoprotein enriched in astrocytes 15 (PEA-15) is known to modulate various cellular processes including cell proliferation, apoptosis, and survival. This study was investigated whether ferulic acid can regulate the levels of PEA-15 and its two phosphorylated forms (Ser 104 and Ser 116) in a cerebral ischemic injury model and in neuronal cells exposed to glutamate. A middle cerebral artery occlusion (MCAO) was performed to induce focal cerebral ischemic injury. Adult male rats were immediately treated with vehicle or ferulic acid (100 mg/kg) at the beginning of the MCAO, and then cerebral cortices were collected 24h after MCAO. The decrease in PEA-15 level after ischemic injury was detected using a proteomic approach. Ferulic acid administration prevented the ischemic injury-induced decrease of PEA-15 level. Moreover, Western blot analysis clearly confirmed that ferulic acid attenuates the ischemic injury-induced decreases in PEA-15, phospho-PEA-15 (Ser 104), and phospho-PEA-15 (Ser 116) levels. Glutamate exposure induced significant reductions in the levels of PEA-15 and the two phospho-PEA-15 (Ser 104 and Ser 116) in cultured hippocampal neuron, while pretreatment with ferulic acid prevented the glutamate toxicity-induced decreases in these proteins levels. The decrease of phospho-PEA-15 protein level indicates that the anti-apoptotic function of PEA-15 was being inhibited. Thus, these results suggest that ferulic acid protects neuronal cells against ischemic injury by maintenance of phospho-PEA-15 protein levels.

Ferulic acid protects neuronal cells from glutamate-induced excitotoxicity and focal cerebral ischemia. This study investigated whether ferulic acid exerts a neuroprotective effect through the activation of Akt and its downstream targets, Bad and 14-3-3. Adult male rats were immediately treated with ferulic acid (100mg/kg, i.v.) after middle cerebral artery occlusion (MCAO). Brains were collected 24h after MCAO and infarct volumes were analyzed using triphenyltetrazolium chloride staining. It was found that ferulic acid treatment significantly reduced infarct volume during MCAO. Ferulic acid attenuated the MCAO injury-induced decrease of phospho-PDK1, phospho-Akt and phospho-Bad levels. However, ferulic acid did not affect the expression of 14-3-3 and Bcl-xL, which exerts an anti-apoptotic effect through interaction with phospho-Bad. Immunoprecipitation analysis demonstrated that the interaction between phospho-Bad and 14-3-3 decreased during MCAO, whereas ferulic acid prevented the injury-induced decrease in these interaction levels. Moreover, ferulic acid prevented the injury-induced increase in cleaved caspase-3 levels. These findings suggest that ferulic acid attenuates cell death during MCAO and that these protective effects are due to inhibition of Akt signaling pathway inactivation and maintenance of the interaction between phospho-Bad and 14-3-3.

Nicotinamide exerts neuroprotective effects against focal cerebral ischemic injury. Phosphoprotein enriched in astrocytes 15 (PEA-15) is prominently expressed in astrocytes that exert broad anti-apoptotic functions. This study investigated whether nicotinamide modulates PEA-15 and levels of two phosphorylated PEA-15 (Serine 104 and 116) in an animal model of middle cerebral artery occlusion (MCAO)-induced injury. Adult male rats were treated with vehicle or nicotinamide (500 mg/kg) 2 hr after the onset of MCAO and cerebral cortices were collected at 24 hr after MCAO. In a proteomic approach, MCAO induced decreases of PEA-15 levels, while nicotinamide treatment attenuated the injury-induced decrease in PEA-15. The results of Western blot analysis suggest that nicotinamide prevented injury-induced reduction in phospho-PEA-15 (Serine 104) and phospho-PEA-15 (Serine 116) levels. The phosphorylation of PEA-15 exerts anti-apoptotic functions, and reduction of PEA-15 phosphorylation leads to apoptotic cell death. These results suggest that nicotinamide exerts a neuroprotective effect by attenuating the injury-induced decreases of PEA-15 and phospho-PEA-15 (Ser 104 and Ser 116) proteins.

Nicotinamide attenuates neuronal cell death related to focal cerebral ischemic injury. This study investigated whether nicotinamide exerts a neuroprotective effect through the activation of Raf- mitogen-activated protein kinase kinase (MEK)-ERK and its downstream targets, including p90 ribosomal S6 kinase (p90RSK) and Bad. Adult male Sprague-Dawley rats were treated with nicotinamide (500 mg/kg) or vehicle 2 hr after the onset of middle cerebral artery occlusion (MCAO). Brains were collected 24 hr after MCAO. In the present study, nicotinamide significantly reduces the volume of infarct regions and decreases the number of positive cells by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining in the cerebral cortex. Nicotinamide prevents injury-induced decrease in Raf-1, MEK1/2, and ERK1/2 phosphorylation. As part of the downstream cascade, nicotinamide inhibits the injury-induced decrease in p90RSK and Bad phosphorylation. Moreover, nicotinamide prevents the injury-induced increase in cleaved caspase-3 levels. These findings suggest that nicotinamide protects neuronal cells against cerebral ischemic injury and that MEK-ERK-p90RSK cascade activation by nicotinamide contributes to these neuroprotective effects.

Nicotinamide protects cortical neuronal cells against cerebral ischemic injury through activation of various cytoprotective mechanisms. Here, this study confirmed the neuroprotective effects of nicotinamide in focal cerebral ischemic injury and investigated whether nicotinamide modulates a crucial survival pathway, Akt and its downstream targets. Adult male rats were treated with vehicle or nicotinamide (500 mg/kg) 2 h after the onset of middle cerebral artery occlusion (MCAO). Brains were collected 24 h after MCAO and infarct volumes were analyzed. Nicotinamide significantly reduced the infarct volume in the cerebral cortex. Potential activation was measured by phosphorylation of PDK1 at Ser241, Akt at Ser473, and Bad at Ser136 using Western blot analysis. Nicotinamide prevented the injury-induced decrease of pPDK1, pAkt, and pBad levels. 14-3-3 levels were not different between vehicle- and nicotinamide-treated animals. However, pBad and 14-3-3 interaction levels decreased during MCAO, but were maintained in the presence of nicotinamide, compared to levels in control animals. These findings suggest that nicotinamide attenuates cell death due to focal cerebral ischemic injury and that neuroprotective effects are mediated through the Akt signaling pathway, thus enhancing neuronal survival.

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.

EGb 761 is a standardized extract of Ginkgo biloba that appears to have a neuroprotective effect against neurodegenerative diseases. Adult male rats were treated with EGb 761 (100 mg/kg) or vehicle prior to middle cerebral artery occlusion (MCAO), and brains were collected 24 h after MCAO. Proteins that were differentially expressed after EGb 761 treatment during cerebral ischemia were detected using two-dimensional gel electrophoresis. Protein spots with more than a 2.5-fold change in intensity between vehicle- and EGb 761-treated groups were identified by mass spectrometry. The levels of peroxiredoxin-2 and protein phosphatase 2A subunit B were significantly decreased in the vehicle-treated group in comparison to the EGb 761-treated group. In contrast, levels of the collapsing response mediator protein 2 (CRMP2) were significantly increased in vehicle-treated animals, while EGb 761 prevented the injury-induced increase of CRMP2. These results suggest that EGb 761 protects neuronal cells against ischemic brain injury through the specific up- and down-modulation of various proteins.

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.

The present study identified the proteins that are differentially expressed during ischemic brain injury. Adult male rats were performed a middle cerebral artery occlusion (MCAO) to induce cerebral ischemia, and brains were collected at 24 hr after MCAO. Protein analysis was performed on the cerebral cortex using two-dimensional gel electrophoresis. Protein spots with a greater than 3 fold change in intensity between the sham and MCAO groups were identified by mass spectrometry. Among these proteins, 60 kDa heat shock protein, dehydropyrimidinase-related protein 2, t-complex protein 1, and Rho GDP dissociation inhibitor levels were significantly increased in MCAO group compared to those of the sham group. In contrast, thioredoxin, peroxiredoxin-2, stathmin, ubiquitin carboxy-terminal hydrolase L1, guanine nucleotide-binding protein alpha, pyridoxal-5'-phosphate phosphatase, and apoplipoprotein A-I levels were significantly decreased in MCAO group. These results suggest that cerebral ischemia induces neuronal cells death by changing expression levels of several proteins.

We previously reported that melatonin prevents neuronal cell death in ischemic brain injury through the activation of Akt and the inhibition of apoptotic cell death. We investigated whether melatonin inhibits the apoptotic signal through the activation of a mammalian target of rapamycin (mTOR) and p70S6 kinase and its downstream target, S6 phosphorylation. 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 male rats were treated with melatonin (5mg/kg) or vehicle prior to middle cerebral artery occlusion (MCAO). Brains were collected at 24h after MCAO and infarct volumes were analyzed. We confirmed that melatonin significantly reduces infarct volume and decreases the number of TUNEL-positive cells in the cerebral cortex. Brain injury induced a decrease in phospho-mTOR and phospho-p70S6 kinase. Melatonin prevented the injury-induced decrease in Akt activation and phosphorylation of mTOR and p70S6 kinases, and the subsequent decrease in S6 phosphorylation. Our results suggest that melatonin prevents cell death resulting from ischemic brain injury and that its neuroprotective effects are mediated by preventing the injury-induced decrease of mTOR and p70S6 kinase phosphorylation.

Tyrosine hydroxylase (TH) is the rate-limiting enzyme of dopamine (DA) biosynthesis, which is up-regulated by vitamin C administration. Nurr1 gene is highly expressed in brain and important for midbrain DAergic cell development and survival. But, the role of vitamin C and/or vitamin E during Nurr1 expression is yet to be known. Further, the synergistic effect of vitamins C and E on TH expression has not yet been explored clinically. Therefore, we studied the effects of single or co- administration of vitamin C (0.5 mM) and vitamin E (1 mM) for 72 hr, on both TH and Nurr1 expression in in vitro primary cultured gestational days (GD) 13.5 rat ventral mesencephalon (VM) by Western blot and immunocytochemistry. Our study revealed highest expressions of both TH and Nurr1 in the vitamin C + vitamin E treated group. TH expression was also increased in the vitamin C treated group than that of the control group, but the vitamin E treated group did not show any statistically significant effect. However, both the vitamin C and the vitamin E treated groups revealed increased expression of Nurr1 protein as compared with the control group. The present experimental data suggest that vitamin C can up-regulate the protein expression of TH, but Nurr1 level was elevated either by vitamin C or by vitamin E administration. Further, vitamin E acts synergistically with vitamin C to elevate TH and Nurr1 expression, which is the most novel finding of our study and for the first time; we reported this result, since there have been no published data on the synergistic effect of both the antioxidant vitamins on TH and Nurr1 expression in VM. As the motor function defect due to the progressive loss of DAergic neuron is the major reason of Parkinsonism, therefore, the results of our study finally suggest the effective role of vitamin C and vitamin E during early treatment of Parkinsonism.

Nitric oxide (NO) is produced by three NO synthases (NOS), iNOS, eNOS, and nNOS. Production of NO by iNOS plays key roles in neurodegeneration, while eNOS is a protective enzyme. This study investigated the neuroprotective effect of melatonin and the levels of NOS isoforms induced by melatonin in ischemic brain injury. Adult male rats were treated with melatonin (5 mg/kg) or vehicle prior to middle cerebral artery occlusion (MCAO). Brains samples were collected at 24 hr after the onset of occlusion. Results confirmed that melatonin significantly reduces infarct area. Western blot analysis was used to evaluate the expression levels of iNOS, eNOS, and nNOS. The level of iNOS and nNOS increased in vehicle-treated animals, while melatonin prevented injury-induced increase of iNOS. In contrast to iNOS levels, eNOS levels decreased in vehicle-treated animals, while melatonin prevented the injury-induced decrease of eNOS. This study provides further evidence that melatonin exerts neuroprotective effects, and the regulation of NOS isoforms by melatonin may contribute to the neuroprotective effects.

Sexual dysfunction is frequently associated with diabetes in males. The present study was designed to evaluate whether streptozotocin-induced diabetes increases apoptotic cell death in rat testis through the regulation of Bcl-2 family proteins. Diabetes was induced by a single intravenous injection of streptozotocin (40 mg/kg body weight) and testis samples were collected after 3 months. The number of positive cells for TUNEL histochemistry was significantly increased in the testicular germ cells of the diabetic group, compared to those of control. The levels of Bcl-2 and Bcl-X(L), anti-apoptotic proteins, were decreased in the diabetic group. In contrast, the levels of Bax and Bad, pro-apoptotic factors, were increased in the diabetic group, compared with the control group. Moreover, the diabetic condition increased the interaction of Bad and Bcl-X(L), and decreased the binding of pBad and 14-3-3. 14-3-3 acts as an anti-apoptotic factor through interaction with Bad. Our findings suggest that streptozotocin-induced diabetes increases apoptotic cell death in testis tissue through the up-and down-regulation of Bcl-2 family proteins and the interaction of Bad and Bcl-X(L).

Estradiol prevents neuronal cell death through the activation of cell survival signals and the inhibition of apoptotic signals. This study investigated whether estradiol modulates the anti-apoptotic signal through the activation of Raf-MEK-ERK and its downstream targets, including 90 ribosomal S6 kinase (p90RSK) and Bad. Adult female rats were ovariectomied and treated with estradiol prior to middle cerebral artery occlusion (MCAO). Brains were collected 24 h after MCAO and infarct volumes were analyzed. We confirmed that estradiol significantly reduces infarct volume and decreases the positive cells of TUNEL staining in the cerebral cortex. Estradiol prevents the injury-induced decrease of Raf-1, MEK1/2, and ERK1/2 phosphorylation. Also, it inhibits the injury-induced decrease of p90RSK and Bad phosphorylation. Further, in the presence of estradiol, the interaction of phospho-Bad and 14-3-3 increased, compared with that of oil-treated animals. Our findings suggest that estradiol prevents cell death due to brain injury and that Raf-MEK-ERK cascade activation and its downstream targets, p90RSK, Bad phosphorylation by estradiol mediated these protective effects.

The present study was designed to evaluate whether ethanol suppresses survival-signaling pathways in rat testes. Ethanol (1.5 g/kg or 3 g/kg i.p., 15% v/v in saline) was administrated to adult male rats for 10 days. Ethanol treatment significantly increased the number of TUNEL-positive cells in rat testes. Potential activation was measured by phosphorylation of Akt and Erk1/2 using Western blot analysis. Ethanol decreased the levels of activated survival kinases, pAkt and pErk1/2. The phosphorylation of Bad at Ser112 and Ser136 was decreased in ethanol-treated animals in comparison to saline-treated animals. Moreover, the interaction of pBad with 14-3-3 was decreased by ethanol exposure. In conclusion, our findings suggest that ethanol induces apoptotic cell death by suppressing the activation of survival kinases and the phosphorylation of their downstream targets in rat testes.

Neural transplantation is one of the most promising treatments for neurodegenerative disorders. Survival rates of embryonic dopamine (DA) neurons following transplantation are low, between 2% and 20% in a number of animal models. To further establish survival changes of the transplanted gestational day 13.5 ventral mesencephalic (VM) cells into left intact adult rat striata so that design strategies of increasing survival of DA neurons, the tyrosine hydroxylase (TH) expression of VM-derived progenitor cells has been examined using immunohistochemistry and Western blot analysis. TH immunostaining revealed that the grafted VM cells developed to mature TH-positive neurons strongly at 3 weeks, peaked at 4 weeks, thereafter, gradually dropped following the degenerative expression of the grafted cells at both 5 and 6 weeks after transplantation. Western blot analysis also showed that the TH proteins were maximally expressed at 4 weeks post-grafting. Our finding suggested that the peak of surviving VM-derived TH positive cells occurred approximately 4 weeks after transplantation.

Stroke is a major cause of disability and death in adults. Diabetes mellitus is a metabolic disorder that strongly increases the risk of severe vascular diseases. This study compared changes in proteins of the cerebral cortex during ischemic brain injury between nondiabetic and diabetic animals.Adult male rats were injected with streptozotocin (40 mg/kg) via the intraperitoneal route to induce diabetes and underwent surgical middle cerebral artery occlusion (MCAO) 4 wk after streptozotocin treatment. Cerebral cortex tissues were collected 24 h after MCAO and cerebral cortex proteins were analyzed by two-dimensional gel electrophoresis and mass spectrometry.Several proteins were identified as differentially expressed between nondiabetic and diabetic animals. Among the identified proteins, we focused on the following metabolism-related enzymes: isocitrate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, adenosylhomocysteinase, pyruvate kinase, and glucose-6-phosphate isomerase (neuroleukin). Expression of these proteins was decreased in animals that underwent MCAO. Moreover, protein expression was reduced to a greater extent in diabetic animals than in nondiabetic animals. Reverse transcription-polymerase chain reaction analysis confirmed that the diabetic condition exacerbates the decrease in expression of metabolism-related proteins after MCAO.These results suggest that the diabetic condition may exacerbate brain damage during focal cerebral ischemia through the downregulation of metabolism-related proteins.

Estradiol prevents neuronal cell death through the activation of cell survival signals and the inhibition of apoptotic signals. This study investigated whether estradiol modulates the anti-apoptotic signal through the phosphorylation of Akt and its downstream target, glycogen synthase kinase 3beta (GSK3beta). Adult female rats were ovariectomized and treated with estradiol prior to middle cerebral artery occlusion (MCAO). Brains were collected 24 h after MCAO and infarct volumes were analyzed. Estradiol administration significantly reduced infarct volume and decreased the positive cells of TUNEL staining in the cerebral cortex. Potential activation was measured by phosphorylation of Akt at Ser(473) and GSK3beta at Ser(9) using Western blot analysis and immunohistochemistry. Estradiol prevented the injury-induced decrease of pAkt and pGSK3beta. Furthermore, pretreatment with estradiol decreased glutamate toxicity-induced cell death in a hippocampal cell line (HT22). Also, estradiol prevented the glutamate toxicity-induced decrease of pAkt and pGSK3beta in HT22 cells. Our findings suggest that estradiol plays a potent protective role against brain injury and that phosphorylation of Akt and GSK3beta by estradiol mediated these protective effects.

Estradiol prevents neuronal cell death through the inhibition of apoptotic signals. This study investigated whether estradiol modulates the anti-apoptotic signal through the activation of Akt and its downstream targets, including forkhead transcription factors FKHR and FHKRL1. Adult female rats were ovariectomied and treated with estradiol prior to middle cerebral artery occlusion (MCAO). Brains were collected 24 h after MCAO and infarct volumes were analyzed. Estradiol administration significantly reduced infarct volume and decreased the positive cells of TUNEL staining in the cerebral cortex. Potential activation was measured by phosphorylation of Akt at Ser473, pFKHR at Ser256, and pFKHRL1 at Thr32 using Western blot analysis and immunohistochemistry. Estradiol prevents the injury-induced decrease of pAkt, pFKHR, and pFKHRL1. Further, in the presence of estradiol, the interaction of pFKHRL1 and 14-3-3 increased, compared to that of oil-treated animals. Our findings suggest that estradiol plays a potent protective role against brain injury and that Akt activation and FKHR phosphorylation by estradiol mediated these protective effects.

The present study was designed to evaluate the effect of ethanol on pituitary adenylate cyclase activating polypeptide (PACAP) and its typ I (PAC1) receptor expression in adult rat testes. Ethanol (3 g/kg i.p., 15% v/v in saline) was administrated to adult male rats for 10 days. Using northern blot analysis, the present study showed the reduction of PACAP mRNA levels in rat testes by ethanol administration. Also, ethanol decreased the expression level of PAC1 receptor in testes. In particular, in situ hybridization clearly showed the decrease of PAC1 receptor mRNA expression in Leydig cells, which produce testosterone. Furthermore, the serum level of testosterone was significantly decreased in the ethanol-treated group. In conclusion, our findings suggest that the decrease of PACAP and PAC1 receptor expression in rat testes by ethanol exposure may partly contribute to the suppression of male reproductive activity.

Ethanol exposure is known to suppress male reproductive activity in laboratory animals and humans. The present study was designed to evaluate whether chronic ethanol exposure decreases proliferative activity or increases apoptosis in the testes. Ethanol (1.5 g/kg or 3 g/kg i.p., 15% v/v in saline) was administrated to adult male rats for 10 days. Proliferating cell nuclear antigen (PCNA) was used as a proliferative marker. Western blot analysis showed that ethanol administration significantly reduced the level of PCNA. Also, immunoreactivity of PCNA-positive cells in the spermatogonia and primary spermatocytes were decreased by ethanol exposure. However, the number of TUNEL-positive cells was significantly increased in the testicular germ cells of ethanol-treated rats. Moreover, ethanol administration significantly increased the level of activated caspase-3 in testes. In conclusion, our findings suggest that ethanol may partly contribute to the suppression of male reproductive activity through a reduction of cell proliferation and an enhancement of cell death in rat testes.

Using in situ hybridization, RNase protection assay and Western blot, we studied the effects of ethanol on the expression levels of GABA(B) receptor mRNA and protein in the cortex and hippocampus from adult rat brain. The results showed that ethanol significantly increased GABA(B1) and GABA(B2) receptor protein expression in the cortex, whereas only GABA(B2) was increased in the hippocampus. GABA(B) receptor agonist baclofen could partially reverse the effect of ethanol. Further studies of the mRNA levels defined that GABA(B1) mRNA levels were significantly increased in the hippocampus, with no significant changes of GABA(B2) mRNA levels. Moreover, GABA(B1) and GABA(B2) receptor mRNA levels were increased on 3-week ethanol treatment. Finally, GABA(B) agonist baclofen and antagonist phaclofen showed significant decreasing effects on GABA(B1) receptor mRNA levels in the cortex, but not in the hippocampus. These results were further confirmed by in situ hybridization. Thus, the present results showed the effects of ethanol on GABA(B) receptors in the cortex and hippocampus, implying the possible role of GABA(B) receptor in ethanol effects. The effects of GABA(B) receptor agonist and antagonist suggested that the possible mechanisms underlying that GABA(B) receptor modulated the behavioral effect induced by ethanol.

In this study, we have examined the possible existence of astrocyte regional heterogeneity in thrombin effect on astrocyte stellation. Neonatal astrocytes were cultured for 2 weeks from six different regions of the neonatal rat brain, including the cerebral cortex, hippocampus, brainstem, midbrain, hypothalamus and cerebellum. Culture medium was changed to DMEM containing 8-CPT-cyclic AMP (cAMP) or isoproterenol plus various concentrations of thrombin for 2 hr. Thrombin effectively blocked both cAMP- and isoproterenol-induced cell stellation in a dose-dependent manner in all regional astrocytes except cerebellar astrocytes. RT-PCR analysis showed that thrombin receptor mRNA was expressed in all regional astrocytes, suggesting that cerebellar astrocytes may maintain a unique signaling pathway downstream of the thrombin receptor.

Diabetic disease is known to suppress male reproductive activity in laboratory animals and humans. The present study was designed to evaluate whether streptozotocin-induced diabetes increases apoptotic cell death in rat testes through activation of the JNK and Bax pathway. Diabetes was induced by a single intravenous injection of streptozotocin (40 mg/kg) and testis samples were collected after 3 months. Compared with controls, body weight and testicular weight were lower in the diabetic group, and the apoptotic index in testicular germ cells was significantly increased. Expression of phospho-JNK and Bax was significantly increased in the diabetic group, and the level of activated caspase-3 was also increased, compared to that of controls. Our findings suggest that streptozotocin-induced diabetes increases apoptotic cell death in rat testes through phosphorylation of JNK and activation of Bax.

The present study was designed to evaluate the effect of ethanol on pituitary adenylate cyclase activating polypeptide (PACAP) expression in adult rat testes. Ethanol (3 g/kg i.p., 15% v/v in saline) was administrated to adult male rats for 10 days. Using northern blot analysis, we elucidated the decrease of PACAP mRNA in rat testes by ethanol administration. The level of PACAP mRNA was decreased by 46.5% in testes of the ethanol-treated animals, compared to that of saline-treated animals. In particular, ethanol exposure decreased the expression of PACAP mRNA and protein in developing germ cells, which are sperm cell progenitors. Thus, our findings suggest that the decrease of PACAP in developing germ cells by ethanol administration may contribute to the suppression of male reproductive activity.

Ferulic acid exhibits neuroprotective effects against focal cerebral ischemia. PI3/K and Akt signaling pathways play an essential role in protecting against cerebral ischemia. Mammalian target of rapamycin (mTOR), a major downstream target of Akt, regulates p70S6 kinase and S6, both of which are involved in ribosomal biogenesis and protein synthesis. I investigated whether ferulic acid regulates mTOR, p70S6 kinase, and S6 phosphorylation during brain ischemic injury. Rats were treated immediately with vehicle or ferulic acid (100mg/kg, i.v.) after middle cerebral artery occlusion (MCAO). Brains tissues were removed at 24h after the onset of MCAO and the cerebral cortex regions were collected. Ferulic acid reduced the MCAO-induced infarct volume. I showed previously that ferulic acid prevents the MCAO injury-induced decrease of Akt phosphorylation. In this study, MCAO injury induced decreases in mTOR, p70S6 kinase, and S6 phosphorylation levels, while ferulic acid attenuated the injury-induced decreases. Immunohistochemical staining demonstrated that ferulic acid prevented the MCAO-induced reduction in the number of positive cells for phosphorylated p70S6 kinase and phosphorylated S6. These findings suggest that ferulic acid has a neuroprotective function against focal cerebral ischemia by modulating p70S6 kinase expression and S6 phosphorylation.

Nicotinamide is an important cofactor in the prevention of brain damage during focal cerebral ischemia. Hippocalcin is a calcium buffer protein that modulates intracellular calcium concentration and attenuates apoptosis. In this study, we investigated whether nicotinamide modulates hippocalcin expression during cerebral ischemia. Male Sprague–Dawley rats were treated with vehicle or nicotinamide (500 mg/kg) 2 h after the onset of middle cerebral artery occlusion (MCAO) and cerebral cortex tissues were collected 24 h after MCAO. Nicotinamide treatment decreased infarct volume in the cerebral cortex of MCAO-operated animals. Our proteomic approach revealed a decrease in hippocalcin expression in vehicle-treated animals during MCAO, which was attenuated by nicotinamide treatment. We used RT-PCR and Western blot analyses to demonstrate that nicotinamide clearly restored the injury-induced decrease in hippocalcin expression. Glutamate toxicity also decreased hippocalcin levels in cultured hippocampal cells, while nicotinamide treatment prevented the glutamate exposure-induced decrease in hippocalcin levels. These results suggest that nicotinamide modulates hippocalcin expression in cerebral ischemic injury and consequently contributes to the prevention of neuronal cell death.

The aim of this study investigated whether nicotinamide affects parvalbumin expression in focal cerebral ischemic injury. Rats were treated with vehicle or nicotinamide (500 mg/kg) 2 hr after middle cerebral artery occlusion (MCAO), and cerebral cortex tissues were collected 24 hr after MCAO. Nicotinamide significantly decreases the volume of infarct areas in the cerebral cortex. A proteomic approach revealed that MCAO induces decreases of parvalbumin levels, while nicotinamide treatment prevents injury-induced decreases in parvalbumin. RT-PCR and Western blot analyses demonstrated that nicotinamide restores injury-induced decreases in parvalbumin. Moreover, immunohistochemical staining confirmed that the numbers of parvalbumin-positive cells were decreased in vehicle-treated animals with MCAO, and that nicotinamide averted this decrease. In cultured hippocampal cells, nicotinamide treatment prevents the glutamate exposure-induced increase in intracellular Ca(2+) concentration and decrease in parvalbumin expression. These results suggest the fact that the maintenance of parvalbumin expression is mediated to the neuroprotective function of nicotinamide against ischemic brain injury.

Intracellular calcium overload is a critical pathophysiological factor in ischemic injury. Hippocalcin is a neuronal calcium sensor protein that buffers intracellular calcium levels and protects cells from apoptotic stimuli. Ferulic acid exerts a neuroprotective effect in cerebral ischemia through its anti-oxidant and anti-inflammation activity. This study investigated whether ferulic acid contributes to hippocalcin expression during cerebral ischemia and glutamate exposure-induced neuronal cell death. Rats were immediately treated with vehicle or ferulic acid (100 mg/kg, i.v.) after middle cerebral artery occlusion (MCAO). Brain tissues were collected 24 h after MCAO and followed by assessment of cerebral infarct. Ferulic acid reduced MCAO-induced infarct regions. A proteomics approach elucidated a decrease in hippocalcin in MCAO-operated animals, ferulic acid attenuates the injury-induced decrease in hippocalcin expression. Reverse transcription-polymerase chain reaction and Western blot analyses confirmed that ferulic acid prevents the injury-induced decrease in hippocalcin. In cultured HT22 hippocampal cells, glutamate exposure increased the intracellular Ca(2+) levels, whereas ferulic acid attenuated this increase. Moreover, ferulic acid attenuated the glutamate toxicity-induced decrease in hippocalcin expression. These findings can suggest the possibility that ferulic acid exerts a neuroprotective effect through modulating hippocalcine expression and regulating intracellular calcium levels.

Ferulic acid plays a neuroprotective role in cerebral ischemia. The aim of this study was to identify the proteins that are differentially expressed following ferulic acid treatment during ischemic brain injury using a proteomics technique. Middle cerebral artery occlusion (MCAO) was performed to induce a focal cerebral ischemic injury in adult male rats, and ferulic acid (100 mg/kg) or vehicle was administered immediately after MCAO. Brain tissues were collected 24 hr after MCAO. The proteins in the cerebral cortex were separated using two-dimensional gel electrophoresis and were identified by mass spectrometry. We detected differentially expressed proteins between vehicle- and ferulic acid-treated animals. Adenosylhomocysteinase, isocitrate dehydrogenase [NAD(+)], mitogen-activated protein kinase kinase 1 and glyceraldehyde-3-phosphate dehydrogenase were decreased in the vehicle-treated group, and ferulic acid prevented the injury-induced decreases in these proteins. However, pyridoxal phosphate phosphatase and heat shock protein 60 were increased in the vehicle-treated group, while ferulic acid prevented the injury-induced increase in these proteins. It is accepted that these enzymes are involved in cellular metabolism and differentiation. Thus, these findings suggest evidence that ferulic acid plays a neuroprotective role against focal cerebral ischemia through the up- and down-modulation of specific enzymes.

Background: Ferulic acid provides a neuroprotective effect during cerebral ischemia through its anti-oxidant function. Protein phosphatase 2A (PP2A) is a serine and threonine phosphatase that contributes broadly to normal brain function. This study investigated whether ferulic acid regulates PP2A subunit B in a middle cerebral artery occlusion (MCAO) animal model and glutamate toxicity-induced neuronal cell death. Methodology/Principal Findings: MCAO was surgically induced to yield permanent cerebral ischemic injury in rats. The rats were treated with either vehicle or ferulic acid (100 mg/kg, i.v.) immediately after MCAO, and cerebral cortex tissues were collected 24 h after MCAO. A proteomics approach, RT-PCR, and Western blot analyses performed to identification of PP2A subunit B expression levels. Ferulic acid significantly reduced the MCAO-induced infarct volume of the cerebral cortex. A proteomics approach elucidated the reduction of PP2A subunit B in MCAO-induced animals, and ferulic acid treatment prevented the injury-induced reduction in PP2A subunit B levels. RT-PCR and Western blot analyses also showed that ferulic acid treatment attenuates the injury-induced decrease in PP2A subunit B levels. Moreover, the number of PP2A subunit B-positive cells was reduced in MCAO-induced animals, and ferulic acid prevented these decreases. In cultured neuronal cells, ferulic acid treatment protected cells against glutamate toxicity and prevented the glutamate-induced decrease in PP2A subunit B. Conclusions/Significance: These results suggest that the maintenance of PP2A subunit B by ferulic acid in ischemic brain injury plays an important role for the neuroprotective function of ferulic acid.

Melatonin has anti-oxidant activity and it exerts a neuroprotective effects during ischemic brain injury. Calcium-buffering proteins including parvalbumin and hippocalcin are involved in neuronal differentiation and maturation through calcium signaling. This study investigated whether melatonin moderates parvalbumin and hippocalcin expression in cerebral ischemia and glutamate toxicity-induced neuronal cell death. Focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO). Male Sprague-Dawley rats were treated with vehicle or melatonin (5 mg/kg) prior to MCAO, and cerebral cortical tissues were collected 24 hr after MCAO. Parvalbumin and hippocalcin levels were decreased in vehicle-treated animal with MCAO, whereas melatonin prevented the ischemic injury-induced reduction in these proteins. In cultured hippocampal cells, glutamate toxicity decreased parvalbumin and hippocalcin levels, while melatonin treatment prevented the glutamate exposure-induced diminished in these proteins levels. Melatonin also attenuated the glutamate toxicity-induced increase in intracellular Ca(2+) levels. These results suggest that the maintenance of parvalbumin and hippocalcin levels by melatonin in ischemic injury contributes to the neuroprotective effect of melatonin against neuronal cell damage.