Identification of proteins in hyperglycemia and stroke animal models.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: These results suggest that the diabetic condition may exacerbate brain damage during focal cerebral ischemia through the downregulation of metabolism-related 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.

Amyloid ß-Peptide Effects on Glucose Regulation Are Dependent on Apolipoprotein E Genotype.

The apolipoprotein E gene (APOE) confers the greatest genetic risk factor for Alzheimer's disease (AD), wherein the ε4 allele confers an elevated risk compared with the ε3 allele. Biological mechanisms that differ across these alleles have been explored in mouse models wherein the murine Apoe gene has undergone targeted replacement with sequences encoding human ApoE3 or ApoE4 (ApoE-TR mice). Such models have indicated that the two variants of ApoE produce differential effects on energy metabolism, including metabolic syndrome. However, glucose regulation has not been compared in ApoE-TR mice with and without amyloid ß-peptide (Aß) accumulation. We crossed ApoE3-TR and ApoE4-TR mice with a transgenic line that accumulates human Aß1-42 In male ApoE3-TR mice, introduction of Aß caused aberrations in glucose tolerance and in membrane translocation of astrocytic glucose transporter 1 (GLUT1). Phosphorylation of Tau at AD-relevant sites was correlated with glucose intolerance. These effects appeared independent of insulin dysregulation and were not observed in females. In ApoE4-TR mice, the addition of Aß had no significant effects because of a trend toward perturbation of the baseline values.

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

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, gamma-enolase, stathmin, thioredoxin, peroxiredoxin-6, hippocalcin, protein phosphatase 2A, adenosylhomocysteinase, ubiquitin carboxy-terminal hydrolase L1, and NAD-specific isocitrate dehydrogenase subunit alpha 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.

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.

Lactic acid bacterial fermentation increases the antiallergic effects of Ixeris dentata.

Ixeris dentata (ID, family Asteraceae), called Seumbakuy in Korea, was fermented with lactic acid bacteria (LAB) and their antiallergic activities were investigated. Fermentation of ID with Bifidobacterium breve or Lactobacillus acidophilus increased its inhibition of degranulation in RBL-2H3 cells induced by the IgE-antigen complex. Oral administration of these extracts to mice inhibited the passive cutaneous anaphylaxis (PCA) reaction induced by the IgE-antigen complex and scratching behaviors induced by compound 48/80. The fermented ID more potently inhibited the PCA reaction and scratching behaviors than the non-fermented one. These extracts also inhibited mRNA expression of TNF-alpha and IL-4, as well as NF-kappaB activation in RBL-2H3 cells induced by the IgE-antigen complex. These findings suggest that LAB fermentation improves ID-mediated inhibition of IgE-induced allergic diseases such as rhinitis and asthma, and that ID works by inhibiting degranulation and NF-kB activation in mast cells and basophils.

Streptozotocin-induced diabetes decreases placenta growth factor (PlGF) levels in rat placenta.

The placenta produces several growth factors, including placenta growth factor (PlGF), which are essential for placenta growth and fetal growth. Diabetic pregnancy induces the abnormal placental growth and fetal development. This study investigated whether diabetes in pregnant rats induces changes in PlGF expression in the placenta. Diabetes was induced by a single intravenous injection of streptozotocin (35 mg/kg body weight) on day 0 of pregnancy, blood and tissue samples were collected on day 20 of pregnancy. In the diabetic group, maternal body weight and fetal weight significantly decreased compared to controls. RT-PCR and Western blot analyses showed that expression of PlGF was significantly decreased in placenta by streptozotocin treatment. Immunohistochemical study showed that the positive signal of PlGF in trophoblast cells was decreased in the diabetic group compared to controls. These findings demonstrate the decline of PlGF in the placenta in diabetic pregnancy.

Hyperglycemia aggravates decreases of PEA-15 and its two phosphorylated forms in cerebral ischemia.

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.

Hyperglycemia decreases expression of 14-3-3 proteins in an animal model of stroke.

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 ß/α, 14-3-3 ζ/δ, 14-3-3 γ, and 14-3-3 ε 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 ß/α, ζ/δ, γ, ε, 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 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 regulates the AKT/GSK-3ß/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-3ß (GSK-3ß) regulates CRMP-2 function through phosphorylation of CRMP-2. Moreover, the pro-apoptotic activity of GSK-3ß 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-3ß, 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-3ß, 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-3ß/CRMP-2 signaling pathway in focal cerebral ischemic injury, thereby protecting against brain injury.

Nicotinamide prevents the down-regulation of MEK/ERK/p90RSK signaling cascade in brain ischemic injury.

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.

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.

Identification of proteins regulated by ferulic acid in a middle cerebral artery occlusion animal model-a proteomics approach.

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.

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.

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.