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Abstract Background Ischemic stroke is a serious neurological disorder caused by blockages in cerebral artery. Protein phosphatase 2A (PP2A) is a phosphatase that performs a critical role in cell signaling and growth. PP2A subunit B acts as a neuroprotective agent in the nerve system. Chlorogenic acid, which is mainly found in roasted coffee, has antioxidant, anti-inflammatory, and anti-apoptotic effects. We hypothesized that chlorogenic acid modulates PP2A subunit B expression in ischemic stroke models and glutamate-mediated neurons. Middle artery occlusion (MCAO) surgery was operated and chlorogenic acid (30 mg/kg) or phosphate buffer saline was treated 2 h after MCAO. The cerebral cortex was collected 24 h after surgery and the change of PP2A subunit B expression was analyzed. Glutamate and/or chlorogenic acid were treated in cultured neurons, further study was performed. Results A decrease in PP2A subunit B expression in MCAO animals was identified. Chlorogenic acid alleviated this decrease due to ischemic injury. Moreover, the number of PP2A subunit B-positive cells in the ischemic cerebral cortex was significantly decreased, chlorogenic acid alleviated this decrease. We also found protective effects of chlorogenic acid in neurons exposed to glutamate. Glutamate decreased the expression of PP2A subunit B and chlorogenic acid mitigated this decrease. Our results elucidated that chlorogenic acid performs neuroprotective functions and attenuates the reduction of PP2A subunit B by brain damage and glutamate-mediated excitotoxicity. Conclusions We showed that chlorogenic acid attenuated the decrease of PP2A subunit B in ischemic injury and neurons exposed to glutamate. Since PP2A subunit B contributes to the protection of brain tissue, we can suggest that chlorogenic acid preserves neurons by modulating PP2A subunit B during ischemic damage.

Abstract Background Cerebral ischemia is a serious neurological disorder that can lead to high morbidity and mortality. Chlorogenic acid is a polyphenol compound with antioxidant that can regulate proteins in cerebral ischemia. Middle cerebral artery occlusion (MCAO) surgery was performed to induce ischemic brain injury and was maintained for 24 h. Chlorogenic acid (30 mg/kg) or vehicle was administrated into the peritoneal cavity 2 h after MCAO surgery. The cerebral cortical tissues were collected for further study and a proteomic approach was performed to identify the proteins changed by chlorogenic acid in the MCAO animals. Results We found that chlorogenic acid alleviated in changes in adenosylhomocysteinase, glycerol-3-phosphate dehydrogenase, eukaryotic translation initiation factor 4A-II, apolipoprotein A-I, and mu-crystallin. These proteins were reduced in MCAO animals with vehicle, and these reductions were attenuated by chlorogenic acid treatment. The mitigation of this reduction by chlorogenic acid was confirmed by the reverse transcription PCR technique. These proteins are associated with energy metabolism, protein synthesis, inflammation, and physiological metabolism. They are involved in the neuroprotective effect of chlorogenic acid. These results showed that chlorogenic acid alleviates the neurological disorders caused by MCAO and regulates the expression of proteins involved in neuroprotection. Conclusions Therefore, our findings provide evidence that chlorogenic acid plays a neuroprotective role in stroke animal models by controlling specific proteins.

Ischemic stroke causes a lack of oxygen and glucose supply to brain, eventually leads to severe neurological disorders. Retinoic acid is a major metabolic product of vitamin A and has various biological effects. The PI3K-Akt signaling pathway is an important survival pathway in brain. Phosphorylated Akt is important in regulating survival and apoptosis. We examined whether retinoic acid has neuroprotective effects in stroke model by regulating Akt and its downstream protein, Bad. Moreover, we investigated the relationship between retinoic acid and Bcl-2 family protein interactions. Animals were intraperitoneally administered vehicle or retinoic acid (5 mg/kg) for four days before surgery and ischemic stroke was induced by middle cerebral artery occlusion (MCAO) surgery. Neurobehavioral tests were performed 24 h after MCAO and cerebral cortical tissues were collected. Cresyl violet staining and TUNEL histochemistry were performed, Western blot and immunoprecipitation analysis were performed to elucidate the expression of various proteins. Retinoic acid reduced neurological deficits and histopathological changes, decreased the number of TUNEL-positive cells, and alleviated reduction of phospho-PDK1, phospho-Akt, and phospho-Bad expression caused by MCAO damage. Immunoprecipitation analysis showed that MCAO damage reduced the interaction between phospho-Bad and 14-3-3, which was attenuated by retinoic acid. Furthermore, retinoic acid mitigated the increase in Bcl-2/Bad and Bcl-xL/Bad binding levels and the reduction in Bcl-2/Bax and Bcl-xL/Bax binding levels caused by MCAO damage. Retinoic acid alleviated MCAO-induced increase of caspase-3 and cleaved caspase-3 expression. We demonstrate that retinoic acid prevented apoptosis against cerebral ischemia through phosphorylation of Akt and Bad, maintenance of phospho-Bad and 14-3-3 binding, and regulation of Bcl-2 family protein interactions. .

Stroke is a leading cause of death and long-term disability which can cause oxidative damage and inflammation of the neuronal cells. Retinoic acid is an active metabolite of vitamin A that has various beneficial effects including antioxidant and anti-inflammatory effects. In this study, we investigated whether retinoic acid modulates oxidative stress and inflammatory factors in a stroke animal model. A middle cerebral artery occlusion (MCAO) was performed on adult male rats to induce focal cerebral ischemia. Retinoic acid (5 mg/kg) or vehicle was injected into the peritoneal cavity for four days before MCAO surgery. The neurobehavioral tests were carried out 24 h after MCAO and cerebral cortex tissues were collected. The cortical damage was assessed by hematoxylin-eosin staining and reactive oxygen species assay. In addition, Western blot and immunohistochemical staining were performed to investigate the activation of glial cells and inflammatory cytokines in MCAO animals. Ionized calcium-binding adapter molecule-1 (Iba-1) and glial fibrillary acidic protein (GFAP) were used as markers of microglial and astrocyte activation, respectively. Tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were used as representative pro-inflammatory cytokines. Results showed that MCAO damage caused neurobehavioral defects and histopathological changes in the ischemic region and increased oxidative stress. Retinoic acid treatment reduced these changes caused by MCAO damage. We detected increases in Iba-1 and GFAP in MCAO animals treated with vehicle. However, retinoic acid alleviated increases in Iba-1 and GFAP caused by MCAO damage. Moreover, MCAO increased levels of nuclear factor-κB and pro-inflammatory cytokines, including TNF-α and IL-1β. Retinoic acid alleviated the expression of these inflammatory proteins. These findings elucidate that retinoic acid regulates microglia and astrocyte activation and modulates pro-inflammatory cytokines. Therefore, this study suggests that retinoic acid exhibits strong antioxidant and anti-inflammatory properties by reducing oxidative stress, inhibiting neuroglia cell activation, and preventing the increase of pro-inflammatory cytokines in a cerebral ischemia.

Epigallocatechin gallate (EGCG) is a polyphenolic component of green tea that has anti-oxidative and anti-inflammatory effects in neurons. Ischemic stroke is a major neurological disease that causes irreversible brain disorders. It increases the intracellular calcium concentration and induces apoptosis. The regulation of intracellular calcium concentration is important to maintain the function of the nervous system. Hippocalcin is a neuronal calcium sensor protein that controls intracellular calcium concentration. We investigated whether EGCG treatment regulates the expression of hippocalcin in stroke animal model and glutamate-induced neuronal damage. We performed middle cerebral artery occlusion (MCAO) to induce cerebral ischemia. EGCG (50 mg/kg) or phosphate buffered saline was injected into the abdominal cavity just before MCAO surgery. The neurobehavioral tests were performed 24 h after MCAO surgery and cerebral cortex tissue was collected. MCAO damage induced severe neurobehavioral disorders, increased infarct volume, and decreased the expression of hippocalcin in the cerebral cortex. However, EGCG treatment improved these deficits and alleviated the decrease in hippocalcin expression in cerebral cortex. In addition, EGCG dose-dependently alleviated neuronal cell death and intracellular calcium overload in glutamate-exposed neurons. Glutamate exposure reduced hippocalcin expression, decreased Bcl-2 expression, and increased Bax expression. However, EGCG treatment mitigated these changes caused by glutamate toxicity. EGCG also attenuated the increase in caspase-3 and cleaved caspase-3 expressions caused by glutamate exposure. The effect of EGCG was more pronounced in non-transfected cells than in hippocalcin siRNA-transfected cells. These findings demonstrate that EGCG protects neurons against glutamate toxicity through the regulation of Bcl-2 family proteins and caspase-3. It is known that hippocalcin exerts anti-apoptotic effect through the modulation of apoptotic pathway. Thus, we can suggest evidence that EGCG has a neuroprotective effect by regulating hippocalcin expression in ischemic brain damage and glutamate-exposed cells.

Abstract Background Chlorogenic acid, a phenolic compound, has potent antioxidant and neuroprotective properties. The ubiquitin–proteasome system is an important regulators of neurodevelopment and modulators of neuronal function. This system is associated with neurodevelopment and neurotransmission through degradation and removal of damaged proteins. Activation of the ubiquitin–proteasome system is a critical factor in preventing cell death. We have previously reported a decrease in the activity of the ubiquitin–proteasome system during cerebral ischemia. This study investigated whether chlorogenic acid regulates the ubiquitin–proteasome system in an animal stroke model. In adult rats, middle cerebral artery occlusion (MCAO) surgery was performed to induce focal cerebral ischemia. Chlorogenic acid (30 mg/kg) or normal saline was injected into the abdominal cavity 2 h after MCAO surgery, and cerebral cortex tissues were collected 24 h after MCAO damage. Results Chlorogenic acid attenuated neurobehavioral disorders and histopathological changes caused by MCAO damage. We identified the decreases in ubiquitin C-terminal hydrolase L1, ubiquitin thioesterase OTUB1, proteasome subunit α type 1, proteasome subunit α type 3, and proteasome subunit β type 4 expression using a proteomics approach in MCAO animals. The decrease in these proteins was alleviated by chlorogenic acid. In addition, the results of reverse transcription-polymerase chain reaction confirmed these changes. The identified proteins were markedly reduced in MCAO damage, while chlorogenic acid prevented these reductions induced by MCAO. The decrease of ubiquitin–proteasome system proteins in ischemic damage was associated with neuronal apoptosis. Conclusions Our results showed that chlorogenic acid regulates ubiquitin–proteasome system proteins and protects cortical neurons from neuronal damage. These results provide evidence that chlorogenic acid has neuroprotective effects and maintains the ubiquitin–proteasome system in ischemic brain injury.

Abstract Background Retinoic acid is a major metabolite of vitamin A and exerts beneficial effects including anti-oxidant and anti-inflammatory activities in neurons. The ubiquitin–proteasome system is an important biological system that regulates cell survival. Ubiquitination regulates protein degradation and plays an important role in oxidative stress. Deubiquitinating enzymes cleave ubiquitin from proteins and control ubiquitination-induced degradation. We detected decreases in ubiquitin carboxy-terminal hydrolase L1, ubiquitin thioesterase OTUB1, and proteasome subunit alpha types 1 and 3 in cerebral ischemic damage. In this study, we investigated whether retinoic acid regulates the expression of deubiquitinating enzymes ubiquitin carboxy-terminal hydrolase L1, ubiquitin thioesterase OTUB1, and proteasome subunit alpha types 1 and 3 in cerebral ischemic injury. Right middle cerebral artery occlusion (MCAO) was performed to induce cerebral ischemic damage in male rats. Retinoic acid (5 mg/kg) or vehicle was intraperitoneally injected every day from 4 days before surgery. Neurological behavioral tests were performed 24 h after MCAO, and right cerebral cortical tissues were collected. Results MCAO damage caused neurological behavioral dysfunction, and retinoic acid alleviated these deficits. The identified proteins decreased in MCAO animals with vehicle, while retinoic acid treatment attenuated these decreases. The results of proteomic study were confirmed by a reverse transcription-PCR technique. Expressions of ubiquitin carboxy-terminal hydrolase L1, ubiquitin thioesterase OTUB1, and proteasome subunit alpha types 1 and 3 were decreased in MCAO animals treated with vehicle. Retinoic acid treatment alleviated these MCAO-induced reductions. The ubiquitin–proteasome system plays an essential role in maintaining cell function and preserving cell shape against ischemic damage. Conclusions These findings suggest that retinoic acid regulates ubiquitin- and proteasome-related proteins including ubiquitin carboxy-terminal hydrolase L1, ubiquitin thioesterase OTUB1, and proteasome subunit alpha types 1 and 3 in a brain ischemia model. Changes in these proteins are involved in the neuroprotective effects of retinoic acid.

Abstract Background Calcium is a critical factor involved in modulation of essential cellular functions. Parvalbumin is a calcium buffering protein that regulates intracellular calcium concentrations. It prevents rises in calcium concentrations and inhibits apoptotic processes during ischemic injury. Quercetin exerts potent antioxidant and anti-apoptotic effects during brain ischemia. We investigated whether quercetin can regulate parvalbumin expression in cerebral ischemia and glutamate toxicity-induced neuronal cell death. Adult male rats were treated with vehicle or quercetin (10 mg/kg) 30 min prior to middle cerebral artery occlusion (MCAO) and cerebral cortical tissues were collected 24 h after MCAO. We used various techniques including Western blot, reverse transcription-PCR, and immunohistochemical staining to elucidate the changes of parvalbumin expression. Results Quercetin ameliorated MCAO-induced neurological deficits and behavioral changes. Moreover, quercetin prevented MCAO-induced a decrease in parvalbumin expression. Conclusions These findings suggest that quercetin exerts a neuroprotective effect through regulation of parvalbumin expression.

Abstract Baicalin is a natural flavonoid that exerts a variety of pharmaceutical effects such as anti-inflammatory and antioxidant. Lipopolysaccharide (LPS) is an endotoxin that releases inflammatory cytokines and induces inflammatory response. This study was investigated the anti-inflammatory mechanism of baicalin against LPS-induced inflammatory response in the hippocampus. Adult mice were randomly grouped into control, LPS-treated, and LPS and baicalin co-treated animals. LPS (250 μg/kg/day) and baicalin (10 mg/kg/day) were administered intraperitoneally for 7 consecutive days. We measured neuroglia cells activation and inflammatory factors activation using Western blot analysis and immunofluorescence staining techniques. Ionized calcium binding adaptor molecule-1 (Iba-1) and glial fibrillary acidic protein (GFAP) are widely used as microglia and astrocyte markers, respectively. LPS treatment increased Iba-1 and GFAP expression, while baicalin co-treatment attenuated this overexpression. Nuclear factor-kappa B (NF-κB) is a key mediator of inflammation. Baicalin co-treatment alleviated LPS-induced increase of NF-κB in the hippocampus. In addition, LPS treatment upregulated pro-inflammatory cytokines including interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). However, baicalin co-treatment prevented LPS-induced increases of IL-1β and TNF-α in the hippocampus. Results from the present study showed that baicalin suppresses LPS-induced neuroinflammation by regulating microglia and astrocyte activation and modulating inflammatory factors in the hippocampus. Thus, these results demonstrate that baicalin has neuroprotective effect by alleviates microglia and astrocyte activation and modulates inflammatory response by suppressing NF-κB expression in hippocampus with neuroinflammation caused by LPS.

Abstract Glutamate induces neurotoxicity during brain development, causing nerve damage. Protein phosphatase 2A (PP2A) is a type of serine/threonine phosphatase that regulates various biological functions. Among the PP2A subunit types, subunit B is abundant in brain tissue and plays an essential role in the nervous system. This study investigated changes in PP2A subunit B expression through glutamate exposure in the cerebral cortex of newborn rats. Sprague-Dawley rat pups (7 days after birth) were injected intraperitoneally with vehicle or glutamate (10 mg/kg). After 4 h of drug treatment, the brain tissue was isolated and fixed for morphological study. In addition, the cerebral cortex was collected for RNA and protein works. We observed severe histopathological changes including swollen neuron and atrophied dendrite in the glutamate exposed cerebral cortex. Glutamate exposure leads to a decrease in PP2A subunit B. Reverse-transcription PCR and Western blot analyses confirmed that glutamate induces a decrease of PP2A subunit B in the cerebral cortex of newborn rats. Moreover, immunohistochemical study showed a decrease in PP2A subunit B positive cells. The reduction of PP2A subunit B expression is considered an indicator of neurodegenerative damage. These results suggest that glutamate exposure causes neuronal damage in the cerebral cortex of new born rats through a decrease in PP2A subunit B.

Abstract Glutamate is a representative excitatory neurotransmitter. However, excessive glutamate exposure causes neuronal cell damage by generating neuronal excitotoxicity. Excitotoxicity in neonates caused by glutamate treatment induces neurological deficits in adults. The 14–3-3 family proteins are conserved proteins that are expressed ubiquitously in a variety of tissues. These proteins contribute to cellular processes, including signal transduction, protein synthesis, and cell cycle control. We proposed that glutamate induces neuronal cell damage by regulating 14–3-3 protein expression in newborn animals. In this study, we investigated the histopathological changes and 14–3-3 proteins expressions as a result of glutamate exposure in the neonatal cerebral cortex. Rat pups at post-natal day 7 were intraperitoneally administrated with vehicle or glutamate (10 mg/kg). Animals were sacrificed 4 h after treatment, and brain tissues were fixed for histological study. Cerebral cortices were isolated and frozen for proteomic study. We observed serious histopathological damages including shrunken dendrites and atypical neurons in glutamate-treated cerebral cortices. In addition, we identified that 14–3-3 family proteins decreased in glutamate-exposed cerebral cortices using a proteomic approach. Moreover, Western blot analysis provided results that glutamate treatment in neonates decreased 14–3-3 family proteins expressions, including the β/α, ζ/δ, γ, ε, τ, and η isoforms. 14–3-3 proteins are involved in signal transduction, metabolism, and anti-apoptotic functions. Thus, our findings suggest that glutamate induces neonatal neuronal cell damage by modulating 14–3-3 protein expression.

Jellyfish stings can result in local tissue damage and systemic pathophysiological sequelae. Despite constant occurrences of jellyfish stings in oceans throughout the world, the toxinological assessment of these jellyfish envenomations has not been adequately reported in quantitative as well as in qualitative measurements. Herein, we have examined and compared the in vivo toxic effects and pathophysiologic alterations using experimental animal models for two representative stinging jellyfish classes, i.e., Cubozoa and Scyphozoa. For this study, mice were administered with venom extracts of either Carybdea brevipedalia (Cnidaria: Cubozoa) or Nemopilema nomurai (Cnidaria: Scyphozoa). From the intraperitoneal (IP) administration study, the median lethal doses leading to the deaths of mice 24 h post-treatment after (LD50) for C. brevipedalia venom (CbV) and N. nomurai venom (NnV) were 0.905 and 4.4697 mg/kg, respectively. The acute toxicity (i.e., lethality) of CbV was much higher with a significantly accelerated time to death value compared with those of NnV. The edematogenic activity induced by CbV was considerably (83.57/25 = 3.343-fold) greater than NnV. For the evaluation of their dermal toxicities, the epidermis, dermis, subcutaneous tissues, and skeletal muscles were evaluated toxinologically/histopathologically following the intradermal administration of the venoms. The minimal hemorrhagic doses (MHD) of the venoms were found to be 55.6 and 83.4 μg/mouse for CbV and NnV, respectively. Furthermore, the CbV injection resulted in extensive alterations of mouse dermal tissues, including severe edema, and hemorrhagic/necrotic lesions, with the minimum necrotizing dose (MND) of 95.42 µg/kg body weight. The skin damaging effects of CbV appeared to be considerably greater, compared with those of NnV (MND = 177.99 µg/kg). The present results indicate that the toxicities and pathophysiologic effects of jellyfish venom extracts may vary from species to species. As predicted from the previous reports on these jellyfish envenomations, the crude venom extracts of C. brevipedalia exhibit much more potent toxicity than that of N. nomurai in the present study. These observations may contribute to our understanding of the toxicities of jellyfish venoms, as well as their mode of toxinological actions, which might be helpful for establishing the therapeutic strategies of jellyfish stings.

Abstract Glutamate leads to neuronal cell damage by generating neurotoxicity during brain development. The objective of this study is to identify proteins that differently expressed by glutamate treatment in neonatal cerebral cortex. Sprague-Dawley rat pups (post-natal day 7) were intraperitoneally injected with vehicle or glutamate (10 mg/kg). Brain tissues were isolated 4 h after drug treatment and fixed for morphological study. Moreover, cerebral cortices were collected for protein study. Two-dimensional gel electrophoresis and mass spectrometry were carried out to identify specific proteins. We observed severe histopathological changes in glutamate-exposed cerebral cortex. We identified various proteins that differentially expressed by glutamate exposure. Identified proteins were thioredoxin, peroxiredoxin 5, ubiquitin carboxy-terminal hydrolase L1, proteasome subunit alpha proteins, isocitrate dehydrogenase, and heat shock protein 60. Heat shock protein 60 was increased in glutamate exposed condition. However, other proteins were decreased in glutamate-treated animals. These proteins are related to anti-oxidant, protein degradation, metabolism, signal transduction, and anti-apoptotic function. Thus, our findings can suggest that glutamate leads to neonatal cerebral cortex damage by regulation of specific proteins that mediated with various functions.

Abstract Lipopolysaccharide (LPS) acts as an endotoxin, releases inflammatory cytokines, and promotes an inflammatory response in various tissues. This study investigated whether LPS modulates neuroglia activation and nuclear factor kappa B (NF-κB)-mediated inflammatory factors in the cerebral cortex. Adult male mice were divided into control animals and LPS-treated animals. The mice received LPS (250 μg/kg) or vehicle via an intraperitoneal injection for 5 days. We confirmed a reduction of body weight in LPS-treated animals and observed severe histopathological changes in the cerebral cortex. Moreover, we elucidated increases of reactive oxygen species and oxidative stress levels in LPS-treated animals. LPS administration led to increases of ionized calcium-binding adaptor molecule-1 (Iba-1) and glial fibrillary acidic protein (GFAP) expression. Iba-1 and GFAP are well accepted as markers of activated microglia and astrocytes, respectively. Moreover, LPS exposure induced increases of NF-κB and pro-inflammatory factors, such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). Increases of these inflammatory mediators by LPS exposure indicate that LPS leads to inflammatory responses and tissue damage. These results demonstrated that LPS activates neuroglial cells and increases NF-κB-mediated inflammatory factors in the cerebral cortex. Thus, these findings suggest that LPS induces neurotoxicity by increasing oxidative stress and activating neuroglia and inflammatory factors in the cerebral cortex.

Abstract Cerebral ischemia is a major cause of neurodegenerative disease. It induces neuronal vulnerability and susceptibility, and leads to neuronal cell death. Resveratrol is a polyphenolic compound that acts as an anti-oxidant. It exerts a neuroprotective effect against focal cerebral ischemic injury. Akt signaling pathway is accepted as a representative cell survival pathway, including proliferation, growth, and glycogen synthesis. This study investigated whether resveratrol regulates Akt/glycogen synthase kinase-3β (GSK-3β) pathway in a middle cerebral artery occlusion (MCAO)-induced ischemic brain injury. Adult male rats were intraperitoneally injected with vehicle or resveratrol (30 mg/kg) and cerebral cortices were isolated 24 h after MCAO. Neurological behavior test, corner test, brain edema measurment, and 2,3,5-triphenyltetrazolium chloride staining were performed to elucidate the neuroprotective effects of resveratrol. Phospho-Akt and phospho-GSK-3β expression levels were measured using Western blot analysis. MCAO injury led to severe neurobehavioral deficit, infraction, and histopathological changes in cerebral cortex. However, resveratrol treatment alleviated these changes caused by MCAO injury. Moreover, MCAO injury induced decreases in phospho-Akt and phospho-GSK-3β protein levels, whereas resveratrol attenuated these decreases. Phosphorylations of Akt and GSK-3β act as a critical role for the suppression of apoptotic cell death. Thus, our finding suggests that resveratrol attenuates neuronal cell death in MCAO-induced cerebral ischemia and Akt/GSK-3β signaling pathway contributes to the neuroprotective effect of resveratrol.

Abstract Hyperglycemia is one of the major risk factors for stroke. Hyperglycemia can lead to a more extensive infarct volume, aggravate neuronal damage after cerebral ischemia. α-Synuclein is especially abundant in neuronal tissue, where it underlies the etiopathology of several neurodegenerative diseases. This study investigated whether hyperglycemic conditions regulate the expression of α-synuclein in middle cerebral artery occlusion (MCAO)-induced cerebral ischemic injury. Male Sprague-Dawley rats were treated with streptozotocin (40 mg/kg) via intraperitoneal injection to induce hyperglycemic conditions. MCAO were performed four weeks after streptozotocin injection to induce focal cerebral ischemia, and cerebral cortex tissues were obtained 24 hours after MCAO. We confirmed that MCAO induced neurological functional deficits and cerebral infarction, and these changes were more extensive in diabetic animals compared to non-diabetic animals. Moreover, we identified a decrease in α-synuclein after MCAO injury. Diabetic animals showed a more serious decrease in α-synuclein than non-diabetic animals. Western blot and reverse-transcription PCR analyses confirmed more extensive decreases in α-synuclein expression in MCAO-injured animals with diabetic condition than these of non-diabetic animals. It is accepted that α- synuclein modulates neuronal cell death and exerts a neuroprotective effect. Thus, the results of this study suggest that hyperglycemic conditions cause more serious brain damage in ischemic brain injuries by decreasing α-synuclein expression.

Abstract Quercetin, a natural flavonoid, copiously exists in vegetable, fruits and tea. Quercetin is beneficial to neurodegenerative disorders via its strong anti-oxidant and anti-inflammatory activities. γ-Enolase is one of the enzymes of glycolytic pathway and is predominantly expressed in neuronal cells. The aim of the present study is to verify whether quercetin modulates the expression of γ-enolase in brain ischemic injury. Adult Sprague-Dawley male rats were subjected to middle cerebral artery occlusion (MCAO) and quercetin (50 mg/kg) or vehicle was administered by intraperitoneal injection at 1 h before MCAO onset. A proteomics study, Western blot analysis, reversetranscription-PCR, and immunofluorescence staining were conducted to investigate the change of γ-enolase expression level. We identified a decline in γ-enolase expression in MCAO-operated animal model using a proteomic approach. However, quercetin treatment significantly attenuated this decline. These results were confirmed using Western blot analysis, reverse transcription-PCR, and immunofluorescence staining techniques. γ-Enolase is accepted as a neuron specific energy synthesis enzyme, and quercetin modulates γ-enolase in a MCAO animal model. Thus, our findings can suggest the possibility that quercetin regulates γ-enolase expression in response to cerebral ischemia, which likely contributes to the neuroprotective effect of quercetin.

Abstract α-Synuclein is abundantly expressed in neuronal tissue, plays an essential role in the pathogenesis of neurodegenerative disorders, and exerts a neuroprotective effect against oxidative stress. Cerebral ischemia causes severe neurological disorders and neuronal dysfunction. In this study, we examined α-synuclein expression in middle cerebral artery occlusion (MCAO)-induced cerebral ischemic injury and neuronal cells damaged by glutamate treatment. MCAO surgical operation was performed on male Sprague-Dawley rats, and brain samples were isolated 24 hours after MCAO. We confirmed neurological behavior deficit, infarction area, and histopathological changes following MCAO injury. A proteomic approach and Western blot analysis demonstrated a decrease in α-synuclein in the cerebral cortices after MCAO injury. Moreover, glutamate treatment induced neuronal cell death and decreased α-synuclein expression in a hippocampal-derived cell line in a dose-dependent manner. It is known that α-synuclein regulates neuronal survival, and low levels of α-synuclein expression result in cytotoxicity. Thus, these results suggest that cerebral ischemic injury leads to a reduction in α-synuclein and consequently causes serious brain damage.

Abstract Ischemic stroke is one of the leading causes of adult disability and death. Hyperglycemia is associated with an increased risk of stroke and poor outcomes after brain injury. Dynamin-like protein I (DLP-1) regulates mitochondrial fission and promotes mitochondrial dynamics. Neurodegenerative diseases are associated with mitochondrial dysfunction, and the downregulation of DLP-1 has been previously identified in a stroke animal model. Here, we investigated the changes in DLP-1 protein expression in an animal model of focal cerebral ischemia with induced hyperglycemia. Streptozotocin (40 mg/kg) was intraperitoneally injected into male rats to induce hyperglycemia, and middle cerebral artery occlusion (MCAO) was surgically induced 4 weeks after streptozotocin treatment. Brain tissue was isolated 24 hours after MCAO, and cerebral cortex samples were used for this study. Proteomics revealed a decrease in DLP-1 expression in MCAO animals when compared with controls, and this downregulation was more prominent in MCAO animals with hyperglycemia. Reverse-transcription polymerase chain reaction and Western blot analyses confirmed that DLP-1 was significantly downregulated in MCAO-injured animals with hyperglycemia compared to those without hyperglycemia. The decrease in DLP-1 indicates mitochondrial morphological changes and dysfunction. Together, these results suggest that the severe decrease of DLP-1 seen after brain injury under hyperglycemic conditions may exacerbate the damage to the brain.

Abstract Diabetes is a major risk factor for stroke and is also associated with worsened outcomes following a stroke. Peroxiredoxin-2 exerts potent neuroprotective effects against oxidative stress. In the present study, we identified altered peroxiredoxin-2 expression in an ischemic stroke model under hyperglycemic conditions. Adult male rats were administrated streptozotocin (40 mg/kg) via intraperitoneal injection to induce diabetes. Middle cerebral artery occlusion (MCAO) was induced surgically 4 weeks after streptozotocin treatment and cerebral cortex tissues were isolated 24 hours after MCAO. Peroxiredoxin-2 expression was evaluated in the cerebral cortex of MCAO-operated animals using a proteomics approach, and was found to be decreased. In addition, the reduction in peroxiredoxin-2 levels was more severe in cerebral ischemia with diabetes compared to animals without diabetes. Reverse-transcriptase PCR and Western blot analyses confirmed the significantly reduced peroxiredoxin-2 expression in MCAO-operated animals under hyperglycemic conditions. It is an accepted fact that peroxiredoxin-2 has antioxidative activity against ischemic injury. Thus, the findings of this study suggest that a more severe reduction in peroxiredoxin-2 under hyperglycemic conditions leads to worsened brain damage during cerebral ischemia with diabetes.

Stroke is the significant cause of human mortality and sufferings depending upon race and demographic location. Melatonin is a potent antioxidant that exerts protective effects in differential experimental stroke models. Several mechanisms have been previously suggested for the neuroprotective effects of melatonin in ischemic brain injury. The aim of this study is to investigate whether melatonin treatment affects the glutamate N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor signaling in cerebral cortex and striatum 24 h after permanent middle cerebral artery occlusion (MCAO). Melatonin (5 mg/kg) attenuated ischemia-induced down regulation of NMDA receptor 2 (NR2a), postsynaptic density-95 (PSD95) and increases NR2a/PSD95 complex association, which further activates the pro-survival PI3K/Akt/GSK3β pathway with mitigated collapsin response mediator protein 2 (CRMP2) phosphorylation. Furthermore, melatonin increases the expression of γ-enolase, a neurotrophic factor in ischemic cortex and striatum, and preserve the expression of presynaptic (synaptophysin and SNAP25) and postsynaptic (p-GluR1845) protein. Our study demonstrated a novel neuroprotective mechanism for melatonin in ischemic brain injury which could be a promising neuroprotective agent for the treatment of ischemic stroke.

Abstract Dynamin-like protein I (DLP-1) is an important mitochondrial fission and fusion protein that is associated with apoptotic cell death in neurodegenerative diseases. In this study, we investigated DLP-1 expression in a focal cerebral ischemia animal model and glutamate-exposed hippocampal-derived cell line. Middle cerebral artery occlusion (MCAO) was surgically induced in adult male rats to induce focal cerebral ischemic injury. Brain tissues were collected 24 hours after the onset of MCAO. MCAO induces an increase in infarct volume and histopathological changes in the cerebral cortex. We identified a decrease in DLP-1 in the cerebral cortices of MCAO-injured animals using a proteomic approach and Western blot analysis. Moreover, glutamate treatment significantly decreased DLP-1 expression in a hippocampalderived cell line. The decrease in DLP-1 indicates mitochondrial dysfunction. Thus, these results suggest that neuronal cell injury induces a decrease in DLP-1 levels and consequently leads to neuronal cell death.

Ischemic stroke is characterized by permanent or transient obstruction of blood flow, which initiates a cascading pathological process, starting from acute ATP loss to subsequent membrane depolarization, glutamate excitotoxicity, and calcium overload. Melatonin is a potent antioxidant that exerts protective effects in different experimental stroke models. In this study, melatonin effects were demonstrated by a proteomic and in silico approach. The proteomic study identified differentially expressed proteins by 2D gel electrophoresis in the striatum 24 h after middle cerebral artery occlusion. Proteomic analysis revealed several proteins with aberrant expression and was validated by western blot and immunofluorescence analysis. Homology modeling was performed to build 3D structures for γ-enolase, thioredoxin (TRX), and heat shock 60 (HSP60) by the template crystal structures using a protein data bank as a sequence database. The structure refinement of each model was achieved by energy minimization via molecular dynamic simulation, and the generated models were further assessed for stability by Procheck and ProSA. The models were processed for docking analysis using AutoDock Vina, and post-docking analysis was determined by discovery studio. The proteomic study showed decreased expression of γ-enolase, TRX, and protein phosphatase 2A subunit B and increased expression of collapsin response mediator protein 2 and HSP60 in the striatum after ischemic injury. Treatment with melatonin modulated the expression profiles of these proteins. This study demonstrated the neuroprotective role of melatonin in the ischemic striatum using a proteomic and in silico approach. Collectively, melatonin may act in a multimechanistic way by modulating the expression of several proteins in the ischemic striatum.

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.

Background: 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. Objectives: 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. Methods: 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. Results: 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. Conclusions: 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. [ABSTRACT FROM AUTHOR]