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 antioxi‑ dant, 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 chlo‑ rogenic acid in neurons exposed to glutamate. Glutamate decreased the expression of PP2A subunit B and chloro‑ genic 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 sug‑ gest that chlorogenic acid preserves neurons by modulating PP2A subunit B during ischemic damage.

Stroke is a major cause of death and long-term disability. Chlorogenic acid is a phenolic compound with a potent neuroprotective effect. γ-enolase is a phosphopyruvate hydratase found in mature neurons and plays an important role in the neuronal survival. This study investigated whether chlorogenic acid regulates the expression of γ-enolase during cerebral ischemia. Middle cerebral artery occlusion (MCAO) was performed to indcue cerebral ischemia. Adult male rats were used and chlorogenic acid (30 mg/kg) or phosphate buffered saline (PBS) was injected intraperitoneally 2 hours after MCAO surgery. Cerebral cortical tissues were collected 24 hours after MCAO surgery. Our proteomic approach identified the reduction of γ-enolase caused by MCAO damage and the mitigation of this reduction by chlorogenic acid treatment. Results of reverse transcription-polymerase chain reaction and Western blot analyses showed decrease in γ-enolase expression in PBS-treated MCAO group. However, chlorogenic acid treatment attenuated this decrease. Results of immunofluorescence staining showed the change of γ-enolase by chlorogenic acid treatment. These results demonstrated that chlorogenic acid regulates the γ-enolase expression during MCAO-induced ischemia. Therefore, we suggest that chlorogenic acid mediates the neuroprotective function by regulating the γ-enolase expression in cerebral ischemia and may be used as a therapeutic agent for brain diseases including stroke.

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.

Background: Epigallocatechin gallate (EGCG) is a flavonoid compound commonly found in green tea. It exhibits antioxidant, anti-inflammatory, and neuroprotective effects in cerebral ischemia. Protein phosphatase 2 A (PP2A) is an important serine/threonine phosphatase enzyme involved in various cellular activities. PP2A subunit B is present abundantly in the brain and plays an important role in the nervous system. We investigated the effect of EGCG on the expression level of PP2A subunit B in cerebral ischemia caused by middle cerebral artery occlusion (MCAO). EGCG (50 mg/kg) or vehicle was injected into the peritoneal cavity prior to MCAO surgery. Neurological behavior tests were performed 24 h after MCAO, and right cerebral cortex tissue was collected. Cerebral ischemia caused serious neurological abnormalities, which were alleviated by EGCG administration. We screened the expression of PP2A subunits containing A, B, and C using reverse-transcription PCR. We confirmed that PP2A subunit B exhibited significant changes in MCAO animals compared to subunits A and C. We continuously examined the expression of PP2A subunit B protein in MCAO animals using Western blot analysis. Results: EGCG alleviated the reduction of PP2A subunit B protein by MCAO damage. In addition, immunohistochemistry demonstrated a decrease in the number of PP2A subunit B-positive cells in the cerebral cortex, and EGCG attenuated this decrease. Maintenance of PP2A subunit B is important for normal brain function. Conclusion Therefore, our findings suggest that EGCG exerts neuroprotective effects against cerebral ischemia through modulation of PP2A subunit B expression.

Background: 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. Objectives: We investigated whether quercetin regulates apoptosis by modulating parvalbumin expression in glutamate induced neuronal damage. Methods: 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. Results: 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. Conclusions 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.

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 phosphoBad and 14-3-3 in ischemic stroke model.

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 dam‑ aged proteins. Activation of the ubiquitin–proteasome system is a critical factor in preventing cell death. We have pre‑ viously 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 pro‑ tects cortical neurons from neuronal damage. These results provide evidence that chlorogenic acid has neuroprotec‑ tive effects and maintains the ubiquitin–proteasome system in ischemic brain injury.

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.

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 transcriptionPCR, 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.

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 transcriptionPCR, 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.