Quercetin Reduces Ischemic Brain Injury by Preventing Ischemia-induced Decreases in the Neuronal Calcium Sensor Protein Hippocalcin.

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.

Quercetin attenuates the injury-induced reduction of γ-enolase expression in a middle cerebral artery occlusion animal model.

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.

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.

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.

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.