Primary Cultured Rat Cortical Cells Research Articles

Neuroprotective effects of antioxidative flavonoids, quercetin, (+)-dihydroquercetin and quercetin 3-methyl ether, isolated from Opuntia ficus-indica var. saboten

The flavonoids quercetin, (+)-dihydroquercetin, and quercetin 3-methyl ether were isolated from the ethyl acetate fractions of the fruits and stems of Opuntia ficus-indica var. saboten. In the present study, we evaluated their protective effects against oxidative neuronal injuries induced in primary cultured rat cortical cells and their antioxidant activities by using three different cell-free bioassays. Quercetin was found to inhibit H 2O 2- or xanthine (X)/xanthine oxidase (XO)-induced oxidative neuronal cell injury, with an estimated IC 50 of 4–5 μg/ml. However, it was no more protective at concentrations of 30 μg/ml and above. (+)-Dihydroquercetin concentration-dependently inhibited oxidative neuronal injuries, but it was less potent than quercetin. On the other hand, quercetin 3-methyl ether potently and dramatically inhibited H 2O 2- and X/XO-induced neuronal injuries, with IC 50 values of 0.6 and 0.7 μg/ml, respectively. All three principles markedly inhibited lipid peroxidation and scavenged 1,1-diphenyl-2-picrylhydrazyl free radicals. In addition, quercetin and quercetin 3-methyl ether were shown to inhibit XO activity in vitro, with respective IC 50 values of 10.67 and 42.01 μg/ml. These results indicate that quercetin, (+)-dihydroquercetin, and quercetin 3-methyl ether are the active antioxidant principles in the fruits and stems of Opuntia ficus-indica var. saboten exhibiting neuroprotective actions against the oxidative injuries induced in cortical cell cultures. Furthermore, quercetin 3-methyl ether appears to be the most potent neuroprotectant of the three flavonoids isolated from this plant.

Synthesis and Evaluation of 4-Hydroxyphenylacetic Acid Amides and 4-Hydroxycinnamamides as Antioxidants

4-Hydroxyphenylacetic acid amides and 4-hydroxycinnamamides were synthesized and their antioxidant and neuroprotective activities were evaluated. Among the prepared compounds, 6f , 6g , 8b, and 9 exhibited potent inhibition of lipid peroxidation in rat brain homogenates, and marked DPPH radical scavenging activities. Furthermore, 6f , 6g , and 9 exhibited neuroprotective action against the oxidative damage induced by the exposure of primary cultured rat cortical cells to H 2O 2, xanthine/xanthine oxidase, or Fe 2+/ascorbic acid. Based on these results, we found that 6f was the most potent antioxidant among the compounds tested.

Protection of cultured rat cortical neurons from excitotoxicity by asarone, a major essential oil component in the rhizomes of Acorus gramineus

Previous reports have shown that the methanol extract and the essential oil from Acori graminei Rhizoma (AGR) inhibited excitotoxic neuronal cell death in primary cultured rat cortical cells. In the present study, an active principle was isolated from the methanol extract by biological activity-guided fractionations and identified as asarone. We evaluated neuroprotective actions and action mechanisms of the isolated asarone as well as the α- and the β-asarone obtained commercially. The isolated asarone inhibited the excitotoxicity induced by the exposure of cortical cultures for 15 min to 300 μM NMDA in a concentration-dependent manner, with the IC 50 of 56.1 μg/ml. The commercially obtained α- and β-asarone exhibited more potent inhibitions of the NMDA-induced excitotoxicity than the isolated asarone. Their respective IC 50 values were 18.2 and 26.5 μg/ml. The excitotoxicity induced by glutamate (Glu) was also inhibited, but with much less potency than the toxicity induced by NMDA. The IC 50 values for the α-, β-, and the isolated asarone were 89.7, 121.7, and 279.5 μg/ml, respectively. Based on the receptor-ligand binding studies using a use-dependent NMDA receptor-channel blocker [ 3H]MK-801, asarone inhibited the specific bindings in a concentration-dependent fashion. These results indicate that asarone, the major essential oil component in AGR, exhibits neuroprotective action against the NMDA- or Glu-induced excitotoxicity through the blockade of NMDA receptor function. The α-asarone was found to exhibit more potent inhibition of [ 3H]MK-801 bindings, which is consistent with its more potent neuroprotective action than the β- or the isolated asarone.

Arctigenin protects cultured cortical neurons from glutamate‐induced neurodegeneration by binding to kainate receptor

We previously reported that arctigenin, a lignan isolated from the bark of Torreya nucifera, showed significant neuroprotective activity against glutamate-induced toxicity in primary cultured rat cortical cells. In this study, the mode of action of arctigenin was investigated using primary cultures of rat cortical cells as an in vitro system. Arctigenin significantly attenuated glutamate-induced neurotoxicity when added prior to or after an excitotoxic glutamate challenge. The lignan protected cultured neuronal cells more selectively from neurotoxicity induced by kainic acid than by N-methyl-D-aspartate. The binding of [(3)H]-kainate to its receptors was significantly inhibited by arctigenin in a competitive manner. Furthermore, arctigenin directly scavenged free radicals generated by excess glutamate and successfully reduced the level of cellular peroxide in cultured neurons. These results suggest that arctigenin exerted significant neuroprotective effects on glutamate-injured primary cultures of rat cortical cells by directly binding to kainic acid receptors and partly scavenging of free radicals.

Mechanisms of cell death in primary cortical neurons and PC12 cells.

Increasing evidence suggests that the regulation of neuronal cell death is complex. In this study we compared the neurotoxic effects of tumor necrosis factor-alpha (TNFalpha), nitric oxide, and thrombin on primary rat cortical cell cultures and the neuronal PC12 cell line. Release of lactate dehydrogenase (LDH) and the intracellular accumulation of nucleosomes were used as indicators of necrosis and apoptosis, respectively. There was significant LDH release in both neuronal cell types, however, the pattern of LDH release was variable and agonist-dependent. In response to the nitric oxide generator, sodium nitroprusside (SNP), cortical cells exhibited pronounced LDH release and dramatic morphologic changes, whereas in differentiated PC12 cells, TNFalpha evoked release of LDH with no associated morphologic changes. Both neuronal cell types, but not undifferentiated PC12 cells, responded to TNFalpha and thrombin with increased apoptosis. Caspase inhibition, but not antioxidant treatment, reduced nucleosome accumulation in primary cortical cells, but not in differentiated PC12 cells. In the differentiated PC12 cells, caspase inhibition reduced TNFalpha-mediated LDH release, but not nucleosome accumulation. These data suggest mechanisms involved in neuronal cell death utilize multiple pathways that vary depending on the neurotoxic insult and are also influenced by subtle differences among neuronal cell phenotypes.

NMDA recepter-mediated neuroprotection by essential oils from the rhizomes of Acorus gramineus

Acori graminei Rhizoma (AGR) is shown to exhibit a number of pharmacological actions including sedation and anticonvulsive action. To further characterize its actions in the CNS, the present study evaluated the effects of essential oils (EO) from AGR on the excitotoxic neuronal cell death induced in primary rat cortical cell cultures. EO inhibited the glutamate-induced excitotoxicity in a concentration-dependent manner, with the IC 50 of 0.241mg/ml. EO exerted more potent neuroprotection against the toxicity induced by NMDA (IC 50 = 0.139 mg/ml). In contrast, the AMPA-induced toxicity was not inhibited by EO. Receptor-ligand binding studies were performed to investigate the neuroprotective action mechanism. EO dramatically inhibited the specific bindings of a use-dependent NMDA receptor-ion channel blocker [ 3H]MK-801, indicating an NMDA receptor antagonist-like action. However, the bindings of [ 3H]MDL 105,519, a ligand selective for the glycine binding site of NMDA receptor, were not considerably inhibited. These results demonstrated that EO extracted from AGR exhibited neuroprotective effects on cultured cortical neurons through the blockade of NMDA receptor activity, and that the glycine binding site appeared not to be the major site of action.

Energetics of drug efficacy at G protein-coupled receptors

Acori graminei Rhizoma (AGR) is shown to exhibit a number of pharmacological actions including sedation and anticonvulsive action. To further characterize its actions in the CNS, the present study evaluated the effects of essential oils (EO) from AGR on the excitotoxic neuronal cell death induced in primary rat cortical cell cultures. EO inhibited the glutamate-induced excitotoxicity in a concentration-dependent manner, with the IC50 of 0.241mg/ml. EO exerted more potent neuroprotection against the toxicity induced by NMDA (IC50 = 0.139 mg/ml). In contrast, the AMPA-induced toxicity was not inhibited by EO. Receptor-ligand binding studies were performed to investigate the neuroprotective action mechanism. EO dramatically inhibited the specific bindings of a use-dependent NMDA receptor-ion channel blocker [3H]MK-801, indicating an NMDA receptor antagonist-like action. However, the bindings of [3H]MDL 105,519, a ligand selective for the glycine binding site of NMDA receptor, were not considerably inhibited. These results demonstrated that EO extracted from AGR exhibited neuroprotective effects on cultured cortical neurons through the blockade of NMDA receptor activity, and that the glycine binding site appeared not to be the major site of action.

Inhibition of l-carnitine uptake into primary rat cortical cell cultures by gaba and gaba uptake blockers

l-carnitine plays a central role in mitochondrial function and is found to be differentially distributed in the brain. We have shown before that the uptake of l-carnitine into cultured rat cortical neurones was temperature-dependent, as well as potently inhibited by factors affecting the sodium gradient as well as by molecules resembling its structure, e.g. d-carnitine, acetyl- l-carnitine and γ-aminobutyric acid (GABA). GABA was the most potent inhibitor of l-carnitine uptake. In the present study we have found that specific GABA uptake blockers, nipecotic acid, cis-4-hydroxynipecotic (HNA), guvacine, 2,4-diaminobutyric acid (DABA) and NO 711 inhibit l-carnitine uptake even more potently than GABA. However, apart from NO 711, they caused about the same maximal inhibition, 67.4% at 50 μM for guvacine, compared to 60.5% by GABA. NO 711 was extremely potent and blocked 80.5% of the l-carnitine uptake. In contrast, the GABA A receptor agonists, isonipecotic acid and isoguvacine, or the antagonist bicuculline, at similar concentrations (50 μM) did not significantly inhibit the uptake of the l-carnitine. However, bicuculline at relatively high concentration (500 μM) was inhibitory (38%). The GABA B receptor agonist, baclofen, or antagonist, phaclofen, were ineffective, although 5-aminovaleric acid did significantly inhibit uptake at both 50 and 500 μM) causing 22 and 48% inhibition respectively. Like bicuculline, it was not as effective as GABA or the specific GABA uptake blockers. The results indicate that the uptake of l-carnitine by rat cortical neurones occurs in part by a process that can be potently inhibited by GABA and GABA uptake blockers.