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Krebs Cycle Intermediates Protective against Oxidative Stress by Modulating the Level of Reactive Oxygen Species in Neuronal HT22 Cells

View Article: PubMed Central - PubMed

ABSTRACT

Krebs cycle intermediates (KCIs) are reported to function as energy substrates in mitochondria and to exert antioxidants effects on the brain. The present study was designed to identify which KCIs are effective neuroprotective compounds against oxidative stress in neuronal cells. Here we found that pyruvate, oxaloacetate, and α-ketoglutarate, but not lactate, citrate, iso-citrate, succinate, fumarate, or malate, protected HT22 cells against hydrogen peroxide-mediated toxicity. These three intermediates reduced the production of hydrogen peroxide-activated reactive oxygen species, measured in terms of 2′,7′-dichlorofluorescein diacetate fluorescence. In contrast, none of the KCIs—used at 1 mM—protected against cell death induced by high concentrations of glutamate—another type of oxidative stress-induced neuronal cell death. Because these protective KCIs did not have any toxic effects (at least up to 10 mM), they have potential use for therapeutic intervention against chronic neurodegenerative diseases.

No MeSH data available.


Related in: MedlinePlus

Modulation of reactive oxygen species (ROS) level by (A) PA, (B) OAA, or (C) AKG against H2O2-induced cytotoxicity. The measurement of the cells loaded with 10 μM DCFH-DA (2′,7′-Dichlorodihydrofluorescin diacetate) for 30 min started at 0 min, and DCF fluorescence levels are shown at 10-min intervals. Various concentrations (1, 2, and 10 mM) of PA, OAA, or AKG were added to the cultures at the same time as DCFH-DA addition. H2O2 (200 μM) was added at 30 min. Values are the means ± SD from four experiments per group. Diamonds, control; circles, H2O2; squares, H2O2 + KCI. Note that the KCI alone groups were not shown in these graphs, because KCIs themselves did not affect ROS levels at any time point.
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antioxidants-06-00021-f003: Modulation of reactive oxygen species (ROS) level by (A) PA, (B) OAA, or (C) AKG against H2O2-induced cytotoxicity. The measurement of the cells loaded with 10 μM DCFH-DA (2′,7′-Dichlorodihydrofluorescin diacetate) for 30 min started at 0 min, and DCF fluorescence levels are shown at 10-min intervals. Various concentrations (1, 2, and 10 mM) of PA, OAA, or AKG were added to the cultures at the same time as DCFH-DA addition. H2O2 (200 μM) was added at 30 min. Values are the means ± SD from four experiments per group. Diamonds, control; circles, H2O2; squares, H2O2 + KCI. Note that the KCI alone groups were not shown in these graphs, because KCIs themselves did not affect ROS levels at any time point.

Mentions: As the neuroprotective effects of the KCIs may have been due to the chemical structure of α-keto acid by direct interaction with H2O2 inside the cells, we next examined whether the KCIs could reduce the level of ROS in HT22 cells (Figure 3). We quantified the levels of ROS with the ROS-sensitive fluorescent indicator DCFH-DA by use of a Spark 10M device (Tecan Japan, Tokyo, Japan). Based on the fluorescence activity, H2O2 (200 μM) increased the levels of ROS by 20–40-fold. ROS levels plateaued at 45–60 min of exposure. PA (Figure 3A) or OAA (Figure 3B) at 1, 2, or 10 mM, and 2 or 10 mM AKG (Figure 3C) significantly lowered intracellular ROS formation; 1 mM AKG did not reduce the ROS level. These results suggest that these KCIs used at low mM levels effectively reduced the ROS level in neural cells.


Krebs Cycle Intermediates Protective against Oxidative Stress by Modulating the Level of Reactive Oxygen Species in Neuronal HT22 Cells
Modulation of reactive oxygen species (ROS) level by (A) PA, (B) OAA, or (C) AKG against H2O2-induced cytotoxicity. The measurement of the cells loaded with 10 μM DCFH-DA (2′,7′-Dichlorodihydrofluorescin diacetate) for 30 min started at 0 min, and DCF fluorescence levels are shown at 10-min intervals. Various concentrations (1, 2, and 10 mM) of PA, OAA, or AKG were added to the cultures at the same time as DCFH-DA addition. H2O2 (200 μM) was added at 30 min. Values are the means ± SD from four experiments per group. Diamonds, control; circles, H2O2; squares, H2O2 + KCI. Note that the KCI alone groups were not shown in these graphs, because KCIs themselves did not affect ROS levels at any time point.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5384184&req=5

antioxidants-06-00021-f003: Modulation of reactive oxygen species (ROS) level by (A) PA, (B) OAA, or (C) AKG against H2O2-induced cytotoxicity. The measurement of the cells loaded with 10 μM DCFH-DA (2′,7′-Dichlorodihydrofluorescin diacetate) for 30 min started at 0 min, and DCF fluorescence levels are shown at 10-min intervals. Various concentrations (1, 2, and 10 mM) of PA, OAA, or AKG were added to the cultures at the same time as DCFH-DA addition. H2O2 (200 μM) was added at 30 min. Values are the means ± SD from four experiments per group. Diamonds, control; circles, H2O2; squares, H2O2 + KCI. Note that the KCI alone groups were not shown in these graphs, because KCIs themselves did not affect ROS levels at any time point.
Mentions: As the neuroprotective effects of the KCIs may have been due to the chemical structure of α-keto acid by direct interaction with H2O2 inside the cells, we next examined whether the KCIs could reduce the level of ROS in HT22 cells (Figure 3). We quantified the levels of ROS with the ROS-sensitive fluorescent indicator DCFH-DA by use of a Spark 10M device (Tecan Japan, Tokyo, Japan). Based on the fluorescence activity, H2O2 (200 μM) increased the levels of ROS by 20–40-fold. ROS levels plateaued at 45–60 min of exposure. PA (Figure 3A) or OAA (Figure 3B) at 1, 2, or 10 mM, and 2 or 10 mM AKG (Figure 3C) significantly lowered intracellular ROS formation; 1 mM AKG did not reduce the ROS level. These results suggest that these KCIs used at low mM levels effectively reduced the ROS level in neural cells.

View Article: PubMed Central - PubMed

ABSTRACT

Krebs cycle intermediates (KCIs) are reported to function as energy substrates in mitochondria and to exert antioxidants effects on the brain. The present study was designed to identify which KCIs are effective neuroprotective compounds against oxidative stress in neuronal cells. Here we found that pyruvate, oxaloacetate, and α-ketoglutarate, but not lactate, citrate, iso-citrate, succinate, fumarate, or malate, protected HT22 cells against hydrogen peroxide-mediated toxicity. These three intermediates reduced the production of hydrogen peroxide-activated reactive oxygen species, measured in terms of 2′,7′-dichlorofluorescein diacetate fluorescence. In contrast, none of the KCIs—used at 1 mM—protected against cell death induced by high concentrations of glutamate—another type of oxidative stress-induced neuronal cell death. Because these protective KCIs did not have any toxic effects (at least up to 10 mM), they have potential use for therapeutic intervention against chronic neurodegenerative diseases.

No MeSH data available.


Related in: MedlinePlus