We examined the effect of AG490 and antioxidants in oxidative stress-induced neuronal death. AG490 rather than antioxidants critically blocked cell death by oxidative stress. Antioxidant activities of AG490 itself was measured to clarify the protection mechanism against oxidative stress compared with reference antioxidants; trolox, NAC, and butylated hydroxytoluene (BHT, positive control for lipid peroxidation).
In the DPPH radical scavenging assay, the antioxidants are able to reduce the stable radical DPPH to the yellow colored diphenyl-picrylhydrazine. The method is based on the reduction of alcoholic DPPH solution in the presence of a hydrogen-donating antioxidant due to the formation of the non-radical form DPPH-H by the reaction. Fig. 6. illustrates a significant decrease in the concentration of DPPH radical due to scavenging activity of AG490 and reference antioxidants. The scavenging effect of AG490 and references on the DPPH radical decreased in the order of NAC (49.0%) > Trolox (47.6%) > AG490 (44.9%) at the concentration of 10 μg/ml.
The blue/green ABTS [2, 2’-azinobis-(3-ethyl-benzothiazoline-6-sulfonic acid)]
radical cation turns to transparent stable ABTS due to the reduction in the presence of antioxidant. AG490 had effective ABTS radical cation scavenging activity in a concentration-dependent manner (1-10 μg/ml). There was a significant decrease in the concentration of ABTS due to the scavenging capacity of AG490. The scavenging effect of AG490 and references on the ABTS decreased in that order: NAC (93.8%) > AG490 (93.4%) > Trolox (67.4%), at the concentration of 10 μg/ml (Fig. 7.).
Metal chelating capacity was significant since it reduced the concentration of the catalyzing transition metal in lipid peroxidation. It was reported that chelating agents are effective as secondary antioxidants because they reduce the redox potential thereby stabilizing the oxidized form of the metal ion. AG490 exhibited about 37.7% chelation of ferrous ion at 30 μg/ml concentration. On the other hand, the percentage of metal chelating capacity of 30 μg/ml of trolox and NAC were found as 25.0% and 7.8%. The metal scavenging effect of those samples decreased in the order of AG490 > Trolox > NAC (Fig.
8.).
For measurements of reductive ability, the ferric ion to ferrous ion transformation was investigated in the presence of AG490 and antioxidants using the method of Oyaizu (1986). The reducing power was increased with increase of antioxidants concentrations.
Reducing power of AG490 and references exhibited the following order: NAC > Trolox >
AG490, at the concentration of 30 μg/ml (Fig. 9.).
C11-BODIPY 581/591 is an analogue of membrane phospholipids. The oxidation processes in membranes of living cells can be assessed by the special shift of C11-BODIPY 581/591 fluorescence upon oxidation. After incorporation of C11-BODIPY 581/591 in the cellular membrane, C11-BODIPY 581/591 changes color red to green due to oxidation of membrane. AG490 delayed the change of color of C11-BODIPY 581/591 incorporated cellular membrane. The percentage of blocking effect of lipid peroxidation of 10 μg/ml of AG490, trolox, NAC, and BHT were found as 268.8, 264.0, 98.0, and 165.9%. The protective effect of lipid peroxidation of those samples decreased in the order: AG490 >
trolox > BHT > NAC, at the concentration of 10 μg/ml (Fig. 10.).
DPPH radical scavenging assay
concentration (µµµµg/ml)
0 5 10 15 20 25 30
A b so rb a n ce ( 5 1 7 n m )
0.00 0.03 0.06 0.09 0.12 0.15 0.18
AG490 Trolox NAC
Fig. 6. DPPH radical scavenging activity The inhibition effects of concentration-dependent antioxidants in DPPH radical scavenging assay (●) AG490; (○) Trolox; (▼) NAC Data are representative of four independent experiments.
ABTS radical cation decolorization assay
concentration (µµµµg/ml)
0 2 4 6 8 10
A b so rb a n ce ( 7 3 4 n m )
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
AG490 Trolox NAC
Fig. 7. ABTS radical cation decolorization assay The inhibition effects of concentration-dependent antioxidants in ABTS radical cation decolorization assay. (●) AG490; (○) Trolox; (▼) NAC Data are representative of four independent experiments.
Metal chelating activity
concentration (µµµµg/ml)
0 5 10 15 20 25 30
A b so rb a n ce ( 5 6 2 n m )
0.0 0.5 1.0 1.5 2.0 2.5 3.0
AG490 Trolox NAC
Fig. 8. Metal chelating activity Ferrous ions chelating effect of different concentrations (1-30µM) of (●) AG490, (○) Trolox, and (▼) NAC Data are representative of four independent experiments
Reducing power
concentration (µµµµg/ml)
0 5 10 15 20 25 30
A b so rb a n ce ( 7 0 0 n m )
0.0 0.3 0.6 0.9 1.2 1.5 1.8 2.1
AG490 Trolox NAC
Fig. 9. Reducing power Total reductive potential of different concentration (1-30µM) of (●) AG490, (○) Trolox, and (▼) NAC Data are representative of four independent experiments.
AG490 Trolox NAC BHT red/green fluorescence ratio(% of CTL) (C11-BODIPY581/591 intensity)
0 50 100 150 200 250
CTL H2O2 1µµµµg/ml + H 3µµµµg/ml +H 10µµµµg/ml +H
* * * *
#
#
#
#
#
# #
## #
Fig. 10. Lipid peroxidation with C-11 BODIPY 581/591 Cortical neurons were loaded with 2μM C11-BODIPY 581/591 for 1h prior to exposure to 50 μM H2O2H. Cells were pre-incubated with AG490 and antioxidants (1-10 μg/ml) for 1h prior to exposure to the oxidant.
Intensity of C11-BODIPY 581/591 fluorescence (the ratio of the decay of red fluorescence and the increase of green fluorescence) was quantified on H2O2-induced oxidation at 4h. Red fluorescence is visualized at around 610 nm when excited at around 580 nm. Green fluorescence is visualized at around 535 nm when excited at around 485 nm. Data are representative of four independent experiments and presented as mean ± SEM (*P<0.05, comparison with control cells, #P<0.05, comparison with oxidative stressed cells)
F. AG490 increased the intracellular GSH level in cortical neurons
Glutathione (GSH) is the electron donor for reaction of peroxides in the glutathione peroxidase (GPx) and functions as a major antioxidant in tissue defense against oxidative stress, including the brain. Intracellular levels of reduced glutathione ( γ-glutamylcysteinylglycine, GSH) are maintained by glutathione reductase, a dimeric cytosolic enzyme that uses NADPH as a cofactor to catalyze the reduction of oxidized glutathione (GSSG) (Floreani et al, 1997).
A single administration of AG490 increased the ratio of GSH/GSSG, a marker of oxidative stress in cultured neurons. Four hours later, total GSH content increased 11.9 ± 2.3% with respect to basal level. The ratio of GSH/total GSH also changed to 105.2 ± 2.5% and the ratio of GSH/GSSG was 145.0% ± 19.2% (Table 1.).
Trolox itself did not influence GSH-related redox system. However, NAC decreased the ratio of GSH/total GSH and GSH/GSSG in our culture system. It is interesting that NAC is a well-known antioxidant, as a precursor of GSH.
Control AG490 Trolox NAC
Total GSH GSH/Total GSH GSH/GSSG
100
Table 1. AG490 altered GSH/GSSG balance in cultured neurons.
AG490 and antioxidants were incubated for 4h in cortical neurons: AG490 (10 μM), NAC (1mM), Trolox (100 μM). Data are representative of six independent experiments and presented as mean ± SEM (*P<0.05, comparison with control cells)
Control AG490 Trolox NAC
Total GSH GSH/Total GSH GSH/GSSG
100
Table 1. AG490 altered GSH/GSSG balance in cultured neurons.
AG490 and antioxidants were incubated for 4h in cortical neurons: AG490 (10 μM), NAC (1mM), Trolox (100 μM). Data are representative of six independent experiments and presented as mean ± SEM (*P<0.05, comparison with control cells)
G. Oxidative stress disturbed the mitochondrial membrane potential (∆Ψm:MMP) in cortical neurons.
Opening of mitochondrial permeability transition pore and loss of mitochondrial membrane potential are linked to oxidative cell death. Therefore we investigated whether AG490 and antioxidants prevent the mitochondrial depolarization caused by H2O2 and BSO.
Dihydrorhodamine is oxidized to rhodamine 123, which is highly fluorescent around 536nm when excited at about 500nm. Rhodamine 123 is lipophilic and positively charged, and tends to accumulate in mitochondria, held there by membrane potential. Cortical neurons remarkably began to lose mitochondrial membrane potential at 2h after exposure to H2O2
and completely abolished it at 4h. Application of AG490 (10 µM) with H2O2 was potently blocked the reduction of mitochondrial membrane potential. We detected less abolishment of mitochondrial membrane potential in cortical neurons exposed to H2O2 with other antioxidants: trolox (100 µM), and NAC (1mM) than abolishment of MMP in cells exposed to H2O2 itself (Fig. 11.). By 1mM BSO application, mitochondrial membrane potential was depolarized around 22h. Unlike the result of H2O2 application, trolox potently blocked the reduction of mitochondrial membrane potential and AG490 restored modestly. However, cortical neurons with NAC and BSO lost MMP earlier than application of BSO itself (Fig.
12.).
Trolox 100uM +H2O2 CTL
2h 4h 6h
H2O250uM
AG490 10uM +H2O2
NAC 1mM +H2O2 (A)
Trolox 100uM +H2O2 CTL
2h 4h 6h
H2O250uM
AG490 10uM +H2O2
NAC 1mM +H2O2 (A)
(B)
hydrogen peroxide following pre-treatment for 30 min with 10 μM AG490 or antioxidants, such as trolox (100 μM), and NAC (1mM). Abolishment of MMP by hydrogen peroxide was detected at 4h and preserved by AG490 and antioxidants. MMP was measured with a ZEISS fluorescence microscope. (B) Relative amount of each group compared to time control group. Data are representative of four independent experiments and presented as mean ± SEM (*P<0.05, comparison with control cells, #P<0.05, comparison with oxidative stressed cells)14h 18h
BSO 1mM
AG490 10uM +BSO
Trolox 100uM +BSO
NAC 1mM +BSO
22h
CTL
(A) 14h 18h
BSO 1mM
AG490 10uM +BSO
Trolox 100uM +BSO
NAC 1mM +BSO
22h
CTL
(A)
(B)
BSO following pre-treatment for 30 min with 10 μM AG490 or antioxidants, such as trolox (100 μM), and NAC (1mM). Abolishment of MMP by BSO was detected at 22h and preserved by AG490 and trolox. MMP was measured with a ZEISS fluorescence microscope.(B) Relative amount of each group compared to time control group. Data are representative of four independent experiments and presented as mean ± SEM (*P<0.05, comparison with control cells, #P<0.05, comparison with oxidative stressed cells)
Ⅳ
Ⅳ
Ⅳ Ⅳ. DISCUSSION
Here, we characterized the neuroprotective effects of tyrphostin B42 (AG490) in cultured primary cortical neurons. AG490, a JAK2 inhibitor decreased the release of lactate dehydrogenase by various oxidants in cortical neurons. AG490 significantly inhibited hydrogen peroxide (H2O2)-induced neuronal death. In the previous study, hydrogen peroxide (H2O2) may directly activate receptors on the outside of the cell and may inactivate protein tyrosine phosphatases (PTPs). Then, hydrogen peroxide activates intracellular kinases including JAK2 (Simon AR, et al, 1998). In this study, JAK2 was activated by 50 µM H2O2
at 5 min and AG490 treated prior to H2O2 decreased H2O2-induced JAK2 phosphorylation (Fig. 3). These results suggested that neuroprotective mechanism of AG490 involves in JAK2 signaling pathway against oxidative stress. In transient focal cerebral ischemia, AG490 prevents the post-ischemic JAK2 and STAT3 phosphorylation and significantly decreases the infarct volume compared to vehicle group. Furthermore, intracerebral injection of siRNA specific for STAT3 lead to suppressed STAT3 mRNA expression and phosphorylation, decreases the infract volume (Satriotomo et al, 2006). In rat astrocyte cultures, AG490 reduces oxidative stress induced by H2O2, which directly activate JAK2/STAT1 (Gorina et al, 2005). H2O2 induced serine (Ser)-727 STAT1 phosphorylation, tyrosine (Tyr)-701 STAT1 phosphorylation, and tyrosine (Tyr)-705 STAT3 phosphorylation. AG490 inhibited Tyr-701 STAT1 and Tyr-705 STAT3 phosphorylation induced by H2O2. AG490 did not prevent H2O2 -induced Ser-727 STAT1 phosphorylation as levels were significantly above control levels.
Although AG490 fully inhibited STAT Tyr phosphorylation, it did not affect STAT1 Ser
phosphorylation induced by H2O2 thus showing that AG490 did not prevent all of the effects of H2O2 in astrocyte cultures. In this point, it is suggested to elicit the nature of the effects of AG490 against oxidative stress and the actual involvement of JAK2/STAT in H2O2-induced cell death.
Cortical neurons were injured during H2O2 metabolism and produced excessive reactive oxygen species (ROS). When ROS was produced maximally by H2O2, AG490 effectively decreased the level of intracellular ROS more than trolox and NAC did (Fig. 4).
Sagara Y. reported that tyrphostins besides AG490 possess structurally and functionally antioxidant activities, such as restoration of intracellular GSH level, directly scavenging action of reactive oxygen species (ROS) and indirect regulation of ROS generation mechanism, and protection from collapsed mitochondrial membrane potential (MMP) against oxidative stress-induced nerve cell death (Sagara Y. et al, 2002). Based on this previous study, I examined protective mechanism of AG490 focused on the production of ROS in oxidative stress-induced neuronal death.
With DPPH radical scavenging assay (Fig. 6) and ABTS radical cation decolorization assay (Fig. 7), AG490 had direct scavenging activity although trolox and NAC were more potent scavenging antioxidants. With metal chelating assay (Fig. 8) and ferric reducing power assay (Fig. 9), as indicators of secondary inhibition on lipid peroxidation, AG490 indirectly inhibited lipid peroxidation less than trolox and NAC did.
However, H2O2 exerts its toxic effects mainly through the ferrous iron-dependent formation of the highly reactive hydroxyl radical (OH-). The Fenton reaction is a one electron non-enzymatic transfer reaction in which transition elements generate hydroxyl radicals from
H2O2 (Almli LM. et al, 2001). When cells were pretreated with desferrioxamine (DFO, a iron chelator) and N, N, N’ ,N’-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN, other metal ions chelator, such as copper and zinc) prior to H2O2 exposure, a reduction in cell death was seen. α-Phenyl-N-tert-butylnitrone (PBN, hydroxyl radical scavenger) also reduced on H2O2 -induced hippocampal neuronal death. Here, we confirmed the effect of AG490 on lipid peroxidation in cortical neurons with C11-BODIPY581/591, a specific fluorescence dye for oxidation of phospholipids. AG490 potently inhibited H2O2-induced lipid peroxidation as well as trolox, a Fenton reaction chain breaking antioxidant did in cortical neurons (Fig. 10).
These results indicated that AG490 structurally and functionally has direct and indirect antioxidant activities though these antioxidant activities, however these were not enough to explain the reason that AG490 was more effective than trolox and NAC against oxidant-induced cell death.
Glutathione (GSH) is one of the most abundant intracellular thiols in the central nervous system and acts as a major cellular antioxidant (Wullner U. et al, 1999). GSH is synthesized in the cytoplasm and then transported into the mitochondria which are the major intracellular source of reactive oxygen species intermediates. Mitochondria lacks catalase and depends on GSH and superoxide dismutase (SOD) to decompose the superoxide radicals that are constantly generated during cell respiration. Therefore a decrease of GSH diminishes the capacity of cells to compensate oxidative stress. Administration of AG490 itself increased the level of total GSH (GSH and GSSG) and the ratio of GSH/GSSH at 4h (Table 1.), but not 1h (data were not shown). Trolox did not influence GSH metabolism, and NAC decreased the ratio of GSH/total GSH and GSH/GSSG at 4h. NAC is a well known
antioxidant as a precursor of intracellular GSH. However, NAC did not have effective antioxidant activity to compensate GSH level in this system.
Mitochondria functions to buffer excessive intracellular free Ca2+ levels in neurons, and generates the ATP that is necessary for normal cell functions. Mitochondrial respiration also generates ROS which can disrupt mitochondrial function. High concentration of H2O2
exposure resulted in gradual increases in intracellular Ca2+ and depolarized mitochondria in neurons (Hoyt KR. et al, 1997). H2O2 (50 µM) exposure induced depolarization of mitochondrial membrane potential at 2h (Fig. 11). AG490 suppressed H2O2-induced reduction of rhodamine 123 intensity, but trolox and NAC did not inhibit the reduction.
Trolox is a broadly used superoxide anion and hydroxyl radical scavenger and a Fenton reaction chain breaking antioxidant. In this study, trolox was confirmed a potent radical scavenging activity through the DPPH and ABTS radical scavenging assay in vitro and reduced intracellular ROS production until neuronal death was progressed. Trolox also showed metal chelating activity and ferric reducing power activity, as secondary indicators on lipid peroxidation and protected oxidant-induced phospholipids peroxidation in cortical neurons. However, it has been suggested that trolox hardly penetrated mitochondrial membrane. Mitochondrial membrane potential abolished by H2O2 was not restored by pretreatment of trolox due to unstable mitochondrial function. On the other hand, trolox preserves mitochondrial membrane potential and decreases intracellular ROS production due to scavenging superoxide anion and hydrogen peroxide generated by BSO exposure.
NAC is known to increase the intracellular stores of glutathione thereby enhancing endogenous antioxidant levels. In this experiment, NAC did not suppress oxidative
stress-induced intracellular ROS production although NAC had a strong reduction activity. This is suggested that NAC did not improve the abolished mitochondrial membrane potential and decreased glutathione level in neuronal mitochondria.
AG490 is a member of tyrphostins, which structurally and functionally shows an antioxidant activity. AG490 restored oxidative stresses-induced depolarization of mitochondrial membrane potential and prevented mitochondrial dysfunction from oxidant exposure in cortical neurons. AG490 also increased the level of reduced glutathione in mitochondria. These results lead to decreased intracellular ROS production.
Taken together, AG490 has a potent protection from various oxidative stresses through the inhibition of JAK2 signaling and the preservation of mitochondrial function, such as polarization of mitochondrial membrane potential and increase of mitochondrial GSH level. Thus AG490, a JAK2 inhibitor leads to decrease oxidative stress-produced toxic ROS in cortical neurons, and protect the neurons against various oxidative stresses.
. CONCLUSION
Ⅴ
Ⅴ
Ⅴ
Ⅴ
Tyrphostin B42, AG490 known as a JAK2 inhibitor, has prominent protective effect against oxidative stressing in vitro neuronal cultures. We examined the protective mechanism of AG490 against oxidative stress. First, AG490 is an inhibitor of protein tyrosine kinases, specific for Janus kinase 2 (JAK2). To elucidate the involvement of JAK2 signaling pathway in the neuroprotection of AG490, we performed western blotting. Phosphorylation of JAK2 was increased by oxidative stress 5 min after oxidative stress application and incubation of AG490 prior to stimuli decreased the phosphorylation of JAK2, therefore JAK2 inhibition might be involved in a protective mechanism of AG490. Second, AG490 had direct scavenging activity of reactive oxygen species (ROS) in antioxidant activity assays in vitro.
Third, AG490 increased the level of reduced glutathione, one of major intracellular antioxidant system, which was associated with the decrease of intracellular ROS generated by oxidative stress. Fourth, AG490 administrated prior to oxidative stimuli blocked the reduction of mitochondrial membrane potential (MMP), which can be explained with restoration of mitochondrial dysfunction.
Taken together, AG490 potently protects cortical neurons against oxidative stress.
AG490 suppresses JAK2/STAT pathway, and preserves the mitochondrial functions, such as restored mitochondrial membrane potential, and increased GSH level, leading to decreased intracellular ROS production.
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