B. METHODS
6. REDUCING POWER
The reducing power of antioxidant was determined according to the method of Oyaizu et al. (1986). The different doses of antioxidant in 1ml of distilled water were mixed with phosphate buffer (2.5ml, 0.2M, pH 6.6) and potassium ferricyanide [K3Fe(CN)6] (2.5ml, 1%). The mixture was incubated at 50 ℃ for 20min. A portion (2.5ml) of TCA (10%) was added to the mixture, which was then centrifuged for 10 min at 1000 * g. The upper layer of solution (2.5ml) was mixed with distilled water (2.5ml) and FeCl3 (0.5ml, 0.1%), and the absorbance was measured at 700 nm in a spectrophotometer. Higher absorbance of the reaction mixture indicated greater reducing power.
7. Membrane lipid peroxidation
Lipid peroxidation was determined using the fluorescent fatty acid analog C11-BODIPY581/591 prepared in DMSO (2mM) and stored under nitrogen at -20 ℃. BODIPY was incorporated into the cell membranes (4 µM in PBS) and incubated for 1h at 37 . The ℃ cells were then washed twice with PBS, and incubated with PBS (control sample), with the oxidant H2O2 or with H2O2 in the presence of antioxidants. At the end of the incubation period (6h), the membrane lipid oxidation was determined by calculating the ratio between the red fluorescence (580/610 nm) decay of BODIPY and the green fluorescence (485/535 nm) increase of the oxidation product, normalized in respect to the protein content.
8. Western blot analysis to confirm JAK/STAT pathway
Phosphorylation of JAK2 and STAT1/3 in cultured cortical neurons was examined by western blotting (Yadav A, et al., 2005). The media were aspirated, and the cells were washed with ice-cold PBS. Subsequently, the cell was lysed in ice-cold Triton lysis buffer (50mM Tris-HCl, pH7.5, 1% triton, 2mM Na3VO4,, 2mM EGTA, 10mM EDTA, 100mM NaF, 1mM Na4P2O7 , 100µg/ml phenylmethylsulfonyl fluoride, and 1µg/ml aprotinin, pepstatin A, and leupeptin). The supernatants collected after removing insoluble debris by centrifuging the samples at 12,000＊g for 10min were used in all studies. Proteins concentrations were determined by Pierce BCA assay kit. 100µg of proteins samples were separated by 8% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF, Millipore) membrane. The membrane was incubated with primary antibodies (phospho-JAK2: 1:1000, phospho-STAT1/3: 1:1000), followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G secondary antibody at a dilution of 1:5000. The blots were detected using a chemiluminescence western blot kit (ECL; Intron, Korea).
9. Measurement of ROS formation
Intracellular ROS formation was measured fluorometrically using 2’,7’-dichlorofluorescein diacetate (H2DCF-DA) (Lebel et al., 1990). The non-fluorescent dye DCF-DA permeated cells easily and hydrolyzed to fluorescent 2’, 7’-dichlorofluorescein (DCF) upon interaction with intracellular ROS. In brief, cultured cells were washed with
Hank’s balanced aqueous salts solution (HBSS) buffer containing 120 mM NaCl, 5 mM KCl, 1.6 mM MgCl2, 2.3 mM CaCl2, 15 mM glucose, 20 mM HEPES, and 10 mM NaOH, loaded with 10µM of DCF-DA and 20% Pluronic F-127 for 30 min, washed again with HBSS. The
DCF fluorescence was analyzed using a Spectra MAX Gemini EM (Molecular devices) at an excitation of 490 nm and an emission of 530 nm and a fluorescence microscope.
10. Measurement of mitochondrial membrane potential (∆Ψm)
∆Ψm was assessed by measuring the accumulation of rhodamine 123 (Molecular
probes, Eugene, OR, USA), a membrane-permeable cationic fluorescent dye (Emaus et al., 1986). Cultured cells were loaded with 2uM rhodamine 123 in HEPES control salt solution (HCSS). The cells were incubated for 30 min at 37 ℃ and washed twice with HCSS. The cells were observed with a fluorescence microscope, the fluorescence intensity of rhodamine 123 was quantified using image-analysis computer software.
11. GSH and GSSG assays
GSH and GSSG were determined enzymatically by using a modification of the procedure of Tietze (Tietze et al., 1969) with modifications (Ishige et al., 2001). The method is based on the determination of a chromophoric product, 2-nitro-5-thiobenzoic acid, resulting from the reaction of 5, 5’-dithiobis-(2-nitrobenzoic acid) with GSH. In this reaction, GSH is oxidized to GSSG, which is then reconverted to GSH in the presence of GR and
NADPH. The rate of 2-nitro-5-thiobenzoic acid formation, which is proportional to the sum of GSH and GSSG present, is followed at 405nm.
Measurement of total glutathione
Cells were washed twice with ice-cold phosphate-buffered saline, collected by scraping, and lysed with 3% sulfosalicylic acid. Lysates were incubated on ice for 10min, and supernatants were collected after centrifugation. Upon neutralization of the supernatant with triethanolamine, the concentration of total glutathione (oxidized and reduced) was determined. Briefly, a neutralized supernatant (25µl)was mixed with 175µl of a reaction mixture containing 143mM sodium phosphate (pH 7.5), 6.3mM Na4EDTA, 6mM 5, 5’-dithiobis(2-nitrobenzoic acid), and 0.25mg/ml NADPH. The reaction was started with adding 1 unit/ml glutathione reductase. Color development was monitored at 405nm in a kinetic mode with a microplate reader. Pure GSH was used to obtain a standard curve. The protein content of each sample was determined using the BCA protein assay kit from Pierce with bovine serum albumin as a standard.
Measurement of GSSG
Sample was prepared rapidly to minimize GSH oxidation. Freshly obtained supernatants were treated with 2-vinylpirydine for destroying reduced form of glutathione (Floreani et al., 1997). Typically, supernatant was added 2-vinylpirydine at room temperature for 60 min. TEA was then added; the mixture was vigorously mixed and the pH checked (generally between 6 and 7). Content of GSSG was determined as described above. GSSG
was quantified from a standard curve obtained by plotting known amounts of GSSG.
Measurement of GSH
GSH present in the sample was calculated as the difference between total GSH and GSSG levels, taking into account the fact that one molecule of GSSG gives rise to two molecules of GSH upon reaction with GR.
12. Statistical analysis
Data were expressed as the mean ± standard error of the mean (S.E.M.). All of the experiments were performed from three to six independent experiments. Statistical analysis among groups was performed using one-way analysis of variance (ANOVA) and Student’s t- test. In all cases, values of p are as follows: *p < 0.05, **p < 0.01.
Ⅲ .
Ⅲ Ⅲ
Ⅲ RESULTS
A. AG490 attenuated neuronal cell death against oxidative stress
The susceptibility of primary cortical neurons to oxidative stress was evaluated by performing lactate dehydrogenase (LDH) release assay following various stimuli: H2O2
(hydrogen peroxide, 50 µM), BSO (L-buthonine-S,R-sulfoxamine, an inhibitor of γ-glutamylcysteine ligase, 1mM), NMDA (N-methyl-D-aspartate, 100 µM), and AA (arachidonic acid, 50 µM) (Fig. 1). There was the significant neuronal death by all stimuli at 24h. Tyrphostin B42 (AG490), a JAK2 inhibitor (10 µM) was pre-treated 30 min before stimuli administration and prevented cortical neurons from oxidative neuronal death. When AG490 (3-30µM) was incubated against H2O2 and BSO, AG490 also prevented cortical neurons in dose-dependent manner from the cytotoxicity of H2O2 and BSO (Fig. 1).
0
LDH release (24h) (% of maximal death)
CTL
LDH release (24h) (% of maximal death)
CTL
LDHrelease (% of maximal death) CTL
50uM H2O2
LDHrelease (% of maximal death) CTL
50uM H2O2
LDHrelease (% of maximal death) CTL
50uM H2O2
Fig. 1. Protective effects of AG490 in oxidative stress-induced neuronal death
(A) Primary cortical neurons were incubated with AG490 (10μM) 30 min before various stimuli, such as H2O2 (50μM), BSO (1mM), NMDA (100μM), and arachidonic acid (AA, 50μM). Neuronal death was estimated by detecting released lactate dehydrogenage (LDH) at 340nm. (B) AG490 (3-30μM) was pre-treated against H2O2 and BSO. Data are representatives of four independent experiments and presented as mean ± SEM (*P<0.05, comparison with control cells, #P<0.05, comparison with oxidative stressed cells)
B. Antioxidants had differential effects on oxidative neuronal cell death
To identify neuroprotective mechanisms of AG490 against reactive oxygen species (ROS)-generated cytotoxicity, we compared the effects of broadly used antioxidants, trolox and NAC. Trolox, an α-tocopherol (vitamin E) derivative, is one of the most powerful antioxidants with relatively high selectivity for scavenging peroxynitrite and hydroxyl radical. NAC (N-acetyl-L-cysteine) is a potent antioxidant that is known to increase the intracellular store of glutathione. The neuronal cells were incubated with each antioxidant for 30min prior to 50µM H2O2 or 1mM BSO treatment. Trolox (30µM-300µM) did not prevent H2O2-induced neuronal cell death, but significantly decreased BSO-induced neuronal cell death in a concentration-dependent manner. NAC (300µM-3mM) attenuated dose-dependently neuronal death against H2O2, but did not do against BSO (Fig. 2).
0
LDHrelease (% of maximal death) CTL
50uM H2O2
LDHrelease (% of maximal death) CTL
50uM H2O2
LDHrelease (% of maximal death) CTL
50uM H2O2
LDHrelease (% of maximal death) CTL
50uM H2O2
LDHrelease (% of maximal death) CTL
50uM H2O2
LDHrelease (% of maximal death) CTL
50uM H2O2
Fig. 2. Effects of antioxidants in oxidative stress-induced neuronal death (A) Primary cortical neurons were treated with antioxidants 30 min prior to 50 μM hydrogen peroxide (H2O2): trolox (30-300 μM), and NAC (300 μM -3 mM). (B) Primary cortical neurons were treated with antioxidants 30 min prior to 1mM BSO. 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)
C. AG490 decreased the phosphorylation of JAK2 against oxidative stress
Activation of the JAK/STAT pathway is known to be restricted to certain oxidative stress stimuli as it is induced by peroxide but not by other types of reactive oxygen species, such as superoxide (Simon et al. 1998).
To elucidate the involvement of Janus kinases 2 (JAK2) signaling pathway in the neuroprotective effect of AG490, we performed western blotting. Cortical neurons were treated for various times (5, 15, 30, and 60 min) with H2O2 and BSO. Tyrosine phosphorylation of JAK2 was increased 5min after stimuli (Fig. 3.). At that time point, AG490 reversed the oxidative stress-induced phosphorylation of JAK2. These results suggested that JAK2 activated by certain oxidants was involved in oxidant-induced neuronal death signaling pathway. AG490, a JAK2 inhibitor suppressed activation of JAK2 and also oxidative stress-induced neuronal death.
5’CTL H AG+H 15’CTL H AG+H 30’CTL H AG+H 60’ CTL H AG+H
Fig. 3. Inhibition of oxidative stress-induced JAK2 phosphorylation by AG490 (A) Primary cortical neurons were incubated for various times (5, 15, 30, and 60 min) with 50 μM hydrogen peroxide following pre-treatment for 30 min with 10 μM AG490. Protein extracts were prepared and subjected to western blotting as described in Material and Methods. Relative amount of each phospho-JAK2 group was compared to normal JAK2 group. (B) Primary cortical neurons were incubated for various times with 1mM BSO following pre-treatment for 30 min with 10 μM AG490. Relative amount of each phospho-JAK2 group was compared to normal phospho-JAK2 group.
D. Oxidative stress-induced intracellular ROS production was regulated by AG490 and antioxidants
Reactive oxygen species (ROS) have been implicated as an important causative factor in cell damage, including apoptosis and necrosis. Therefore we examined whether free radicals are involved in oxidative stress-induced neurotoxicity. The intracellular ROS level was quantified with H2-DCFDA, which can be converted by ROS into DCF and then easily visualized by strong fluorescence at around 530 nm when excited at around 480 nm.
Cortical neurons were incubated with 50µM H2O2 in the serum-free medium. The intracellular ROS level peaked at 4h-6h after exposure to H2O2. When the pretreatment with AG490 (10 µM), trolox (100 µM), and NAC (1mM) was carried out a half hour before H2O2
application, the change of ROS level was detected comparison with only H2O2 application.
As a result, AG490 potently blocked the production of ROS until we observed. Trolox delayed the production of intracellular ROS until 6h after H2O2 application. After 6h, trolox did not resist the increase of ROS level any more. In our system, cortical neurons with NAC application continuously maintained high level of the intracellular ROS at all times (Fig. 4.).
When 1mM BSO was incorporated with serum-free medium in cultured neurons, the intracellular ROS production was increased at 18h. Unlike the result of H2O2 application, ROS level against BSO was well regulated by pre-treatment of 100 µM trolox for long time.
AG490 had a little decrease effect of ROS production compared to trolox. NAC induced the increase of the intracellular ROS generation by BSO in cortical neurons, same as the result of ROS generation by H2O2 (Fig. 5.).
2h 4h 6h
H2O2 50uM
AG490 10uM +H2O2
Trolox 100uM +H2O2
NAC 1mM +H2O2 CTL
(A) 2h 4h 6h
H2O2 50uM
AG490 10uM +H2O2
Trolox 100uM +H2O2
NAC 1mM +H2O2 CTL (A)
(B)
(A) Primary cortical neurons were incubated for 2, 4, and 6h with 50 μM hydrogen peroxide following pre-treatment for 30 min with 10 μM AG490 and antioxidants, such as trolox (100 μM), and NAC (1mM). The intracellular ROS generated by hydrogen peroxide was increased at 4h and decreased by AG490 and trolox except for NAC. Images were taken 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)
Fig. 5. ROS generation against buthionin sulfoxamine (BSO) in cortical neurons (A) Primary cortical neurons were incubated for 14, 18, and 22h with 1mM buthionin sulfoxamine following pre-treatment for 30 min with 10 μM AG490 and antioxidants, such as trolox (100 μM), and NAC (1mM). The intracellular ROS generated by buthionin sulfoxamine was increased at 18h and decreased by AG490 and trolox except for NAC.
Images were taken 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)
E. Antioxidant activities in vitro
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)