DISCUSSION

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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- 국문 요약 -

산화적 산화적

산화적 산화적 스트레스에 스트레스에 스트레스에 의한 스트레스에 의한 의한 의한 신경세포사멸에 신경세포사멸에 신경세포사멸에 대한 신경세포사멸에 대한 대한 대한 AG490 보호기전 보호기전 보호기전 보호기전

아주대학교 대학원의학과 김아영

(지도교수: 백 은 주)

자유 라디칼의 증가로 인한 산화적 스트레스는 신경세포사멸에 있어서 중요한 역할을 한다. 자유 라디칼은 세포내부에 존재하는 분자들을 산화시키고, 세포막의 안정화를 저해하며, 미토콘드리아의 기능을 저해시킨다. 본 연구에서는 Janus kinases 2 (JAK2)의 특이적 억제제인 AG490을 이용하여, 일차배양 신경세포 의 산화적 스트레스로 인한 사멸에서의 보호효과를 관찰하고 보호경로에 대해서 조사해보고자 한다.

본 연구의 결과로, 일차배양 신경세포에서 AG490은 과산화수소 (H2O2), L-buthionin-S,R-sulfoxamine (BSO), N-methyl-D-aspartate (NMDA), arachidonic acid (AA)와 같은 다양한 산화제에 의해 일어나는 세포사멸을 상당히 감소시킨다. 특 히, AG490은 과산화수소에 의한 신경세포 사멸을 강력하게 저해한다. 이러한 AG490 보호효과 경로를 이해하기 위해하여 JAK2 신호전달경로와 산화적 스트레

스의 관계를 알아보고, 산화적 스트레스에 의해 생성된 활성산소의 조절을 관찰 하며, 미토콘드리아의 기능의 변화를 관찰하였다. AG490은 특이적인 산화제에 의 해서 인산화된 JAK2의 인산화를 억제시키고, 약물 자체가 항산화제 활성을 가지 고 있음을 알게 되었다. 약물의 구조적 특징에 의한 항산화제 활성 이외에도 AG490은 세포내의 글루타티온 (GSH)의 양을 증가시켜서 산화적 스트레스로 유 도되는 활성산소를 감소시켰다. 또한, AG490은 산화적 스트레스에 의해서 유도되 는 미토콘드리아 막전위의 감소를 막아서 신경세포의 사멸을 억제한다.

이러한 결과들을 통해서 JAK2 억제제인 AG490은 산화적 스트레스로 유 발된 신경세포 사멸에 있어 강력한 보호작용을 함을 알 수 있었다. AG490의 세 포 보호 작용은 JAK2 신호전달을 차단하고, 활성산소의 생성을 감소시키며 미토 콘드리아 기능을 유지시킴으로서 나타나는 현상이라 할 수 있다.

핵심어: AG490, 산화적 스트레스, 세포사멸