치밀이음부 구조단백질인 Occludin에 대한 활성산소종의 영향
The Changes of Occludin in Tight Junction of Blood-Brain Barrier by ROS
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뇌에서 혈액뇌장벽을 형성하는 내피세포는 치밀이음부를 통해 뇌의 항상성을 유지하고 있다. 치밀이음부의 단백질 중의 하나인 occludin 은 뇌혈관장벽 (BBB) 의 기능을 유지하는 중요한 단백질로 인식되고 있다. 본 실험에서는 소의 뇌에서 배양된 BBB 내피세포에서 활성산소종의 하나인 H2O2 에 의해 일어나는 occludin 단백질의 변화를 관찰하였다. H2O2 에 의해 TEER 가 감소하는 것은 occludin 의 재분포에 의한 것이었다. 세포독성은 4 시간내에서는 1 mM H2O2 이하에서는 나타나지 않았다. Confocal laser microscope 으로 관찰한 결과, H2O2 에 의해 occludin 은 치밀이음부에서 중간중간이 사라져 감소해 있었고, 이러한 양상은 H2O2 의 용량과 노출시간에 비례하였다. 그러나 Western blot 결과, occludin의 총량은 증가하였다. 투과전자현미경 관찰을 통해 H2O2 가 세포사이의 결합의 구조에 뚜렷한 변화를 미치지 않는 것을 알 수 있었다. 이를 통해 H2O2 에 의한 BBB 기능소실은 occludin 이 치밀이음부에서 부분적으로 사라지는 것에 의하지만, 세포는 기능손상을 복구하기위한 방편으로 이 단백질의 생산을 더욱 증가시키는 것으로 생각된다.
Abstract
Cerebral microvessel endothelial cells that form blood-brain barrier (BBB) have tight junction for maintaining brain homeostasis. Occludin, one of tight junction protein, is crucial for BBB function. H2O2 induced occludin changes and effects in bovine brain BBB endothelial cells were examined in this study. The decrease of transendothelial electrical resistance (TEER) by H2O2 was due to disruption of occludin localization. Cytotoxicity test revealed that H2O2 did not cause cell death below 1 mM H2O2 within 4 hr. H2O2 caused intermittent disruption and loss of occludin at tight junctions and occludin disappeared with dose dependent manner from tight junction in confocal laser microscopy. But Western blot revealed that the total amounts of occludin increased by H2O2 administration. Transmission electron microscopy revealed that the ultrastructure of tight junction was not changed by H2O2. These data suggest that functional disruption of BBB by H2O2 was due to the localized loss of occludin in tight junction, but the expression of occludin increased in order to compensate the disrupted function in BBB.
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