초록
연구목적： Nitric oxide(NO)는 뇌졸중 및 여러 가지 신경변성 질환의 병리생리에 중요한 역할을 한다. 본 연구는 백서 뇌로부터 분리한 별아교세포에서 세균내독소 lipopolysaccharide(LPS)에 의한 NO 생성과정이 MAP kinase cascades와 전사 활성인자 NF-kB에 의해 중재됨을 밝히고자 하는 것이다. 방 법： NO 생성은 Greese’s reagents와 Western blot analysis를 사용하여 측정하였고 Erk, JNK1, p38은 in vitro immunecomplex kinase assay로 측정했다. NF-kB의 전사적 활성은 electrophoretic mobility shift assay로 결정하였다. 결 과： LPS는 nitric oxide(NO) 생성과 iNOS 단백질의 발현 증가를 유도하였다. LPS에 의한 NO 생성은 MAP kinases인 Erk 및 JNK/SAPK의 효소적 활성억제제인 PD 098059와 curcumin에 의하여 억제되었으나 p38 활성억제제인 SB 203580에 의해서는 영향을 받지 않았다. 또한 LPS는 MAP kinases인 Erk, JNK/SAPK 및 p38의 효소적 활성을 증가시켰다. Ras 활성억제제도 LPS 자극에 의한 NO 생성을 감소시켰다. LPS에 의한 별아교세포 자극은 전사 활성인자 NFkB의 활성을 유도하였으나 이 활성은 PD 098059와 curcumin에 의하여 억제되었다. PKA는 JNK 활성의 억제기전을 통하여 별아교세포에서 LPS의 의한 NO 생성을 감소시켰다. 결 론： 이 결과는 별아교세포에서 LPS에 의한 NO 생성과정이 MAP kinases와 전사 활성인자 NF-kB에 의해 활성화됨을 시사한다.
Objecives：Nitric oxide(NO) plays an important role in pathophysiology of stroke and various neurodegenerative diseases. This study is designed to elucidate the mechanisms by which signaling pathway of LPS-stimulated NO generation in rat prmary astrocytes may be mediated by MAP kinase cascades and transcriptional activation of NF-kB. Method：The generation of NO from primary rat neonatal astrocytes was measured by using Greese’s reagents and Western blot analysis with including Erk, JNK1 and p38 was assessed by in vitro immunecomplex kinase assay. Activation of transcriptional activator, NF-kB, was determined by using electrophoretic mobility shift assay. Results：Treatment of cultured rat primary neonatal astorcytes with LPS results in the generation of NO as well as increase in the expression of inducible nitric oxide synthase(iNOS). LPS-induced NO generation is inhibited by the addition of inhibitors of MEK and JNK1/SPAK, PD 98059 and curcumin. LPS also imcreases the phosphotransferase activity of Erk as well as JNK1 and increases the phosphorylation of p38. Inhibition of Ras results in decrease of LPS-inudced NO generation. cAMP decreases the LPS-induced NO generation via inhibition of JNK1. Furthermore LPS activates transcriptional activator, NF-kB which is inhibited by the addition of inhibitors of MEK and JNK. Conclusion：These data suggest that MAP kinases, especially Erk and JNK1, may mediate the signaling cascade of LPS-induced NO generation in rat primary astrocytes via activation of transcriptional factor, NF-kB.