초록
Cobrotoxin의 항염증 치료 기전에 대해 연구하기 위하여 성상세포에 LPS 및 SNP로 염증을 유도한 후 NF-${\kappa}B$와 DNA의 결합 능력, NF-kB Dependent Luciferase 발현, Astrocyte의 세포활성, NF-${\kappa}B$ 구성 단백질인 P50, P-$1{\kappa}B$, $1{\kappa}BB$ 및 염증(炎症)관련 유전자(遺傳子)인 Cox-2, iNOS, cPLA2의 발현과 GSH와 DTT로 sulf-hydryl기를 환원시 NF-${\kappa}B$와 DNA의 결합 능력, NF-${\kappa}B$ 구성 단백질인 P50 발현에 미치는 영향과 Cobrotoxin의 성상세포 내 유입 등을 관찰하여 다음과 같은 결론을 얻었다. 1. LPS 로 염증을 유발한 후 NF-${\kappa}B$와 DNA의 결합 능력을 관찰한 결과 Cobrotoxin 0.1${\mu}g/m{\ell}$ 처리군, Astrocyte 내에서의 Cobrotoxin 0.1, 0.5${\mu}g/m{\ell}$ 처리군에서 모두 대조군에 비하여 유의한 억제를 나타내었다. 2. LPS로 염증을 유발한 후 Astrocyte 내에서 NF-${\kappa}B$ Dependent Luciferase 발현을 살펴본 결과 Cobrotoxin 모든 처리군에서 대조군에 비하여 유의한 억제를 나타내었다. 3. SNP로 염증을 유발한 후 Cobrotoxin이 NF-${\kappa}B$ 구성 단백질인 P50, P-$1{\kappa}B$, $1{\kappa}B$ 발현에 미치는 영향을 살펴본 결과 P50와 $1{\kappa}B$는 Cobrotoxin 0.1, 0.5 및 $1{\mu}g/m{\ell}$ 모든 처리군에서 대조군에 비하여 유의한 억제를 나타내었고, P-$1{\kappa}B$는 Cobrotoxin $0.1{\mu}g/m{\ell}$ 처리군에서 대조군에 비하여 억제를, Cobrotoxin 0.5, $1{\mu}g/m{\ell}$ 처리군에서 각각 대조군에 비하여 유의한 억제를 나타내었다. 4. LPS로 염증을 유발한 후 Cobrotoxin이 NF-${\kappa}B$ 구성 단백질인 P50, P-$1{\kappa}B$, $1{\kappa}B$ 발현에 미치는 영향을 살펴본 결과 P50와 $1{\kappa}B$는 Cobrotoxin 0.5, $1{\mu}g/m{\ell}$ 처리군에서 각각 대조군에 비하여 유의한 억제를 나타내었다. 5. SNP로 염증을 유발한 후 Cobrotoxin이 염증(炎症) 관련 유전자(遺傳子)인 Cox-2, iNOS, cPLA2 발현에 미치는 영향을 살펴본 결과 Cox-2, iNOS, cPLA2 모두 Cobrotoxin $1{\mu}g/m{\ell}$ 처리군에서 대조군에 비하여 유의한 억제를 나타내었다. 6. LPS로 염증을 유발한 후 Cobrotoxin이 염증(炎症) 관련 견전자(遣傳子)인 Cox-2, iNOS, cPLA2 발현에 미치는 영향을 살펴본 결과 Cox-2와 cPLA2의 경우 Cobrotoxin 0.1, 0.5 및 $1{\mu}g/m{\ell}$ 모든 처리군에서, iNOS의 경우 Cobrotoxin 0.5, $1{\mu}g/m{\ell}$ 처리군에서 대조군에 비하여 유의한 억제를 나타내었다. 7. Astrocyte내에서 SNP로 염증을 유발한 후 GSH와 DTT로 sulf-hydryl기를 환원하여 NF-${\kappa}B$와 BNA의 결합 능력을 관찰한 결과 Cobrotoxin $0.5{\mu}g/m{\ell}$과 DTT 1mM Cobrotoxin $0.5{\mu}g/m{\ell}$과 DTT 5mM의 동시처리군은 각각 Cobrotoxin $0.5{\mu}g/m{\ell}$ 처리군에 비하여 유의한 증가를 나타내었다. 8. Astrocyte 내에서 LPS로 염증을 유발한 후 GSH와 DTT로 sulf-hydryl기를 환원하여 NF-${\kappa}B$와 DNA의 결합 능력을 관찰한 결과 cobrotoxin $0.5{\mu}g/m{\ell}$과 DTT 1mM Cobrotoxin $0.5{\mu}g/m{\ell}$과 DTT 5mM의 동시처리군과 Cobrotoxin $0.5{\mu}g/m{\ell}$과 GSH 1mM Cobrotoxin $0.5{\mu}g/m{\ell}$과 GSH 5mM의 동시처리군 모두에서 Cobrotoxin $0.5{\mu}g/m{\ell}$ 처리군에 비하여 유의한 증가를 나타내었다. 9. Astrocyte 내에서 SNP로 염증을 유발한 후 GSH와 DTT로 sulf-hydryl기 환원시 NF-${\kappa}B$ 구성 단백질인 P50 발현에 미치는 영향을 관찰한 결과 SNP, Cobrotoxin $1{\mu}g/m{\ell}$와 DTT 1 또는 5mM 동시처리군과 SNP, Cobrotoxin $1{\mu}g/m{\ell}$와 GSH 1 또는 5mM 동시처리군 모두에서 Cobrotoxin $1{\mu}g/m{\ell}$ 처리군에 비하여 발현의 유의한 증가를 나타내었다. 10. Cobrotoxin의 세포 내 유입 확인을 살펴본 결과 Astrocyte 내에서 Cobrotoxin이 세포내로 유입되는 것으로 나타났다. 이상의 결과로 보아 Cobrotoxin 처리는 성상 세포 들을 대상을 LPS 및 SNP로 유도된 NF-${\kappa}B$ 관련 염증 기전과 iNOS, COX-2, cPLA2와 같은 염증관련 유전자의 발현 및 NO, PGE2,에 유의한 변동을 나타내었고, 이들 결과는 Cobrotoxin의 항염증 효과 및 그 치료기전에 대하여 입증한 것이며, 향후 안전성 연구를 바탕으로 신경계 및 심혈관계 염증치료에 약침개발과 같은 적극적인 활용이 기대된다.
Objectives : The purpose of this study was to investigate the anti-inflammatory effect of Cobrotoxin on binding affinity of cobrotoxin with P50, $IKK{\alpa}$ and $IKK{\beta}$, activities of NF-${\kappa}B$, Cell viability of astrocyte, expressions of protein molecules of NF-${\kappa}B$ such as P50, P-$1{kappa}B$, $1{\kappa}B$ and iflammation related genes such as Cox-2, iNOS, cPLA2 in the SNP or LPS induced Inflammatory pathway of Rats' astrocytes. Methods : In this study, The expression of cytosolic phospholipase A2, Nitric oxcide, Cyclooxygenase-2 and inducible nitrogen oxide synthase was determined by western blotting with corresponding antibodies, and the generation of NF-${\kappa}B$ was assayed by EMSA method in astrocytes of rats. The Cell viability of astrocytes was determined by MTT assay, and Binding affinity of Cobrotoxin with P50, $IKK{\alpha}$ and $IKK{\beta}$ was assayed by Surface plasmon resonance analysis, and NF-${\kappa}B$ dependent luciferase activity was determined by luciferase analysis, and Uptake of cobrotoxin in astrocytes was identified by Confocal laser scanning microscope Results : 1. Compared with control, LPS-induced NF-${\kappa}B$ DNA binding activity was decreased significantly by 0.1, $0.5{\mu}g/m{\ell}$ of Cobrotoxin in Astrocyte. 2. Compared with control, LPS-induced NF-kB dependent luciferase expression was decreased significantly by 0.1, 0.5 and $1{\mu}g/m{\ell}$ of Cobrotoxin in Astrocyte. 3. Compared with control, SNP induced P50, $I{\kappa}B$ expressions in astrocyte were decreased significantly by 0.1, 0.5 and $1{\mu}g/m{\ell}$ of Cobrotoxin and P-$1{\kappa}B$ expression was decreased significantly by 0.5 and $1{\mu}g/m{\ell}$ of Cobrotoxin. 4. Compared with control, LPS induced P50, $1{\kappa}B$ expressions in astrocyte were decreased significantly by 0.5 and $1{\mu}g/m{\ell}$ of Cobrotoxin. 5. Compared with control, SNP induced Cox-2, iNOS, CPLA2 expressions in astrocyte were decreased significantly by $1{\mu}g/m{\ell}$ of Cobrotoxin. 6. Compared with control, LPS induced Cox-2, cPLA2 expressions in astrocyte were decreased significantly by 0.1, 0.5, $1{\mu}g/m{\ell}$ of Cobrotoxin and iNOS expression was decreased significantly by 0.5, $1{\mu}g/m{\ell}$ of Cobrotoxin. 7. Compared with $0.5{\mu}g/m{\ell}$ of Cobrotoxin, SNP-induced NF-${\kappa}B$ DNA bindins activity in astrocyte was increased significantly by Cobrotoxin $0.5{\mu}g/m{\ell}$ with DTT 1mM and Cobrotoxin $0.5{\mu}g/m{\ell}$ with DTT 5mM. 8. Compared with $0.5{\mu}g/m{\ell}$ of Cobrotoxin, LPS-induced NF-${\kappa}B$ DNA binding activity in astrocyte was increased significantly by Cobrotoxin $0.5{\mu}g/m{\ell}$ with DTT 1mM, Cobrotoxin $0.5{\mu}g/m{\ell}$ with DTT 5mM, Cobrotoxin $0.5{\mu}g/m{\ell}$with GSH 1mM and Cobrotoxin $0.5{\mu}g/m{\ell}$ with GSH 5mM 9. Compared with $0.1{\mu}g/m{\ell}$ of cobrotoxin, SNP induced P50 expressions in astrocyte were increased significantly by Cobrotoxin $0.5{\mu}g/m{\ell}$ with DTT 1mM, Cobrotoxin $0.5{\mu}g/m{\ell}$ with DTT 5mM Cobrotoxin $0.5{\mu}g/m{\ell}$ with GSH 1mM and Cobrotoxin $0.5{\mu}g/m{\ell}$ with GSH 5mM. 10. The uptake of the labeled cobrotoxin into the cells was shown under a confocal laser scanning microscope. cobrotoxin was uptaken into the membrane and nucleus of astrocytes. Conclusions : In summary, the present results demonstrate that cobrotoxin directly binds to sulfhydryl group of p50 and IKKS resulting In the reduction of translocation of p50 and IkB release, thereby inhibits activation of NF-${\kappa}B$, and suggest that pico to nanomolar range of cobrotoxin could inhibit the expression of genes in the NF-${\kappa}B$ signal pathway.
(Department of Acupuncture & Moxibustion, College of Oriental Medicine, Kyung-Won University)