복령다당체에 의한 큰포식세포 활성화
Activation of macrophage by PCSC22 isolated from Poria cocos sclerotium
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복령균핵(sclerotium of Poria cocos)은 투여시 진정효과와 이뇨작용, 항경련작용, 항염증작용 등이 있는 것으로 밝혀졌다. 최근의 항종양작용도 있다는 보고로 미루어, 이러한 작용들은 면역계 활성을 통해 숙주의 방어 기전을 강화시켜주는 것으로 추측된다. 복령균핵으로부터 정제된 다당체인 PCSC22가 큰포식세포를 활성화시켜 일산화질소(nitric oxide, NO)의 생성을 증가시키는 것으로 알려져 있다. p38 kinase는 inducible NO synthase (iNOS)의 유전자 발현에 중요한 신호전달 단백질 중 하나이다. 본 실험에서는 복령균핵이 큰포식세포를 활성화시 키는 기전을 밝혀내기 위하여 iNOS를 이용한 큰포식세포 면역형광염색을, p38의 변화를 보기위한 Western immunoblot assay를 실시하였다. PCSC22를 마우스 큰포식세포주인 RAW 264.7 세포에 24시간 처리한 결과, 용량 의존적으로 NO 생성을 증가 시켰다. iNOS에 면역형광염색을 실시한 결과, 세포질에서 소량 발현되던 iNOS 단백 질이 PCSC22를 처리한 후에는 다량 발현됨을 알 수 있었다. 또한 Western immunoblot assay를 실시한 결과 PCS22를 처리한 RAW264.7세포에서 p38 kinase의 인산화가 유도되었으며, p38 kinase의 특이적 억제제인 SB203580을 전처리 하였을 때 PCSC22에 의한 NO 생성이 억제됨을 알 수 있었다. 결론적으로, PCSC22의 큰포식 세포에 대한 작용은 p38 kinase 경로의 활성화를 통하여 iNOS 유전자의 발현을 자극하며, 이러한 큰포식세포의 활 성화가 항암활성과 관련이 있는 것으로 사료된다.
Abstract
The sclerotium of Poria cocos Wolf, which grows on the roots of pine trees, has long been used as a sedative, diuretic, and anti-inflammatory agent. The accumulating data revealed that certain ingredients of the sclerotium of Poria cocos showed anti-tumor activities. Although the mechanism of anti-tumor activity is not known, the polysaccharides may potentiate the host defense mechanism through the activation of immune system. In the present study we show that PCSC22, a polysaccharide isolated from the sclerotium of Poria cocos with one percent sodium carbonate, significantly induces nitric oxide (NO). Immunohistochemical staining of inducible NO synthase (iNOS) showed that the increase of NO was due to the induction of iNOS production. To further study the mechanism responsible for the induction of iNOS gene expression, we investigated the effect of PCSC22 on the activation of p38 kinase, which is important in the gene expression of inflammatory cytokines including iNOS. Western blot assay showed that PCSC22 produced phosphorylation of p38 kinase. In conclusion, we demonstrate that PCSC stimulates macrophages to express iNOS gene through the activation of p38 kinase.
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