加減凉膈散이 RBL-2H3 비만세포와 OVA/alum에 감작된 생쥐에 미치는 항알레르기 효과
Anti-allergic Effects of Gagam-YangGyeokSan on RBL-2H3 Mast Cells and OVA/alum Sensitized Mice
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Objectives : 가감양격산은 한의학 임상에서 알레르기 치료에 이용되고 있지만, 정확한 작용 기전 및 타겟 세포는 아직 밝혀지지 않았다. 이 연구는 가감양격산의 비만세포에서의 항알레르기 효과를 알아보기 위하여 설계되었다. Methods : 본 연구에서는 real-time PCR과 ELSA를 이용하여 IL-4, IL-13 생산을 측정하였다. 또한 GATA-1, GATA-2, NF-AT1, NF-AT2, AP-1 and NF-κB p65과 같은 전사인자의 발현을 western blotting법으로 분석하였다. 그리고 OVA로 자극한 생쥐에서 IgE, IL-4, IL-13을 측정하였다. Results : RBL-2H3 비만세포에 가감양격산을 처리한 결과 PI로 유도된 IL-4와 IL-13을 포함하는 Th2 사이토카인에 용량에 따라 억제효과가 있음을 밝혀냈다. IL-4의 mRNA 유전자 발현은 100㎍/㎖ 용량에서 가감양격산에 의해 현저하게 억제되었다. 그리고 IL-13의 mRNA 유전자 발현은 200㎍/㎖ 용량에서는 현저하게 억제되었으나, 50㎍/㎖, 100㎍/㎖ 용량에서는 대조군과 차이를 보이지 않았다. IL-4와 IL-13 유전자 발현을 포함하는 전사인자의 western blot 결과는 GATA-1, GATA-2, NF-AT2, c-Jun, NF-κB p65 를 포함하는 비만세포 전사인자의 발현을 억제하나 c-Fos는 억제하지 않는 것으로 나타났다. OVA를 감작한 생쥐에서는 IgE, IL-4, IL-13을 억제하였다. Conclusions : 우리는 가감양격산이 비만세포에서 GATA-1, GATA-2, NF-AT2, c-Jun, NF-κB p65와 같은 전사인자를 조절하는 것을 통해 IL-4, IL-13과 같은 Th2 사이토카인을 억제하여 항알레르기 효과를 발휘하는 것으로 사료된다.
Abstract
Objectives Gagamyanggyeoksan (G-YGS) has been used to suppress allergic reaction, however, the cellular target of G-YGS and its mode of action remain unclear. The present study was designed to investigate the effect of extracted G-YGS on the PMA and lonomycin (PI)-induced activation of RBL-2H3. Methods For this investigation, We examined IL-4, IL-13 mRNA expression by Real-Time PCR, IL-4, IL-13 production by ELISA analysis and manifestations of GATA-1, GATA-2, NF-AT1, NF-AT2, AP-1 and NF-κB p65 transcription factors by western blotting, OVA-specific IgE, IL-4, IL-13 by mouse be sensitive to OVA. Results Here we showed that treatment of RBL-2H3 mast cells with G-YGS, suppressed PI-induced production of Th2 cytokines including IL-4 and IL-13 in a dose dependent manner. The mRNA expression of IL-4 were completely abolished by G-YGS at the concentration of 100 ㎍/ml. Data from a stable cell lines consistently expressing IL-4. And the mRNA expression of IL-13 were abolished by G-YGS at the 200 ㎍/ml. But there is no difference between the 50 ㎍/ml, the 100 ㎍/ml and the comparison. Results from the western blot analysis of transcription factors involving IL-4 and IL-13 expression indicated that it prominently decreased the expression of mast cell specific transcricption factors including GATA-1, GATA-2,NF-AT2, c-Jun, NF-κB p65 but not c-Fos. And G-YGS suppressed IgE, IL-4, IL-13 in mouse be sensitive to OVA. Conclusions We suggested the anti-allergic activities of G-YGS might be mediated by down-regulation of Th2 cytokines such as IL-4 and IL-13 through the regulation of transcription factors as GATA-1, GATA-2, NF-AT2, c-Jun, NF-κB p65.
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