세포수준에서 NF-kB 전사 인자 활성도의 새로운 측정법 확립과 NF-kB 전사 인자를 매개로 하는 길경 사포닌의 아폽토시스 유도 및 항염증 효과에 대한 작용기전 연구

다국어 초록 (Multilingual Abstract)

길경의 성분으로는 10여종의 triterpene saponin을 약 2% 함유하고 있으며, 이 saponin중에는 platycodigenin의 배당체로 platycodin A, C, D, D2와 2종의 monoacetate, platycodin D3 가 보고되었다.이러한 길경 saponins에 대한 생리활성을 조사해 본 결과, saponin구조에 따라 각각의 활성이 다르다고 보고하고 있으며, 이러한 사포닌 중에서 platycodin D, D3라는 물질은 염증을 유도하는 COX-2를 간접적으로 저해를 시키는 것으로 보고 하고 있으며, 기도의 mucin를 증가시킴으로써 다양한 기도관련 질병을 치료제로서 가능성을 보이고 있다. 이러한 만성적인 항염증 효과에 대한 생리활성를 지닌 사포닌들은 또한, 특정 암세포에 대한 항암제 효과도 가지는 것으로 보고한다. 사포닌계열에 대한 항암효과가 나타나고 있다는 보고서는 수 없이 많지만, 그러한 항암효과에 대한 자세한 기전은 명쾌히 설명해 주지는 못하는 실정이다. 피부 암세포주에 아폽토시스(apoptosis)를 구명하기 위해서 Platycodin D를 처리한 후에 DNA을 추출하고 Agarose gel을 이용하여 DNA Fragmentation을 확인하고, TUNEL Assay를 수행하여 확실한 아폽토시스를 확인하였다. Western blotting을 이용하여 관여되는 세포의 단백질을 확인하였고 Platycodin D에 의한 아폽토시스는 Extrinsic Apoptosis라는 것을 구명하였다. 또한, 우리는 길경에 있는 Platycoin D, D3라는 성분이 각질형성세포의 확산을 억제하고 마우스에서 유도된 Papillomas를 감소시키는 효과가 있다는 연구결과를 얻을 수가 있었다.

다국어 초록 (Multilingual Abstract)

The nuclear factor (NF)-kB family of eukaryotic transcription factors plays an important role in the regulation of immune responses, embryo and cell lineage development, cell apoptosis, cell-cycle progression, inflammation, and oncogenesis. A wide range of stimuli, including cytokines, mitogens, environmental particles, toxic metals, and viral or bacterial products, activate NF-kB, mostly through IkB (IKK)-dependent phosphorylation and subsequent degradation of its inhibitor, the IkB family of proteins. Activated NF-kB translocates into the nucleus where it modulates the expression of a variety of genes, including those encoding cytokines, growth factors, acute phase response proteins, cell adhesion molecules, other transcription factors, and several cell apoptosis regulators.
First, a new cell-based assay system for monitoring NF-kB was developed to determine the influence of activated NF-kB in human HaCaT cells. The pNF-kB-SEAP-NPT plasmid that permits expression of the secretory alkaline phosphatase (SEAP) reporter gene in response to the NF-kB and contains the neomycin phosphotransferase (NPT) gene for geneticin resistance in host cells was constructed and transfected into the human keratinocyte cell line HaCaT. This assay system could be used to determine the quantitative measurement of NF-kB activity in human skin and allow to screen of anti-inflammatory agents from various synthetic chemicals and natural products for dermatological purpose.
Second, the possible role of NF-kB activation on the synthesis of melanotrophic factors from the keratinocytes was presented by determining the activities of NF-kB induced by melanogenic inhibitors (MIs) in human HaCaT keratinocytes transfected with pNF-kB-SEAP-NPT plasmid. MIs such as niacinamide, kojic acid, hydroquinone, resorcinol, arbutin, and glycolic acid were preincubated with transfectant HaCaT cells for 3 h and then irradiated with ultraviolet B (UVB). NF-kB activation was measured with the SEAP reporter gene assay using a fluorescence detection method. Of the MIs tested, kojic acid (IC50 = 60 ？？M) was found to be the most potent inhibitor of UVB-upregulating NF-kB activation in transfectant HaCaT cells, which is followed by niacinamide (IC50 = 540 ？？M). Pretreatment of the transfectant HaCaT cells with the MIs, especially kojic acid or niacinamide, effectively lowered NF-kB binding measured by electrophoretic mobility shift assay. Furthermore, these two inhibitors remarkably reduced the secretion of IL-6, one of the melanotrophic factors, triggered by UV-irradiation of the HaCaT cells. MIs were suggested as potential inhibitor of NF-kB activation in human keratinocytes. These observations suggested that MIs might act at least partially through the modulation of the synthesis of melanotrophic factors in keratinocytes in vivo.