Anti-inflammatory Effects of Bee Venom on Cytokines Expression in LPS-induced Human Monocytic Cells Line, THP-1 = Human monocytic cells line, THP-1에서 LPS-유래 cytokines 발현에 대한 봉독의 항염증 효과

다국어 초록 (Multilingual Abstract)

The research study focuses on the identification of the molecular mechanism of anti-inflammatory effects with bee venom (BV), which has been used for the treatment of various inflammatory diseases in Oriental medicine. This experiment especially examined the effects of BV on the cytokine production induced by lipopolysaccharide (LPS) on the Human acute monocytic leukemia cell line (THP-1). We investigated the effects of BV on the expression of pro-inflammatory cytokines in vitro with RT-PCR and microarray in LPS treated THP-1 cells. BV decreased the pro-flamatory cytokine production and the levels of cytokines were reduced by 34%-TNF-α, 64.4%-TGF-β, 86.3%-IL-1β, 80.3%-IL-6, 66%-IL-8, and 45.3%-IL-10 when compared to that of LPS induced THP-1 cells. Further the results were analyzed by using the microarray and exhibited similar results to that of the RT-PCR. These results suggest that BV has an anti-inflammatory effect by inhibiting the pro-inflammatory cytokine expression, possibly through suppression of the Map Kinase (MAPK) mechanism. These possible mechanisms were also proved by using the Map Kinase inhibitor (PD 98059). Based on these results we concluded that BV possesses an anti-inflammatory activity on the human monocyte THP-1cell line.

국문 초록 (Abstract)

봉독은 기원전부터 인체의 질병치료에 이용되어 왔으며 최근까지의 연구에 따르면 소염, 진통, 진경, 면역기능증가, 항암작용 등이 있는 것으로 밝혀져있다. 이 연구는 봉독의 항염증 효과에 대한 분자수준의 기전을 밝히기 위한 것으로 특히 Human acute monocytic leukemia cell line (THP-1)에 lipopolysaccharide (LPS)로 유도된 cytokine에 대한 봉독의 효과를 검증하였고 이 결과를 gene chip을 통해서 증명함으로써 그 효과를 검증했다.
봉독은 염증을 촉진시키는 cytokines의 발현을 억제하는 효과가 있어 LPS로 유도된 THP-1 cells의 cytokines 량과 비교할 때 각각 34% (TNF-α), 64.4% (TGF-β), 86.3% (IL-1β), 80.3% (IL-6), 66% (IL-8), 45.3% (IL-10)로 cytokines 생성량이 감소하였다. 또한 이 결과는 microarray를 사용하여 분석되었으며 RT-PCR의 결과와 일치하였다. 이는 봉독이 하나의 가정으로 볼때 Map Kinase (MAPK)를 억제하여 간접적으로 염증촉진 cytokine의 발현을 억제함으로써 항염증효과를 나타내는 것으로 보인다. 이상의 결과에 의거하여 봉독은 human monocyte THP-1cell line에 항염증 작용을 한다고 결론내릴 수 있다.

목차 (Table of Contents)

• Ⅰ. Introduction = 1
• Ⅱ. Materials and Methods = 6
• 1. Cell culture conditions = 6
• 2. PMA treatment & Cell viability assay & Macrophage differentiation and stimulation = 6
• 3. Reverse transcription (RT) - PCR analysis = 8
• 4. Gene expression profiling studies = 10
• 4-1. RNA preparation = 10
• 4-2. Labeling and purification = 11
• 4-3. Hybridization and data export = 11
• 4-4. Raw data preparation and statistic analysis = 12
• Ⅲ. Results = 13
• 1. Analysis of cell survival by Bee Venom on monocyte-derived macrophage = 13
• 2. Inhibitory effect of Bee Venom by various proinflammatory cytokines mRNA activity = 15
• 2-1. Inhibition of TNF-α expression by Bee Venom = 16
• 2-2. Inhibition of TGF-β expression by Bee Venom = 18
• 2-3. Inhibition of IL-8 expression by Bee Venom = 20
• 2-4. Inhibition of IL-6 expression by Bee Venom = 22
• 2-5. Inhibition of IL-1β expression by Bee Venom = 24
• 2-6. Inhibition of IL-10 expression by Bee Venom = 26
• 3. Chemokine and cytokine expression analysis by using sentrix Human-6-V2 expression bead chips analysis = 28
• Ⅳ. Discussion and Conclusion = 32
• References = 37
• Abstract = 46
• 국문초록 = 48