2019 Fiscal Year Annual Research Report
Development of a high-throughout drug recovery assay of stress for natural compounds screening
| Project/Area Number |
18K17993
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| Research Institution | National Institute of Advanced Industrial Science and Technology |
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Principal Investigator |
Saiki Papawee 国立研究開発法人産業技術総合研究所, 生命工学領域, 研究員 (10803934)
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| Project Period (FY) |
2018-04-01 – 2020-03-31
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| Keywords | stress / IL-6 / IL-10 / bioluminescence |
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| Outline of Annual Research Achievements |
Based on mouse experiment in the first year, I selected IL-6 and IL-10 for new screening assay. First, I established IL-6 and IL-10 promoter assay which can monitor with reference gene as Glyceraldehyde 3-phosphate dehydrogenase (gapdh) promoter in living single cell. It could determine IL-6 and IL-10 levels continuously in real-time within two steps. We evaluated IL-6 and IL-10 reporter expression in LPS-induced RAW 264.7 cells with well-known anti-inflammatory compounds such as quercetin, xanthones, b-D-glucan and dexamethasone. As the results, this new assay could use for determination of IL-6 and IL-10 reporter expression in LPS-induced RAW 264.7 cells for anti-inflammation activity. Next, I developed new and stable RAW 264.7 derived dual-color IL-6/gapdh and IL-10/gapdh reporters. This assay allowed us to easily determine relative IL-6 and IL-10 levels with 96-well plate within one step. I evaluated the relative IL-6 and IL-10 levels in the LPS-induced stable cells testing 52 natural products by real-time bioluminescence monitoring and time-point determination using a microplate luminometer. By these assay, we found 1`S-1`-acetoxychavicol from greater galangal that could decrease relative IL-6 and IL-6/IL-10 values and induced the relative IL-10. These suggested that the use of these stable cells by real-time monitoring could serve as a screening assay for anti-inflammatory activity and may be used to discover new drugs against inflammatory disease and stress.
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