Novel and Stable Dual-Color IL-6 and IL-10 Reporters Derived from RAW 264.7 for Anti-Inflammation Screening of Natural Products.

Our previous study suggested that the interleukin (IL)-6 and IL-10 could serve as good biomarkers for chronic inflammatory disease. We previously established an IL-6 and IL-10 reporters assay that could examine reporter activity along with the reference gene in LPS-induced RAW 264.7 cells. In this study, we described 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. We 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. The relative IL-6 and IL-6/IL-10 values decreased by the crude ethanol extracts from nutmeg and by 1'S-1'-acetoxychavicol from greater galangal using real-time bioluminescence monitoring. At the same time, the relative IL-10 was induced. The relative IL-6 and IL-6/IL-10 decreased by crude ethanol extracts from nutmeg and 1'S-1'-acetoxychavicol acetate at 6 h. Only crude ethanol extract from nutmeg induced IL-10 at 6 h. We 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 chronic inflammatory disease.