Abstract

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.

Highlights

  • Nitric oxide (NO) has a most versatile role in the immune system

  • These results showed that crude ethanol extracts from oregano, laurel, and long pepper we found that S-10 -Acetoxychavicol acetate from greater galangal could reduce IL-6 level and increase at 75 μg/mL and 1′S-1′-Acetoxychavicol acetate at 5, 25, and 100 μM from greater galangal could

  • We generated new stable RAW 264.7 derived IL-6/gapdh and IL-10/gapdh reporters cell lines. We evaluated these stable cells with LPS and found that LPS response to IL-6 and IL-10 levels in stable cells are concentration-dependent

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Summary

Introduction

Nitric oxide (NO) has a most versatile role in the immune system. It is involved in several diseases such as infectious disease, autoimmune disease, tumors [1], stress-related diseases [2], chronic inflammatory diseases, stroke, diabetes, neurodegenerative disorders [3], and inflammatory bowel disease [4]. IL-10 could inhibit anti-inflammatory IL-6 in activated macrophage cell lines [6]. We established IL-6 and IL-10 reporters that could determine reporter activity along with gapdh reporter in LPS-induced RAW 264.7 cells. It needs transfection of dual IL-6/gapdh and IL-10/gapdh reporters. We could determine relative IL-6/gapdh and IL-10/gapdh with 96-well plate within one step It will make determination of IL-6 and IL-10 are less cost and time by these assays. Our assay led to discover a new compound for balancing pro-inflammatory and anti-inflammatory activity

Results and Discussion
Cell Culture
Cell Proliferation Assay
Statistical Analysis
Conclusions
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