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Current Drug Metabolism

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ISSN (Print): 1389-2002
ISSN (Online): 1875-5453

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Research Article

Establishment of a High Content Image Platform to Measure NF-κB Nuclear Translocation in LPS-induced RAW264.7 Macrophages for Screening Anti-inflammatory Drug Candidates

Author(s): Yan-Yu Zhang, Yun-Da Yao, Qi-Qing Cheng, Yu-Feng Huang and Hua Zhou*

Volume 23, Issue 5, 2022

Published on: 15 June, 2022

Page: [394 - 414] Pages: 21

DOI: 10.2174/1389200223666220411121614

Price: $65

Abstract

Background: High Content Image (HCI), an automatic imaging and analysis system, provides a fast drug screening method by detecting the subcellular distribution of protein in intact cells.

Objective: This study established the first standardized HCI platform for lipopolysaccharide (LPS)-induced RAW264.7 macrophages to screen anti-inflammatory compounds by measuring nuclear factor-κB (NF-κB) nuclear translocation.

Methods: The influence of the cell passages, cell density, LPS induction time and concentration, antibody dilution, serum, dimethyl sulfoxide, and analysis parameters on NF-κB nuclear translocation and HCI data quality was optimized. The BAY-11-7085, the positive control for inhibiting NF-κB, and the Western blot assay were separately employed to verify the stability and reliability of the platform. Lastly, the effect of BHA on NO release, iNOS expression, IL-1β, IL-6, and TNF-α mRNA in LPS-induced RAW264.7 cells was detected.

Results: The optimal conditions for measuring NF-κB translocation in LPS-induced RAW264.7 cells by HCI were established. Cells that do not exceed 22 passages were seeded at a density of 10 k cells/well and pretreated with compounds following 200 ng/mL LPS for 40 min. Parameters including the nuclear area of 65 μm2, cell area of 80 μm2, collar of 0.9 μm, and sensitivity of 25% were recommended for image segmentation algorithms in the analysis workstation. Benzoylhypaconine from aconite was screened for the first time as an anti-inflammatory candidate by the established HCI platform. The inhibitory effect of benzoylhypaconine on NF-κB translocation was verified by Western blot. Furthermore, benzoylhypaconine reduced the release of NO, inhibited the expression of iNOS, and decreased the mRNA levels of IL-1β, IL-6, and TNF-α.

Conclusion: The established HCI platform could be applied to screen anti-inflammatory compounds by measuring the NF-κB nuclear translocation in LPS-induced RAW264.7 cells.

Keywords: High content image, lipopolysaccharide, RAW264.7 macrophages, nuclear factor-κB nuclear translocation, anti-inflammation, BAY 11-7085, benzoylhypaconine.

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