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

摘要

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-kappa B (NF-kappa B) 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-kappa B nuclear translocation and HCI data quality was optimized. The BAY-11-7085, the positive control for inhibiting NF-kappa B, 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-1 beta, IL-6, and TNF-alpha mRNA in LPS-induced RAW264.7 cells was detected. @@@ Results: The optimal conditions for measuring NF-kappa B 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 mu m(2), cell area of 80 mu m(2), collar of 0.9 mu m, 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-kappa B 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-1 beta, IL-6, and TNF-alpha. @@@ Conclusion: The established HCI platform could be applied to screen anti-inflammatory compounds by measuring the NF-kappa B nuclear translocation in LPS-induced RAW264.7 cells.

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