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A Fluorescence-based Protocol for Preliminary Screening of Protein Synthesis Inhibitors from Natural Sources
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A Fluorescence-based Protocol for Preliminary Screening of Protein Synthesis Inhibitors from Natural Sources

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Inhibition of reactive oxygen species down-regulates protein synthesis in RAW 264.7

Kenya Kimura1, Sachiko Ito, Masato Nagino

  • 1Department of Immunology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi 466-8550, Japan.

Biochemical and Biophysical Research Communications
|May 20, 2008
PubMed

Insights

Lipopolysaccharide (LPS) triggers endoplasmic reticulum (ER) stress in macrophages. N-acetyl-L-cysteine, an ROS inhibitor, rapidly induces ER stress markers and suppresses TNF-alpha, suggesting protein synthesis shutoff.

Area of Science:

  • Immunology
  • Cell Biology
  • Molecular Biology

Background:

  • Macrophages play a crucial role in immune responses.
  • Endoplasmic reticulum (ER) stress is implicated in cellular dysfunction.
  • Lipopolysaccharide (LPS) is a potent immune stimulant.

Purpose of the Study:

  • To investigate ER stress responses in macrophages stimulated with LPS.
  • To explore the role of reactive oxygen species (ROS) in LPS-induced ER stress.
  • To determine the effect of ROS inhibition on TNF-alpha production.

Main Methods:

  • Stimulation of RAW 264.7 macrophage cell line with LPS.
  • Treatment with N-acetyl-L-cysteine (ROS inhibitor).
  • Measurement of eIF2alpha phosphorylation, ATF4, GADD34, GADD153 expression, and TNF-alpha production.

Main Results:

  • LPS induced ER stress markers (eIF2alpha phosphorylation, ATF4, GADD34, GADD153) in a time- and dose-dependent manner.
  • N-acetyl-L-cysteine rapidly induced eIF2alpha phosphorylation and GADD34 expression.
  • N-acetyl-L-cysteine significantly suppressed LPS-induced TNF-alpha production, correlating with early eIF2alpha phosphorylation.

Conclusions:

  • LPS induces ER stress in macrophages.
  • ROS play a significant role in modulating LPS-induced ER stress and TNF-alpha production.
  • Early ER stress, potentially mediated by ROS, can lead to protein synthesis shutoff, impacting inflammatory responses.

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