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1.
Free Radic Biol Med ; 158: 53-59, 2020 10.
Article in English | MEDLINE | ID: mdl-32682927

ABSTRACT

Glutathione (GSH) plays critical roles in the inflammatory response by acting as the master substrate for antioxidant enzymes and an important anti-inflammatory agent. In the early phase of the inflammatory response of macrophages, GSH content is decreased due to the down regulation of the catalytic subunit of glutamate cysteine ligase (GCLC). In the current study we investigated the underlying mechanism for this phenomenon. In human THP1-differentiated macrophages, GCLC mRNA had a half-life of 4 h under basal conditions, and it was significantly reduced to less than 2 h upon exposure to lipopolysaccharide (LPS), suggesting an increased decay of GCLC mRNA in the inflammatory response. The half-life of GCLC protein was >10 h under basal conditions, and upon LPS exposure the degradation rate of GCLC protein was significantly increased. The pan-caspase inhibitor Z-VAD-FMK but not the proteasome inhibitor MG132, prevented the down regulation of GCLC protein caused by LPS. Both caspase inhibitor Z-LEVD-FMK and siRNA of caspase-5 abrogated LPS-induced degradation of GCLC protein. In addition, supplement with γ-GC, the GCLC product, efficiently restored GSH content and suppressed the induction of NF-κB activity by LPS. In conclusion, these data suggest that GCLC down-regulation in the inflammatory response of macrophages is mediated through both increased mRNA decay and caspase-5-mediated GCLC protein degradation, and γ-GC is an efficient agent to restore GSH and regulate the inflammatory response.


Subject(s)
Glutamate-Cysteine Ligase , Glutathione , Caspases/metabolism , Down-Regulation , Glutamate-Cysteine Ligase/genetics , Glutamate-Cysteine Ligase/metabolism , Glutathione/metabolism , Humans , Macrophages/metabolism
2.
Methods Mol Biol ; 2144: 237-244, 2020.
Article in English | MEDLINE | ID: mdl-32410040

ABSTRACT

4-Hydroxenonenal (HNE) is one of the major α,ß-unsaturated aldehyde products of lipid peroxidation. HNE can form conjugates with macromolecules, including protein, and thereby alter their function. HNE and its conjugation with proteins are increased in aging and age-related diseases. To elucidate how HNE is involved in these aging-related pathophysiological changes, it is necessary to assess HNE modification of proteins. Here a simple and convenient Western-blot based method is presented to detect HNE modification of proteins in tissues of aging mice.


Subject(s)
Aging/metabolism , Aldehydes/metabolism , Lipid Peroxidation/genetics , Molecular Biology/methods , Animals , Mice , Oxidative Stress/genetics , Proteins/metabolism
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