L929 cell conditioned medium protects RAW264.7 cells from oxidative injury through inducing antioxidant enzymes
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1Research Laboratory of Free Radical Medicine, The First Military Medical University, Guangzhou, People's Republic of China. zyjyx@fimmu.edu.cn
Abstract:
We have previously found that L929 cell conditioned medium (L929-CM) could protect mouse peritoneal macrophages from oxidative injury. To uncover the mechanism further, we investigated the effect of L929-CM on the oxidative injury caused by tbOOH to RAW264.7 cell lines. The results showed that L929-CM could protect RAW264.7 cells from oxidative injury (presented by cell morphology and cell survival rate), and L929-CM could also improve total superoxide dismutase (SOD), selenium-dependent and non-selenium-dependent glutathione peroxidase (SeGPx and non-SeGPx) activities in RAW264.7 cells. RT-PCR analysis showed that, L929-CM could induce plasma glutathione peroxidase (PLGPx) mRNA expression, while there was no inducing effect of L929-CM on phospholipid hydroperoxide glutathione peroxidase (PHGPx) mRNA expression in RAW264.7 cells. 5 microg/ml actinomycin D, 30 microg/ml cycloheximide (de novo protein synthesis inhibitor) and 50 microg/ml acetovanilone (intracellular superoxide anion production inhibitor) had no effects in attenuating the induction of PLGPx expression by L929-CM.
Insights
L929 cell conditioned medium protects RAW264.7 cells from oxidative stress by enhancing antioxidant enzyme activity and inducing plasma glutathione peroxidase mRNA expression. This study reveals a key mechanism for L929-CM
Area of Science:
- Cell Biology
- Biochemistry
- Oxidative Stress Research
Background:
- L929 cell conditioned medium (L929-CM) previously demonstrated protective effects on mouse peritoneal macrophages against oxidative injury.
- Understanding the underlying mechanisms of this protection is crucial for developing novel therapeutic strategies.
Purpose of the Study:
- To investigate the protective effects of L929-CM against tert-butyl hydroperoxide (tbOOH)-induced oxidative injury in RAW264.7 macrophage cell lines.
- To elucidate the molecular mechanisms by which L929-CM confers protection, focusing on antioxidant enzyme activity and gene expression.
Main Methods:
- RAW264.7 cells were treated with L929-CM and subsequently exposed to tbOOH to assess oxidative injury.
- Cell morphology and survival rates were evaluated.
- Activities of total superoxide dismutase (SOD), selenium-dependent glutathione peroxidase (SeGPx), and non-selenium-dependent glutathione peroxidase (non-SeGPx) were measured.
- Reverse transcription polymerase chain reaction (RT-PCR) was used to analyze the mRNA expression of plasma glutathione peroxidase (PLGPx) and phospholipid hydroperoxide glutathione peroxidase (PHGPx).
- The effects of actinomycin D, cycloheximide, and acetovanilone on PLGPx induction were examined.
Main Results:
- L929-CM significantly protected RAW264.7 cells from tbOOH-induced oxidative injury, as evidenced by improved cell morphology and survival rates.
- L929-CM treatment led to increased activities of total SOD, SeGPx, and non-SeGPx in RAW264.7 cells.
- RT-PCR analysis revealed that L929-CM induced PLGPx mRNA expression but did not affect PHGPx mRNA expression.
- Inhibitors of de novo protein synthesis and intracellular superoxide anion production did not attenuate the L929-CM-induced PLGPx mRNA expression.
Conclusions:
- L929-CM confers significant protection to RAW264.7 cells against oxidative stress.
- The protective mechanism involves the enhancement of key antioxidant enzyme activities and the induction of PLGPx mRNA expression.
- These findings highlight the potential of L929-CM as a protective agent against oxidative damage in macrophages.
