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L929 cell conditioned medium protects RAW264.7 cells from oxidative injury through inducing antioxidant enzymes
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.