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. 2018 Jan 30;14(1):30.
doi: 10.1186/s12917-018-1360-0.

Cortisol modulates inflammatory responses in LPS-stimulated RAW264.7 cells via the NF-κB and MAPK pathways

Junsheng Dong  1   2 , Jianji Li  1   2 , Luying Cui  1   2 , Yefan Wang  1   2 , Jiaqi Lin  1   2 , Yang Qu  1   2 , Heng Wang  3   4
Affiliations

Cortisol modulates inflammatory responses in LPS-stimulated RAW264.7 cells via the NF-κB and MAPK pathways

Junsheng Dong et al. BMC Vet Res. .

Abstract

Background: The uteruses of most dairy cattle are easily infected by bacteria, especially gram-negative bacteria, following parturition. Macrophages are important cells of the immune system and play a critical role in the inflammatory response. In addition, cortisol levels become significantly increased due to the stress of parturition in dairy cattle, and cortisol is among the most widely used and effective therapies for many inflammatory diseases. In this study, we assessed the anti-inflammatory effects and potential molecular mechanisms of cortisol using a Lipopolysaccharide (LPS)-induced RAW264.7 macrophage cell line.

Results: Cortisol significantly suppressed the production of prostaglandin E2 (PGE2) and decreased the gene and protein expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) in a dose-dependent manner. Moreover, cortisol inhibited the mRNA expression of pro-inflammatory cytokines including tumor necrosis factor alpha (TNFα), interleukin-1β (IL-1β), and interleukin-6 (IL-6) and decreased IL-1β secretion in an LPS-treated RAW264.7 macrophage cell line. Moreover, we found that cortisol suppressed nuclear factor-kappa B (NF-κB) signaling in RAW264.7 macrophages stimulated with LPS. This suppression was mediated by the inhibition of IκBα degradation and NF-κB p65 phosphorylation. In addition, cortisol also suppressed the phosphorylation of mitogen-activated protein kinases (MAPK) such as extracellular signal-regulated kinase (ERK1/2), p38 MAPK, and c-Jun N-terminal kinase/stress-activated protein kinase (JNK).

Conclusions: These results suggest that high cortisol levels can attenuate LPS-induced inflammatory responses in the RAW264.7 macrophage cell line by regulating the NF-κB and MAPK signaling pathways.

Keywords: Anti-inflammatory; Cortisol; LPS; MAPKs; Macrophage; NF-κB.

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The authors declare that they have no competing interests.

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Figures

Fig. 1
Fig. 1
Effects of different concentrations of cortisol on RAW264.7 cell viability as measured by the CCK-8 assay. The data shown are means ± SEM (n = 6). *p < 0.05 and **p < 0.01 vs. control group
Fig. 2
Fig. 2
Effect of cortisol on PGE2 and cytokine production in LPS-stimulated RAW 264.7 macrophages. RAW264.7 cells were co-treated with cortisol (0, 5, 15 and 30 ng/mL) and LPS (1 μg/mL) for 0, 6, 12, and 24 h. Levels of PGE2 (a), IL-1β (b), IL-6 (c), and TNFα (d) in culture supernatants were measured by ELISA. The data presented are the means±SEM. ** p < 0.01 vs. the control group; # p < 0.05, ## p < 0.01 vs. the LPS group
Fig. 3
Fig. 3
Effects of cortisol on the mRNA and protein expression levels of iNOS and COX-2 in LPS-stimulated RAW264.7 cells. a and b Cells were co-treated with cortisol (5,15 and 30 ng/mL) and LPS (1 μg/mL) for 0, 6, 12, and 24 h. RNA was isolated and analyzed by RT-PCR. c and d Cells were co-treated with cortisol (5, 15 and 30 ng/mL) and LPS (1 μg/mL) for 24 h. Total proteins were isolated and analyzed by Western blot. The data presented are the means±SEM. ** p < 0.01 vs. the control group; # p < 0.05, ## p < 0.01 vs. the LPS group
Fig. 4
Fig. 4
Effects of cortisol on IL-1β (a), IL-6 (b) and TNFα (c) mRNA expression in LPS-stimulated RAW264.7 cells. RAW264.7 cells were co-treated with cortisol (5, 15 and 30 ng/mL) and LPS (1 μg/mL) for 0, 6, 12, and 24 h. RNA was isolated and analyzed by RT-PCR. The data presented are the means±SEM. **p < 0.01 vs. the control group; # p < 0.05, ## p < 0.01 vs. the LPS group
Fig. 5
Fig. 5
Inhibitory effects of cortisol on LPS-stimulated NF-κB p65 and IκBα phosphorylation in RAW 264.7 cells. a Cells were stimulated with LPS (1 μg/mL) alone for 0, 15, 30 and 45 min. b Cells were co-treated with cortisol (5, 15 and 30 ng/mL) and LPS (1 μg/mL) for 30 min. Total proteins were isolated and subjected to Western blotting. The data presented are the means±SEM. ** p < 0.01 vs. the control group; # p < 0.05, ## p < 0.01 vs. the LPS group
Fig. 6
Fig. 6
Inhibitory effects of cortisol on MAPK phosphorylation in RAW264.7 macrophages. a Cells were stimulated with LPS (1 μg/mL) alone for 0, 15, 30 and 45 min. b Cells were co-treated with cortisol (5, 15 and 30 ng/mL) and LPS (1 μg/mL) for 30 min. Total proteins were isolated and subjected to Western blotting. The data presented are the means±SEM. ** p < 0.01 vs. the control group; # p < 0.05, ## p < 0.01 vs. the LPS group
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