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Anti-inflammatory effects of progesterone in lipopolysaccharide-stimulated BV-2 microglia.

Lei B, Mace B, Dawson HN, Warner DS, Laskowitz DT, James ML - PLoS ONE (2014)

Bottom Line: Progesterone decreased LPS-mediated phosphorylation of p38, c-Jun N-terminal kinase and extracellular regulated kinase MAPKs.These progesterone effects were inhibited by its antagonist mifepristone.In conclusion, progesterone exhibits pleiotropic anti-inflammatory effects in LPS-stimulated BV-2 microglia by down-regulating proinflammatory mediators corresponding to suppression of NF-κB and MAPK activation.

View Article: PubMed Central - PubMed

Affiliation: Multidisciplinary Neuroprotection Laboratories, Duke University Medical Center, Durham, North Carolina, United States of America; Department of Anesthesiology, Duke University Medical Center, Durham, North Carolina, United States of America.

ABSTRACT
Female sex is associated with improved outcome in experimental brain injury models, such as traumatic brain injury, ischemic stroke, and intracerebral hemorrhage. This implies female gonadal steroids may be neuroprotective. A mechanism for this may involve modulation of post-injury neuroinflammation. As the resident immunomodulatory cells in central nervous system, microglia are activated during acute brain injury and produce inflammatory mediators which contribute to secondary injury including proinflammatory cytokines, and nitric oxide (NO) and prostaglandin E2 (PGE2), mediated by inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2), respectively. We hypothesized that female gonadal steroids reduce microglia mediated neuroinflammation. In this study, the progesterone's effects on tumor necrosis factor alpha (TNF-α), iNOS, and COX-2 expression were investigated in lipopolysaccharide (LPS)-stimulated BV-2 microglia. Further, investigation included nuclear factor kappa B (NF-κB) and mitogen activated protein kinase (MAPK) pathways. LPS (30 ng/ml) upregulated TNF-α, iNOS, and COX-2 protein expression in BV-2 cells. Progesterone pretreatment attenuated LPS-stimulated TNF-α, iNOS, and COX-2 expression in a dose-dependent fashion. Progesterone suppressed LPS-induced NF-κB activation by decreasing inhibitory κBα and NF-κB p65 phosphorylation and p65 nuclear translocation. Progesterone decreased LPS-mediated phosphorylation of p38, c-Jun N-terminal kinase and extracellular regulated kinase MAPKs. These progesterone effects were inhibited by its antagonist mifepristone. In conclusion, progesterone exhibits pleiotropic anti-inflammatory effects in LPS-stimulated BV-2 microglia by down-regulating proinflammatory mediators corresponding to suppression of NF-κB and MAPK activation. This suggests progesterone may be used as a potential neurotherapeutic to treat inflammatory components of acute brain injury.

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Progesterone attenuated LPS-stimulated TNF-α production.Cells were treated in serum-free DMEM. (A) Pretreatment of progesterone for 1 h dose-dependently suppressed LPS (30 ng/ml, 4 h)-mediated TNF-α secretion in cell supernants. *P<0.05 compared with LPS group, n = 7. P8, P7 and P6: progesterone at 10−8, 10−7 and 10−6 M. (B) The representative immunoblot demonstrated time course changes for transmembrane TNF-α (tmTNFα) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, a loading control) with LPS (30 ng/ml) stimulation from 0 to 24 h. (C) Pretreatment of progesterone for 1 h dose-dependently decreased LPS (30 ng/ml, 4 h)-stimulated tmTNF-α expression. The representative immunoblot (D) and summary of band densitometric quantifications (E) showed that progesterone receptor antagonist mifepristone reversed progesterone’s effect on tmTNF-α expression induced by 4 h LPS stimulation. *P<0.05 compared to LPS group, n = 3. P7L: progesterone 10−7 M+LPS; M9PL: mifepristone 10−9 M+progesterone 10−7 M+LPS; M8PL: mifepristone 10−8 M+progesterone 10−7 M+LPS; M7PL: mifepristone 10−7 M+progesterone 10−7 M+LPS.
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pone-0103969-g002: Progesterone attenuated LPS-stimulated TNF-α production.Cells were treated in serum-free DMEM. (A) Pretreatment of progesterone for 1 h dose-dependently suppressed LPS (30 ng/ml, 4 h)-mediated TNF-α secretion in cell supernants. *P<0.05 compared with LPS group, n = 7. P8, P7 and P6: progesterone at 10−8, 10−7 and 10−6 M. (B) The representative immunoblot demonstrated time course changes for transmembrane TNF-α (tmTNFα) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, a loading control) with LPS (30 ng/ml) stimulation from 0 to 24 h. (C) Pretreatment of progesterone for 1 h dose-dependently decreased LPS (30 ng/ml, 4 h)-stimulated tmTNF-α expression. The representative immunoblot (D) and summary of band densitometric quantifications (E) showed that progesterone receptor antagonist mifepristone reversed progesterone’s effect on tmTNF-α expression induced by 4 h LPS stimulation. *P<0.05 compared to LPS group, n = 3. P7L: progesterone 10−7 M+LPS; M9PL: mifepristone 10−9 M+progesterone 10−7 M+LPS; M8PL: mifepristone 10−8 M+progesterone 10−7 M+LPS; M7PL: mifepristone 10−7 M+progesterone 10−7 M+LPS.

Mentions: To determine progesterone’s role in cytokine production, 4 h of LPS (30 ng/ml) exposure was used to stimulate BV-2 cell cytokine production in the presence of escalating doses of progesterone. Pretreatment of progesterone dose-dependently attenuated LPS-induced soluble TNF-α (sTNF-α) production in the supernatant. Significant sTNF-α decrease (approximately 20%) occurred with 10−7 and 10−6 M progesterone (Figure 2A). In time course experiments, as the precursor of sTNF-α, transmembrane TNF-α (mTNF-α) expression on BV-2 cells was found to increase at 1 h, reached maximum at 4 h, and then gradually decreased to 1-h levels by 24 h after LPS addition (Figure 2B). Progesterone also reduced LPS-induced mTNF-α expression in a dose dependent manner with similar maximal reduction at both 10−7 and 10−6 M (Figure 2C). Since 10−7 M is within the physiological concentration range of progesterone in blood in the female mouse [37], it was used for further experiments. Finally, mifepristone, a progesterone receptor antagonist, reversed progesterone’s effect on mTNF-α expression starting at 10−9 M (Figure 2D, 2E).


Anti-inflammatory effects of progesterone in lipopolysaccharide-stimulated BV-2 microglia.

Lei B, Mace B, Dawson HN, Warner DS, Laskowitz DT, James ML - PLoS ONE (2014)

Progesterone attenuated LPS-stimulated TNF-α production.Cells were treated in serum-free DMEM. (A) Pretreatment of progesterone for 1 h dose-dependently suppressed LPS (30 ng/ml, 4 h)-mediated TNF-α secretion in cell supernants. *P<0.05 compared with LPS group, n = 7. P8, P7 and P6: progesterone at 10−8, 10−7 and 10−6 M. (B) The representative immunoblot demonstrated time course changes for transmembrane TNF-α (tmTNFα) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, a loading control) with LPS (30 ng/ml) stimulation from 0 to 24 h. (C) Pretreatment of progesterone for 1 h dose-dependently decreased LPS (30 ng/ml, 4 h)-stimulated tmTNF-α expression. The representative immunoblot (D) and summary of band densitometric quantifications (E) showed that progesterone receptor antagonist mifepristone reversed progesterone’s effect on tmTNF-α expression induced by 4 h LPS stimulation. *P<0.05 compared to LPS group, n = 3. P7L: progesterone 10−7 M+LPS; M9PL: mifepristone 10−9 M+progesterone 10−7 M+LPS; M8PL: mifepristone 10−8 M+progesterone 10−7 M+LPS; M7PL: mifepristone 10−7 M+progesterone 10−7 M+LPS.
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pone-0103969-g002: Progesterone attenuated LPS-stimulated TNF-α production.Cells were treated in serum-free DMEM. (A) Pretreatment of progesterone for 1 h dose-dependently suppressed LPS (30 ng/ml, 4 h)-mediated TNF-α secretion in cell supernants. *P<0.05 compared with LPS group, n = 7. P8, P7 and P6: progesterone at 10−8, 10−7 and 10−6 M. (B) The representative immunoblot demonstrated time course changes for transmembrane TNF-α (tmTNFα) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, a loading control) with LPS (30 ng/ml) stimulation from 0 to 24 h. (C) Pretreatment of progesterone for 1 h dose-dependently decreased LPS (30 ng/ml, 4 h)-stimulated tmTNF-α expression. The representative immunoblot (D) and summary of band densitometric quantifications (E) showed that progesterone receptor antagonist mifepristone reversed progesterone’s effect on tmTNF-α expression induced by 4 h LPS stimulation. *P<0.05 compared to LPS group, n = 3. P7L: progesterone 10−7 M+LPS; M9PL: mifepristone 10−9 M+progesterone 10−7 M+LPS; M8PL: mifepristone 10−8 M+progesterone 10−7 M+LPS; M7PL: mifepristone 10−7 M+progesterone 10−7 M+LPS.
Mentions: To determine progesterone’s role in cytokine production, 4 h of LPS (30 ng/ml) exposure was used to stimulate BV-2 cell cytokine production in the presence of escalating doses of progesterone. Pretreatment of progesterone dose-dependently attenuated LPS-induced soluble TNF-α (sTNF-α) production in the supernatant. Significant sTNF-α decrease (approximately 20%) occurred with 10−7 and 10−6 M progesterone (Figure 2A). In time course experiments, as the precursor of sTNF-α, transmembrane TNF-α (mTNF-α) expression on BV-2 cells was found to increase at 1 h, reached maximum at 4 h, and then gradually decreased to 1-h levels by 24 h after LPS addition (Figure 2B). Progesterone also reduced LPS-induced mTNF-α expression in a dose dependent manner with similar maximal reduction at both 10−7 and 10−6 M (Figure 2C). Since 10−7 M is within the physiological concentration range of progesterone in blood in the female mouse [37], it was used for further experiments. Finally, mifepristone, a progesterone receptor antagonist, reversed progesterone’s effect on mTNF-α expression starting at 10−9 M (Figure 2D, 2E).

Bottom Line: Progesterone decreased LPS-mediated phosphorylation of p38, c-Jun N-terminal kinase and extracellular regulated kinase MAPKs.These progesterone effects were inhibited by its antagonist mifepristone.In conclusion, progesterone exhibits pleiotropic anti-inflammatory effects in LPS-stimulated BV-2 microglia by down-regulating proinflammatory mediators corresponding to suppression of NF-κB and MAPK activation.

View Article: PubMed Central - PubMed

Affiliation: Multidisciplinary Neuroprotection Laboratories, Duke University Medical Center, Durham, North Carolina, United States of America; Department of Anesthesiology, Duke University Medical Center, Durham, North Carolina, United States of America.

ABSTRACT
Female sex is associated with improved outcome in experimental brain injury models, such as traumatic brain injury, ischemic stroke, and intracerebral hemorrhage. This implies female gonadal steroids may be neuroprotective. A mechanism for this may involve modulation of post-injury neuroinflammation. As the resident immunomodulatory cells in central nervous system, microglia are activated during acute brain injury and produce inflammatory mediators which contribute to secondary injury including proinflammatory cytokines, and nitric oxide (NO) and prostaglandin E2 (PGE2), mediated by inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2), respectively. We hypothesized that female gonadal steroids reduce microglia mediated neuroinflammation. In this study, the progesterone's effects on tumor necrosis factor alpha (TNF-α), iNOS, and COX-2 expression were investigated in lipopolysaccharide (LPS)-stimulated BV-2 microglia. Further, investigation included nuclear factor kappa B (NF-κB) and mitogen activated protein kinase (MAPK) pathways. LPS (30 ng/ml) upregulated TNF-α, iNOS, and COX-2 protein expression in BV-2 cells. Progesterone pretreatment attenuated LPS-stimulated TNF-α, iNOS, and COX-2 expression in a dose-dependent fashion. Progesterone suppressed LPS-induced NF-κB activation by decreasing inhibitory κBα and NF-κB p65 phosphorylation and p65 nuclear translocation. Progesterone decreased LPS-mediated phosphorylation of p38, c-Jun N-terminal kinase and extracellular regulated kinase MAPKs. These progesterone effects were inhibited by its antagonist mifepristone. In conclusion, progesterone exhibits pleiotropic anti-inflammatory effects in LPS-stimulated BV-2 microglia by down-regulating proinflammatory mediators corresponding to suppression of NF-κB and MAPK activation. This suggests progesterone may be used as a potential neurotherapeutic to treat inflammatory components of acute brain injury.

Show MeSH
Related in: MedlinePlus