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Signaling function of Na,K-ATPase induced by ouabain against LPS as an inflammation model in hippocampus.

Kinoshita PF, Yshii LM, Vasconcelos AR, Orellana AM, Lima Lde S, Davel AP, Rossoni LV, Kawamoto EM, Scavone C - J Neuroinflammation (2014)

Bottom Line: The involvement of the inflammatory transcription factor NF-κB in the OUA effect was indicated by its prevention of LPS-induced nuclear translocation of the NF-κB subunit, RELA (p65), as well as the decreased cytosol levels of the NF-κB inhibitor, IKB, in the hippocampus.Our results suggest that a low dose of OUA has an important anti-inflammatory effect in the rat hippocampus.This effect was associated with decreased GFAP induction by LPS in the dentate gyrus, a brain area linked to adult neurogenesis.

View Article: PubMed Central - PubMed

Affiliation: Molecular Neuropharmacology Laboratory, Department of Pharmacology, Institute of Biomedical Science, University of São Paulo, 05508-900, São Paulo, Brazil. paula.f.kinoshita@gmail.com.

ABSTRACT

Background: Ouabain (OUA) is a newly recognized hormone that is synthesized in the adrenal cortex and hypothalamus. Low doses of OUA can activate a signaling pathway by interaction with Na,K-ATPase, which is protective against a number of insults. OUA has central and peripheral anti-inflammatory effects. Lipopolysaccharide (LPS), via toll-like receptor 4 activation, is a widely used model to induce systemic inflammation. This study used a low OUA dose to evaluate its effects on inflammation induced by LPS injection in rats.

Methods: Adult male Wistar rats received acute intraperitoneal (ip) OUA (1.8 μg/kg) or saline 20 minutes before LPS (200 μg/kg, ip) or saline injection. Some of the animals had their femoral artery catheterized in order to assess arterial blood pressure values before and after OUA administration. Na,K-ATPase activity, cytokine mRNA levels, apoptosis-related proteins, NF-κB activation brain-derived neurotrophic factor BDNF, corticosterone and TNF-α levels were measured.

Results: OUA pretreatment decreased mRNA levels of the pro-inflammatory cytokines, inducible nitric oxide synthase (iNOS) and IL-1β, which are activated by LPS in the hippocampus, but with no effect on serum measures of these factors. None of these OUA effects were linked to Na,K-ATPase activity. The involvement of the inflammatory transcription factor NF-κB in the OUA effect was indicated by its prevention of LPS-induced nuclear translocation of the NF-κB subunit, RELA (p65), as well as the decreased cytosol levels of the NF-κB inhibitor, IKB, in the hippocampus. OUA pretreatment reversed the LPS-induced glial fibrillary acidic protein (GFAP) activation and associated inflammation in the dentate gyrus. OUA also prevented LPS-induced increases in the hippocampal Bax/Bcl2 ratio suggesting an anti-apoptotic action in the brain.

Conclusion: Our results suggest that a low dose of OUA has an important anti-inflammatory effect in the rat hippocampus. This effect was associated with decreased GFAP induction by LPS in the dentate gyrus, a brain area linked to adult neurogenesis.

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Effects of pretreatment of ouabain (OUA) (1.8 μg/kg) on lipopolysaccharide (LPS)-induced (200 μg/kg) p65 (RELA) subunit NF-κB nuclear translocation and IκB (IKB) degradation. Pretreatment of OUA blocks the LPS-induced NF-κB activation. (A) Representative Western blotting autoradiographs and densitometric analysis (arbitrary units, AU) of p65 nuclear/β-ACTIN ratio, *P < 0.05 versus control and OUA + LPS. (B) Representative Western blotting autoradiographs and densitometric analysis (arbitrary units, AU) of IKB cytosolic/β-ACTIN ratio, *P < 0.05 versus control, OUA, OUA + LPS. Data are presented as mean ± SEM from five individual experiments. One-way ANOVA followed by Newman-Keuls post-test.
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Fig3: Effects of pretreatment of ouabain (OUA) (1.8 μg/kg) on lipopolysaccharide (LPS)-induced (200 μg/kg) p65 (RELA) subunit NF-κB nuclear translocation and IκB (IKB) degradation. Pretreatment of OUA blocks the LPS-induced NF-κB activation. (A) Representative Western blotting autoradiographs and densitometric analysis (arbitrary units, AU) of p65 nuclear/β-ACTIN ratio, *P < 0.05 versus control and OUA + LPS. (B) Representative Western blotting autoradiographs and densitometric analysis (arbitrary units, AU) of IKB cytosolic/β-ACTIN ratio, *P < 0.05 versus control, OUA, OUA + LPS. Data are presented as mean ± SEM from five individual experiments. One-way ANOVA followed by Newman-Keuls post-test.

Mentions: Western blotting assays showed that LPS (ip) administration increases both nuclear RELA (p65) translocation and cytosolic IKB degradation, which could lead to NF-κB activation in the rat hippocampus. Although OUA itself did not change either nuclear p65 translocation or cytosolic IKB, it did block LPS-induced RELA (p65) and IKB effects in the rat hippocampus (Figure 3A and B). The inhibition of LPS-induced NF-κB translocation by OUA likely underlies the OUA inhibition of LPS-induced iNos, Il-1β and increased Bax/Bcl-2 ratio.Figure 3


Signaling function of Na,K-ATPase induced by ouabain against LPS as an inflammation model in hippocampus.

Kinoshita PF, Yshii LM, Vasconcelos AR, Orellana AM, Lima Lde S, Davel AP, Rossoni LV, Kawamoto EM, Scavone C - J Neuroinflammation (2014)

Effects of pretreatment of ouabain (OUA) (1.8 μg/kg) on lipopolysaccharide (LPS)-induced (200 μg/kg) p65 (RELA) subunit NF-κB nuclear translocation and IκB (IKB) degradation. Pretreatment of OUA blocks the LPS-induced NF-κB activation. (A) Representative Western blotting autoradiographs and densitometric analysis (arbitrary units, AU) of p65 nuclear/β-ACTIN ratio, *P < 0.05 versus control and OUA + LPS. (B) Representative Western blotting autoradiographs and densitometric analysis (arbitrary units, AU) of IKB cytosolic/β-ACTIN ratio, *P < 0.05 versus control, OUA, OUA + LPS. Data are presented as mean ± SEM from five individual experiments. One-way ANOVA followed by Newman-Keuls post-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4307894&req=5

Fig3: Effects of pretreatment of ouabain (OUA) (1.8 μg/kg) on lipopolysaccharide (LPS)-induced (200 μg/kg) p65 (RELA) subunit NF-κB nuclear translocation and IκB (IKB) degradation. Pretreatment of OUA blocks the LPS-induced NF-κB activation. (A) Representative Western blotting autoradiographs and densitometric analysis (arbitrary units, AU) of p65 nuclear/β-ACTIN ratio, *P < 0.05 versus control and OUA + LPS. (B) Representative Western blotting autoradiographs and densitometric analysis (arbitrary units, AU) of IKB cytosolic/β-ACTIN ratio, *P < 0.05 versus control, OUA, OUA + LPS. Data are presented as mean ± SEM from five individual experiments. One-way ANOVA followed by Newman-Keuls post-test.
Mentions: Western blotting assays showed that LPS (ip) administration increases both nuclear RELA (p65) translocation and cytosolic IKB degradation, which could lead to NF-κB activation in the rat hippocampus. Although OUA itself did not change either nuclear p65 translocation or cytosolic IKB, it did block LPS-induced RELA (p65) and IKB effects in the rat hippocampus (Figure 3A and B). The inhibition of LPS-induced NF-κB translocation by OUA likely underlies the OUA inhibition of LPS-induced iNos, Il-1β and increased Bax/Bcl-2 ratio.Figure 3

Bottom Line: The involvement of the inflammatory transcription factor NF-κB in the OUA effect was indicated by its prevention of LPS-induced nuclear translocation of the NF-κB subunit, RELA (p65), as well as the decreased cytosol levels of the NF-κB inhibitor, IKB, in the hippocampus.Our results suggest that a low dose of OUA has an important anti-inflammatory effect in the rat hippocampus.This effect was associated with decreased GFAP induction by LPS in the dentate gyrus, a brain area linked to adult neurogenesis.

View Article: PubMed Central - PubMed

Affiliation: Molecular Neuropharmacology Laboratory, Department of Pharmacology, Institute of Biomedical Science, University of São Paulo, 05508-900, São Paulo, Brazil. paula.f.kinoshita@gmail.com.

ABSTRACT

Background: Ouabain (OUA) is a newly recognized hormone that is synthesized in the adrenal cortex and hypothalamus. Low doses of OUA can activate a signaling pathway by interaction with Na,K-ATPase, which is protective against a number of insults. OUA has central and peripheral anti-inflammatory effects. Lipopolysaccharide (LPS), via toll-like receptor 4 activation, is a widely used model to induce systemic inflammation. This study used a low OUA dose to evaluate its effects on inflammation induced by LPS injection in rats.

Methods: Adult male Wistar rats received acute intraperitoneal (ip) OUA (1.8 μg/kg) or saline 20 minutes before LPS (200 μg/kg, ip) or saline injection. Some of the animals had their femoral artery catheterized in order to assess arterial blood pressure values before and after OUA administration. Na,K-ATPase activity, cytokine mRNA levels, apoptosis-related proteins, NF-κB activation brain-derived neurotrophic factor BDNF, corticosterone and TNF-α levels were measured.

Results: OUA pretreatment decreased mRNA levels of the pro-inflammatory cytokines, inducible nitric oxide synthase (iNOS) and IL-1β, which are activated by LPS in the hippocampus, but with no effect on serum measures of these factors. None of these OUA effects were linked to Na,K-ATPase activity. The involvement of the inflammatory transcription factor NF-κB in the OUA effect was indicated by its prevention of LPS-induced nuclear translocation of the NF-κB subunit, RELA (p65), as well as the decreased cytosol levels of the NF-κB inhibitor, IKB, in the hippocampus. OUA pretreatment reversed the LPS-induced glial fibrillary acidic protein (GFAP) activation and associated inflammation in the dentate gyrus. OUA also prevented LPS-induced increases in the hippocampal Bax/Bcl2 ratio suggesting an anti-apoptotic action in the brain.

Conclusion: Our results suggest that a low dose of OUA has an important anti-inflammatory effect in the rat hippocampus. This effect was associated with decreased GFAP induction by LPS in the dentate gyrus, a brain area linked to adult neurogenesis.

Show MeSH