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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) in the lipopolysaccharide (LPS) (200 μg/kg) inflammation model on the mRNA levels ofIl-1β, iNos, Bax, andBcl2. Pretreatment of OUA can decrease the mRNA levels of pro-inflammatory cytokines and pro-apoptotic proteins. Representative semiquantitative RT- PCR photographs and densitometric analysis (arbitrary units, AU) of the specific bands. mRNA levels are presented as ratios of the target gene to Gapdh expression. Data are presented as mean ± SEM from five individual experiments. One-way ANOVA followed by Newman-Keuls post-test. (A)Il-1β mRNA,*P < 0.05 versus control and OUA and **P < 0.01 versus control and OUA and OUA + LPS. (B)iNos mRNA, *P < 0.05 versus control and OUA + LPS. (C)Bax/Bcl2 ratio,**P < 0.001 versus OUA, control and OUA + LPS and *P < 0.05 versus OUA.
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Fig2: Effects of pretreatment of ouabain (OUA) (1.8 μg/kg) in the lipopolysaccharide (LPS) (200 μg/kg) inflammation model on the mRNA levels ofIl-1β, iNos, Bax, andBcl2. Pretreatment of OUA can decrease the mRNA levels of pro-inflammatory cytokines and pro-apoptotic proteins. Representative semiquantitative RT- PCR photographs and densitometric analysis (arbitrary units, AU) of the specific bands. mRNA levels are presented as ratios of the target gene to Gapdh expression. Data are presented as mean ± SEM from five individual experiments. One-way ANOVA followed by Newman-Keuls post-test. (A)Il-1β mRNA,*P < 0.05 versus control and OUA and **P < 0.01 versus control and OUA and OUA + LPS. (B)iNos mRNA, *P < 0.05 versus control and OUA + LPS. (C)Bax/Bcl2 ratio,**P < 0.001 versus OUA, control and OUA + LPS and *P < 0.05 versus OUA.

Mentions: iNos mRNA levels, an important indicator of inflammation, increased in the LPS group in comparison with the control group as expected. OUA pretreatment decreased the LPS induction of iNos mRNA activation, returning levels to that of controls. OUA alone had no impact on iNos mRNA level (Figure 2A). Il-1β mRNA also increased in the LPS-treated group, which OUA pretreatment attenuated. OUA itself had no impact on Il-1β mRNA level (Figure 2B).Figure 2


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) in the lipopolysaccharide (LPS) (200 μg/kg) inflammation model on the mRNA levels ofIl-1β, iNos, Bax, andBcl2. Pretreatment of OUA can decrease the mRNA levels of pro-inflammatory cytokines and pro-apoptotic proteins. Representative semiquantitative RT- PCR photographs and densitometric analysis (arbitrary units, AU) of the specific bands. mRNA levels are presented as ratios of the target gene to Gapdh expression. Data are presented as mean ± SEM from five individual experiments. One-way ANOVA followed by Newman-Keuls post-test. (A)Il-1β mRNA,*P < 0.05 versus control and OUA and **P < 0.01 versus control and OUA and OUA + LPS. (B)iNos mRNA, *P < 0.05 versus control and OUA + LPS. (C)Bax/Bcl2 ratio,**P < 0.001 versus OUA, control and OUA + LPS and *P < 0.05 versus OUA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Effects of pretreatment of ouabain (OUA) (1.8 μg/kg) in the lipopolysaccharide (LPS) (200 μg/kg) inflammation model on the mRNA levels ofIl-1β, iNos, Bax, andBcl2. Pretreatment of OUA can decrease the mRNA levels of pro-inflammatory cytokines and pro-apoptotic proteins. Representative semiquantitative RT- PCR photographs and densitometric analysis (arbitrary units, AU) of the specific bands. mRNA levels are presented as ratios of the target gene to Gapdh expression. Data are presented as mean ± SEM from five individual experiments. One-way ANOVA followed by Newman-Keuls post-test. (A)Il-1β mRNA,*P < 0.05 versus control and OUA and **P < 0.01 versus control and OUA and OUA + LPS. (B)iNos mRNA, *P < 0.05 versus control and OUA + LPS. (C)Bax/Bcl2 ratio,**P < 0.001 versus OUA, control and OUA + LPS and *P < 0.05 versus OUA.
Mentions: iNos mRNA levels, an important indicator of inflammation, increased in the LPS group in comparison with the control group as expected. OUA pretreatment decreased the LPS induction of iNos mRNA activation, returning levels to that of controls. OUA alone had no impact on iNos mRNA level (Figure 2A). Il-1β mRNA also increased in the LPS-treated group, which OUA pretreatment attenuated. OUA itself had no impact on Il-1β mRNA level (Figure 2B).Figure 2

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