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Baclofen, a GABABR agonist, ameliorates immune-complex mediated acute lung injury by modulating pro-inflammatory mediators.

Jin S, Merchant ML, Ritzenthaler JD, McLeish KR, Lederer ED, Torres-Gonzalez E, Fraig M, Barati MT, Lentsch AB, Roman J, Klein JB, Rane MJ - PLoS ONE (2015)

Bottom Line: ALI increased total protein, tumor necrosis factor α (TNF-α and interleukin-1 receptor associated protein (IL-1R AcP), in the bronchoalveolar lavage fluid (BALF).Protective effects of baclofen treatment on ALI were possibly mediated by inhibition of TNF-α- and IL-1β-mediated inflammatory signaling.GABABR2 agonists may play a potential therapeutic role in ALI.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Louisville, Louisville, Kentucky, United States of America.

ABSTRACT
Immune-complexes play an important role in the inflammatory diseases of the lung. Neutrophil activation mediates immune-complex (IC) deposition-induced acute lung injury (ALI). Components of gamma amino butyric acid (GABA) signaling, including GABA B receptor 2 (GABABR2), GAD65/67 and the GABA transporter, are present in the lungs and in the neutrophils. However, the role of pulmonary GABABR activation in the context of neutrophil-mediated ALI has not been determined. Thus, the objective of the current study was to determine whether administration of a GABABR agonist, baclofen would ameliorate or exacerbate ALI. We hypothesized that baclofen would regulate IC-induced ALI by preserving pulmonary GABABR expression. Rats were subjected to sham injury or IC-induced ALI and two hours later rats were treated intratracheally with saline or 1 mg/kg baclofen for 2 additional hours and sacrificed. ALI was assessed by vascular leakage, histology, TUNEL, and lung caspase-3 cleavage. ALI increased total protein, tumor necrosis factor α (TNF-α and interleukin-1 receptor associated protein (IL-1R AcP), in the bronchoalveolar lavage fluid (BALF). Moreover, ALI decreased lung GABABR2 expression, increased phospho-p38 MAPK, promoted IκB degradation and increased neutrophil influx in the lung. Administration of baclofen, after initiation of ALI, restored GABABR expression, which was inhibited in the presence of a GABABR antagonist, CGP52432. Baclofen administration activated pulmonary phospho-ERK and inhibited p38 MAPK phosphorylation and IκB degradation. Additionally, baclofen significantly inhibited pro-inflammatory TNF-α and IL-1βAcP release and promoted BAL neutrophil apoptosis. Protective effects of baclofen treatment on ALI were possibly mediated by inhibition of TNF-α- and IL-1β-mediated inflammatory signaling. Interestingly, GABABR2 expression was regulated in the type II pneumocytes in lung tissue sections from lung injured patients, further suggesting a physiological role for GABABR2 in the repair process of lung damage. GABABR2 agonists may play a potential therapeutic role in ALI.

No MeSH data available.


Related in: MedlinePlus

Baclofen administration alters signaling kinase activation in ALI.(A) Lung tissue homogenates from three animal groups: control, lung injury (LI), or LI followed by baclofen administration (LI+B), were subjected to immunoblot analysis with anti-I B, anti-pp38 MAPK, and anti-GAPDH antisera. (B and C) IκB, pp38 MAPK, and GAPDH immunoblots from above three animal groups were scanned and band densities were quantified using ImageJ software. Phosphorylated p38 MAPK and IκB band densities were normalized to their respective GAPDH band intensity. Data is expressed as mean ± SEM (n = 3 animals per group). (D) Lung tissue homogenates from three animal groups: control (lane 1), lung injury (LI) (lanes 2 and 3), or LI followed by baclofen administration (LI+B) (lanes 4, 5, and 6) were subjected to immunoblot analysis with anti-pERK MAPK and anti-GAPDH antisera. (E) pERK MAPK and GAPDH immunoblots from above three animal groups with 3 animals per group were scanned and band densities were quantified using ImageJ software. Phosphorylated ERK MAPK band densities were normalized to their respective GAPDH band intensity. Data is expressed as mean ± SEM (n = 3 animals per group).
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pone.0121637.g007: Baclofen administration alters signaling kinase activation in ALI.(A) Lung tissue homogenates from three animal groups: control, lung injury (LI), or LI followed by baclofen administration (LI+B), were subjected to immunoblot analysis with anti-I B, anti-pp38 MAPK, and anti-GAPDH antisera. (B and C) IκB, pp38 MAPK, and GAPDH immunoblots from above three animal groups were scanned and band densities were quantified using ImageJ software. Phosphorylated p38 MAPK and IκB band densities were normalized to their respective GAPDH band intensity. Data is expressed as mean ± SEM (n = 3 animals per group). (D) Lung tissue homogenates from three animal groups: control (lane 1), lung injury (LI) (lanes 2 and 3), or LI followed by baclofen administration (LI+B) (lanes 4, 5, and 6) were subjected to immunoblot analysis with anti-pERK MAPK and anti-GAPDH antisera. (E) pERK MAPK and GAPDH immunoblots from above three animal groups with 3 animals per group were scanned and band densities were quantified using ImageJ software. Phosphorylated ERK MAPK band densities were normalized to their respective GAPDH band intensity. Data is expressed as mean ± SEM (n = 3 animals per group).

Mentions: IC-induced ALI results in increased lung cell apoptosis which is inhibited by baclofen treatment. Therefore, we wanted to identify signaling kinases that were activated in the lung after ALI in baclofen treated and untreated animals. Lung tissue homogenates from three animal groups with 3 animals per group were immunoblotted with, IκB, phospho-p38 MAPK, and pERK antisera. Pro-inflammatory NF-κB activation in the lung has been documented in IC-induced ALI [38, 39] and degradation of IκB, an inhibitor of NFκB, has been used in this model to document increased NFκB activation [30]. In Fig 7A we demonstrated lung IκB expression was significantly decreased after ALI compared to control, consistent with IκB degradation and NFκB activation (Fig 7A and 7B) [30, 38]. Baclofen treatment after induction of ALI preserved IκB expression (Fig 7A and 7B) suggestive of blockade of NFκB activation. In addition, in Fig 7A we demonstrated that ALI significantly increased p38 MAPK phosphorylation in the lung tissue compared to control (Fig 7A and 7C) which was significantly inhibited by baclofen treatment after induction of ALI (Fig 7A and 7C). Furthermore, ERK phosphorylation was significantly enhanced in animals treated with baclofen after induction of ALI, as compared to control or animals subjected to ALI (Fig 7D and 7E). These results suggest that baclofen administration inhibits NFκB activation (by inhibiting IκB degradation) and p38 MAPK activation while promoting ERK activation.


Baclofen, a GABABR agonist, ameliorates immune-complex mediated acute lung injury by modulating pro-inflammatory mediators.

Jin S, Merchant ML, Ritzenthaler JD, McLeish KR, Lederer ED, Torres-Gonzalez E, Fraig M, Barati MT, Lentsch AB, Roman J, Klein JB, Rane MJ - PLoS ONE (2015)

Baclofen administration alters signaling kinase activation in ALI.(A) Lung tissue homogenates from three animal groups: control, lung injury (LI), or LI followed by baclofen administration (LI+B), were subjected to immunoblot analysis with anti-I B, anti-pp38 MAPK, and anti-GAPDH antisera. (B and C) IκB, pp38 MAPK, and GAPDH immunoblots from above three animal groups were scanned and band densities were quantified using ImageJ software. Phosphorylated p38 MAPK and IκB band densities were normalized to their respective GAPDH band intensity. Data is expressed as mean ± SEM (n = 3 animals per group). (D) Lung tissue homogenates from three animal groups: control (lane 1), lung injury (LI) (lanes 2 and 3), or LI followed by baclofen administration (LI+B) (lanes 4, 5, and 6) were subjected to immunoblot analysis with anti-pERK MAPK and anti-GAPDH antisera. (E) pERK MAPK and GAPDH immunoblots from above three animal groups with 3 animals per group were scanned and band densities were quantified using ImageJ software. Phosphorylated ERK MAPK band densities were normalized to their respective GAPDH band intensity. Data is expressed as mean ± SEM (n = 3 animals per group).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4388838&req=5

pone.0121637.g007: Baclofen administration alters signaling kinase activation in ALI.(A) Lung tissue homogenates from three animal groups: control, lung injury (LI), or LI followed by baclofen administration (LI+B), were subjected to immunoblot analysis with anti-I B, anti-pp38 MAPK, and anti-GAPDH antisera. (B and C) IκB, pp38 MAPK, and GAPDH immunoblots from above three animal groups were scanned and band densities were quantified using ImageJ software. Phosphorylated p38 MAPK and IκB band densities were normalized to their respective GAPDH band intensity. Data is expressed as mean ± SEM (n = 3 animals per group). (D) Lung tissue homogenates from three animal groups: control (lane 1), lung injury (LI) (lanes 2 and 3), or LI followed by baclofen administration (LI+B) (lanes 4, 5, and 6) were subjected to immunoblot analysis with anti-pERK MAPK and anti-GAPDH antisera. (E) pERK MAPK and GAPDH immunoblots from above three animal groups with 3 animals per group were scanned and band densities were quantified using ImageJ software. Phosphorylated ERK MAPK band densities were normalized to their respective GAPDH band intensity. Data is expressed as mean ± SEM (n = 3 animals per group).
Mentions: IC-induced ALI results in increased lung cell apoptosis which is inhibited by baclofen treatment. Therefore, we wanted to identify signaling kinases that were activated in the lung after ALI in baclofen treated and untreated animals. Lung tissue homogenates from three animal groups with 3 animals per group were immunoblotted with, IκB, phospho-p38 MAPK, and pERK antisera. Pro-inflammatory NF-κB activation in the lung has been documented in IC-induced ALI [38, 39] and degradation of IκB, an inhibitor of NFκB, has been used in this model to document increased NFκB activation [30]. In Fig 7A we demonstrated lung IκB expression was significantly decreased after ALI compared to control, consistent with IκB degradation and NFκB activation (Fig 7A and 7B) [30, 38]. Baclofen treatment after induction of ALI preserved IκB expression (Fig 7A and 7B) suggestive of blockade of NFκB activation. In addition, in Fig 7A we demonstrated that ALI significantly increased p38 MAPK phosphorylation in the lung tissue compared to control (Fig 7A and 7C) which was significantly inhibited by baclofen treatment after induction of ALI (Fig 7A and 7C). Furthermore, ERK phosphorylation was significantly enhanced in animals treated with baclofen after induction of ALI, as compared to control or animals subjected to ALI (Fig 7D and 7E). These results suggest that baclofen administration inhibits NFκB activation (by inhibiting IκB degradation) and p38 MAPK activation while promoting ERK activation.

Bottom Line: ALI increased total protein, tumor necrosis factor α (TNF-α and interleukin-1 receptor associated protein (IL-1R AcP), in the bronchoalveolar lavage fluid (BALF).Protective effects of baclofen treatment on ALI were possibly mediated by inhibition of TNF-α- and IL-1β-mediated inflammatory signaling.GABABR2 agonists may play a potential therapeutic role in ALI.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Louisville, Louisville, Kentucky, United States of America.

ABSTRACT
Immune-complexes play an important role in the inflammatory diseases of the lung. Neutrophil activation mediates immune-complex (IC) deposition-induced acute lung injury (ALI). Components of gamma amino butyric acid (GABA) signaling, including GABA B receptor 2 (GABABR2), GAD65/67 and the GABA transporter, are present in the lungs and in the neutrophils. However, the role of pulmonary GABABR activation in the context of neutrophil-mediated ALI has not been determined. Thus, the objective of the current study was to determine whether administration of a GABABR agonist, baclofen would ameliorate or exacerbate ALI. We hypothesized that baclofen would regulate IC-induced ALI by preserving pulmonary GABABR expression. Rats were subjected to sham injury or IC-induced ALI and two hours later rats were treated intratracheally with saline or 1 mg/kg baclofen for 2 additional hours and sacrificed. ALI was assessed by vascular leakage, histology, TUNEL, and lung caspase-3 cleavage. ALI increased total protein, tumor necrosis factor α (TNF-α and interleukin-1 receptor associated protein (IL-1R AcP), in the bronchoalveolar lavage fluid (BALF). Moreover, ALI decreased lung GABABR2 expression, increased phospho-p38 MAPK, promoted IκB degradation and increased neutrophil influx in the lung. Administration of baclofen, after initiation of ALI, restored GABABR expression, which was inhibited in the presence of a GABABR antagonist, CGP52432. Baclofen administration activated pulmonary phospho-ERK and inhibited p38 MAPK phosphorylation and IκB degradation. Additionally, baclofen significantly inhibited pro-inflammatory TNF-α and IL-1βAcP release and promoted BAL neutrophil apoptosis. Protective effects of baclofen treatment on ALI were possibly mediated by inhibition of TNF-α- and IL-1β-mediated inflammatory signaling. Interestingly, GABABR2 expression was regulated in the type II pneumocytes in lung tissue sections from lung injured patients, further suggesting a physiological role for GABABR2 in the repair process of lung damage. GABABR2 agonists may play a potential therapeutic role in ALI.

No MeSH data available.


Related in: MedlinePlus