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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

Prevention of IC deposition-induced lung apoptosis by administration of baclofen.(A) Lung tissue sections from three animal groups: control, lung injury (LI), or LI followed by baclofen administration (LI+B), were subjected to terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining to study the changes in apoptosis in various groups. Shown are representative images with 20X images and zoomed images are shown below. N = 3 animals per group were subjected to TUNEL staining. (B) 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 and 5), were subjected to immunoblot analysis with anti-cleaved caspase-3 and anti-GAPDH antisera. A representative blot is shown from n = 3 independent experiments performed with 3 animals per group. (C) Cleaved caspase-3 and GAPDH immunoblots were scanned and band densities were quantified using ImageJ software. Cleaved caspase-3 band density is normalized to its respective GAPDH band intensity. Data is expressed as mean ± SEM (n = 3 animals per group).
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pone.0121637.g004: Prevention of IC deposition-induced lung apoptosis by administration of baclofen.(A) Lung tissue sections from three animal groups: control, lung injury (LI), or LI followed by baclofen administration (LI+B), were subjected to terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining to study the changes in apoptosis in various groups. Shown are representative images with 20X images and zoomed images are shown below. N = 3 animals per group were subjected to TUNEL staining. (B) 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 and 5), were subjected to immunoblot analysis with anti-cleaved caspase-3 and anti-GAPDH antisera. A representative blot is shown from n = 3 independent experiments performed with 3 animals per group. (C) Cleaved caspase-3 and GAPDH immunoblots were scanned and band densities were quantified using ImageJ software. Cleaved caspase-3 band density is normalized to its respective GAPDH band intensity. Data is expressed as mean ± SEM (n = 3 animals per group).

Mentions: Having documented amelioration of lung damage and induction of BAL neutrophil apoptosis after baclofen treatment, we next wanted to determine levels of apoptosis in the lung tissue sections and lung tissue homogenates in the presence and absence of baclofen administration. Fig 4A demonstrates TUNEL staining of lung tissue sections from the three groups of animals with 3 animals per group. The evaluation of lung tissue sections revealed increased TUNEL staining in ALI tissue sections compared to control lungs. Baclofen after IC deposition demonstrated a decrease in TUNEL positivity (Fig 4A). Lung tissue homogenates from the 3 groups of animals with 3 animals per group were immunoblotted for cleaved caspase-3, a marker of apoptosis. Cleaved caspase-3 was detected in the lung from ALI animals, while little or no cleaved caspase-3 was present in controls or in animals receiving baclofen. These results indicate that baclofen administration prevents ALI induced apoptosis in lung tissue.


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)

Prevention of IC deposition-induced lung apoptosis by administration of baclofen.(A) Lung tissue sections from three animal groups: control, lung injury (LI), or LI followed by baclofen administration (LI+B), were subjected to terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining to study the changes in apoptosis in various groups. Shown are representative images with 20X images and zoomed images are shown below. N = 3 animals per group were subjected to TUNEL staining. (B) 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 and 5), were subjected to immunoblot analysis with anti-cleaved caspase-3 and anti-GAPDH antisera. A representative blot is shown from n = 3 independent experiments performed with 3 animals per group. (C) Cleaved caspase-3 and GAPDH immunoblots were scanned and band densities were quantified using ImageJ software. Cleaved caspase-3 band density is normalized to its respective GAPDH band intensity. Data is expressed as mean ± SEM (n = 3 animals per group).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4388838&req=5

pone.0121637.g004: Prevention of IC deposition-induced lung apoptosis by administration of baclofen.(A) Lung tissue sections from three animal groups: control, lung injury (LI), or LI followed by baclofen administration (LI+B), were subjected to terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining to study the changes in apoptosis in various groups. Shown are representative images with 20X images and zoomed images are shown below. N = 3 animals per group were subjected to TUNEL staining. (B) 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 and 5), were subjected to immunoblot analysis with anti-cleaved caspase-3 and anti-GAPDH antisera. A representative blot is shown from n = 3 independent experiments performed with 3 animals per group. (C) Cleaved caspase-3 and GAPDH immunoblots were scanned and band densities were quantified using ImageJ software. Cleaved caspase-3 band density is normalized to its respective GAPDH band intensity. Data is expressed as mean ± SEM (n = 3 animals per group).
Mentions: Having documented amelioration of lung damage and induction of BAL neutrophil apoptosis after baclofen treatment, we next wanted to determine levels of apoptosis in the lung tissue sections and lung tissue homogenates in the presence and absence of baclofen administration. Fig 4A demonstrates TUNEL staining of lung tissue sections from the three groups of animals with 3 animals per group. The evaluation of lung tissue sections revealed increased TUNEL staining in ALI tissue sections compared to control lungs. Baclofen after IC deposition demonstrated a decrease in TUNEL positivity (Fig 4A). Lung tissue homogenates from the 3 groups of animals with 3 animals per group were immunoblotted for cleaved caspase-3, a marker of apoptosis. Cleaved caspase-3 was detected in the lung from ALI animals, while little or no cleaved caspase-3 was present in controls or in animals receiving baclofen. These results indicate that baclofen administration prevents ALI induced apoptosis in lung tissue.

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