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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 inhibits release of pro-inflammatory mediators in BAL supernatants of lung injured rats.(A) Bronchoalveolar lavage fluid (BALF) was centrifuged to separate BAL cells from BAL supernatant. BAL supernatant was collected from three animal groups: control (lane 1), lung injury (LI) (lane 2, 3, 4), or LI followed by baclofen administration (LI+B) (lanes 5 and 6), and the proteins were separated by SDS-PAGE followed by immunoblot analysis for TNF-α. (B) TNF-α Immunoblots were scanned and band densities were quantified using ImageJ software. Arbitrary densitometric values obtained were normalized to protein concentration for each sample. Data is expressed as mean ± SEM (n = 5 animals per group). (C) BAL supernatants from three animal groups: control, lung injury (LI), LI followed by baclofen administration (LI+B) were subjected to proteomic analysis to identify and quantitate a BALF protein that was present in lung injured animals and was regulated by baclofen treatment. Interleukin-1 receptor accessory protein beta (IL-R AcP) was identified in all three animal groups tested. Spectral counts for IL-1R AcP were plotted against control, LI, LI+B groups. Data is expressed as mean ± SEM (n = 3 per group). (D) Mass spectrometry data was validated by immunoblotting BAL supernatants from three animal groups: control, lung injury (LI), or LI followed by baclofen administration (LI+B) with anti-IL-1R AcP antibody. (E) IL-1R AcP immunoblots were scanned and band densities were quantified using ImageJ software. Arbitrary densitometric values obtained were normalized to protein concentration for each sample. Data is expressed as mean ± SEM (n = 5 animals per group).
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pone.0121637.g003: Baclofen inhibits release of pro-inflammatory mediators in BAL supernatants of lung injured rats.(A) Bronchoalveolar lavage fluid (BALF) was centrifuged to separate BAL cells from BAL supernatant. BAL supernatant was collected from three animal groups: control (lane 1), lung injury (LI) (lane 2, 3, 4), or LI followed by baclofen administration (LI+B) (lanes 5 and 6), and the proteins were separated by SDS-PAGE followed by immunoblot analysis for TNF-α. (B) TNF-α Immunoblots were scanned and band densities were quantified using ImageJ software. Arbitrary densitometric values obtained were normalized to protein concentration for each sample. Data is expressed as mean ± SEM (n = 5 animals per group). (C) BAL supernatants from three animal groups: control, lung injury (LI), LI followed by baclofen administration (LI+B) were subjected to proteomic analysis to identify and quantitate a BALF protein that was present in lung injured animals and was regulated by baclofen treatment. Interleukin-1 receptor accessory protein beta (IL-R AcP) was identified in all three animal groups tested. Spectral counts for IL-1R AcP were plotted against control, LI, LI+B groups. Data is expressed as mean ± SEM (n = 3 per group). (D) Mass spectrometry data was validated by immunoblotting BAL supernatants from three animal groups: control, lung injury (LI), or LI followed by baclofen administration (LI+B) with anti-IL-1R AcP antibody. (E) IL-1R AcP immunoblots were scanned and band densities were quantified using ImageJ software. Arbitrary densitometric values obtained were normalized to protein concentration for each sample. Data is expressed as mean ± SEM (n = 5 animals per group).

Mentions: To examine potential mechanisms underlying baclofen’s protective effects on ALI we subjected BALF supernatants from control, I and LI+B treated animal groups with 5 animals per group to immunoblot analysis with anti-TNF-α antibody. Fig 3A and 3B demonstrates that TNF-α release in BALF was significantly induced after ALI as compared to control. Baclofen treatment significantly inhibited TNF-α release in BALF. The immunoblot data shown in Fig 3A was normalized to the total protein loaded in each condition as the immunoblot was performed on BALF supernatants hence an internal loading control such as β-actin or GAPDH could not be performed. Thus TNF-α arbitrary densitometric values were normalized to protein concentration for each condition.


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 inhibits release of pro-inflammatory mediators in BAL supernatants of lung injured rats.(A) Bronchoalveolar lavage fluid (BALF) was centrifuged to separate BAL cells from BAL supernatant. BAL supernatant was collected from three animal groups: control (lane 1), lung injury (LI) (lane 2, 3, 4), or LI followed by baclofen administration (LI+B) (lanes 5 and 6), and the proteins were separated by SDS-PAGE followed by immunoblot analysis for TNF-α. (B) TNF-α Immunoblots were scanned and band densities were quantified using ImageJ software. Arbitrary densitometric values obtained were normalized to protein concentration for each sample. Data is expressed as mean ± SEM (n = 5 animals per group). (C) BAL supernatants from three animal groups: control, lung injury (LI), LI followed by baclofen administration (LI+B) were subjected to proteomic analysis to identify and quantitate a BALF protein that was present in lung injured animals and was regulated by baclofen treatment. Interleukin-1 receptor accessory protein beta (IL-R AcP) was identified in all three animal groups tested. Spectral counts for IL-1R AcP were plotted against control, LI, LI+B groups. Data is expressed as mean ± SEM (n = 3 per group). (D) Mass spectrometry data was validated by immunoblotting BAL supernatants from three animal groups: control, lung injury (LI), or LI followed by baclofen administration (LI+B) with anti-IL-1R AcP antibody. (E) IL-1R AcP immunoblots were scanned and band densities were quantified using ImageJ software. Arbitrary densitometric values obtained were normalized to protein concentration for each sample. Data is expressed as mean ± SEM (n = 5 animals per group).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4388838&req=5

pone.0121637.g003: Baclofen inhibits release of pro-inflammatory mediators in BAL supernatants of lung injured rats.(A) Bronchoalveolar lavage fluid (BALF) was centrifuged to separate BAL cells from BAL supernatant. BAL supernatant was collected from three animal groups: control (lane 1), lung injury (LI) (lane 2, 3, 4), or LI followed by baclofen administration (LI+B) (lanes 5 and 6), and the proteins were separated by SDS-PAGE followed by immunoblot analysis for TNF-α. (B) TNF-α Immunoblots were scanned and band densities were quantified using ImageJ software. Arbitrary densitometric values obtained were normalized to protein concentration for each sample. Data is expressed as mean ± SEM (n = 5 animals per group). (C) BAL supernatants from three animal groups: control, lung injury (LI), LI followed by baclofen administration (LI+B) were subjected to proteomic analysis to identify and quantitate a BALF protein that was present in lung injured animals and was regulated by baclofen treatment. Interleukin-1 receptor accessory protein beta (IL-R AcP) was identified in all three animal groups tested. Spectral counts for IL-1R AcP were plotted against control, LI, LI+B groups. Data is expressed as mean ± SEM (n = 3 per group). (D) Mass spectrometry data was validated by immunoblotting BAL supernatants from three animal groups: control, lung injury (LI), or LI followed by baclofen administration (LI+B) with anti-IL-1R AcP antibody. (E) IL-1R AcP immunoblots were scanned and band densities were quantified using ImageJ software. Arbitrary densitometric values obtained were normalized to protein concentration for each sample. Data is expressed as mean ± SEM (n = 5 animals per group).
Mentions: To examine potential mechanisms underlying baclofen’s protective effects on ALI we subjected BALF supernatants from control, I and LI+B treated animal groups with 5 animals per group to immunoblot analysis with anti-TNF-α antibody. Fig 3A and 3B demonstrates that TNF-α release in BALF was significantly induced after ALI as compared to control. Baclofen treatment significantly inhibited TNF-α release in BALF. The immunoblot data shown in Fig 3A was normalized to the total protein loaded in each condition as the immunoblot was performed on BALF supernatants hence an internal loading control such as β-actin or GAPDH could not be performed. Thus TNF-α arbitrary densitometric values were normalized to protein concentration for each condition.

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