Effects of Liver x receptor agonist treatment on signal transduction pathways in acute lung inflammation

Abstract

Background: Liver x receptor alpha (LXRalpha) and beta (LXRbeta) are members of the nuclear receptor super family of ligand-activated transcription factors, a super family which includes the perhaps better known glucocorticoid receptor, estrogen receptor, thyroid receptor, and peroxisome proliferator-activated receptors. There is limited evidence that LXL activation may reduces acute lung inflammation. The aim of this study was to investigate the effects of T0901317, a potent LXR receptor ligand, in a mouse model of carrageenan-induced pleurisy.

Methods: Injection of carrageenan into the pleural cavity of mice elicited an acute inflammatory response characterized by: accumulation of fluid containing a large number of neutrophils (PMNs) in the pleural cavity, infiltration of PMNs in lung tissues and subsequent lipid peroxidation, and increased production of nitrite/nitrate (NOx), tumor necrosis factor-alpha, (TNF-alpha) and interleukin-1beta (IL-1beta). Furthermore, carrageenan induced the expression of iNOS, nitrotyrosine and PARP, as well as induced apoptosis (TUNEL staining and Bax and Bcl-2 expression) in the lung tissues.

Results: Administration of T0901317, 30 min after the challenge with carrageenan, caused a significant reduction in a dose dependent manner of all the parameters of inflammation measured.

Conclusions: Thus, based on these findings we propose that LXR ligand such as T0901317, may be useful in the treatment of various inflammatory diseases.

Figure 1

Effect of T0901317 on histological alterations of lung tissue 4 h after carrageenan-induced injury and on PMN infiltration in the lung. Lung sections taken from carrageenan-treated mice pre-treated with vehicle demonstrated edema, tissue injury (b, d) as well as infiltration of the tissue with neutrophils (see b1). Carrageenan-treated animals pre-treated with T0901317 (20 mg/kg i.p.) (c, d) demonstrated reduced lung injury and neutrophil infiltration. Original magnification: × 125. Section from a sham animals demonstrating the normal architecture of the lung tissue (a, d). The figure is representative of at least 3 experiments performed on different experimental days. MPO activity, index of PMN infiltration, was significantly elevated at 4 h after carrageenan (CAR) administration in vehicle-treated mice (e), if compared with sham mice (e). T0901317 significantly reduced in a dose dependent manner MPO activity in the lung (e). The figure is representative of at least 3 experiments performed on different experimental days. Data are expressed as mean ± s.e.m. from n = 10 mice for each group. ND: not detectable. *P < 0.01 versus sham group. °P < 0.01 versus carrageenan.

Figure 2

Effect of T0901317 on carrageenan-induced iNOS expression and NO formation in the lung. Lung sections taken from carrageenan-treated mice pre-treated with vehicle showed positive staining for iNOS, localized mainly in inflammatory cells (b, e). The degree of positive staining for iNOS was markedly reduced in tissue sections obtained from mice pre-treated with 20 mg/kg T0901317 (c, e). Original magnification: × 125. Lung sections taken from sham mice showed no staining for iNOS (a, e). The figure is representative of at least 3 experiments performed on different experimental days. A significant increase in iNOS (d, d1) expression, assayed by Western blot analysis, was detected in lungs obtained from mice subjected to carrageenan-induced pleurisy, if compared with lung from sham mice (d, d1). Pre-treatment with T0901317 20 mg/kg significantly attenuated iNOS (d, d1) expression in the lung tissues. A representative blot of lysates obtained from 5 animals per group is shown and densitometry analysis of all animals is reported. The results in panel d1 are expressed as mean ± s.e.m. from n = 5/6 lung tissues for each group. ND: not detectable. *P < 0.01 versus sham group. °P < 0.01 versus carrageenan.

Figure 3

Effect of T0901317 on carrageenan-induced nitrotyrosine formation and lipid peroxidation and PARP activation in the lung. No staining for nitrotyrosine is present in lung section from sham mice (a, g). Lung sections taken from carrageenan-treated mice pre-treated with vehicle showed positive staining for nitrotyrosine, localized mainly in inflammatory cells (b, g). There was a marked reduction in the immunostaining for nitrotyrosine in the lungs of carrageenan-treated mice pre-treated with 20 mg/kg T0901317 (c, g). Malondialdehyde (MDA) levels, an index of lipid peroxidation, were significantly increased in lung tissues 4 h after carrageenan (CAR) administration (h), if compared with lung from sham mice (h). T0901317 significantly reduced in a dose dependent manner the carrageenan-induced elevation of MDA tissues levels (h). Lung sections taken from carrageenan-treated mice pre-treated with vehicle showed positive staining for PAR (e, g). There was a marked reduction in the immunostaining for PAR in the lungs of carrageenan-treated mice pre-treated with 20 mg/kg T0901317 (f, g). Lung section from sham mice showed no staining for PAR (d, g). The figure is representative of at least 3 experiments performed on different experimental days. Data are expressed as mean ± s.e.m. from n = 10 mice for each group. ND: not detectable *P < 0.01 versus sham group. °P < 0.01 versus carrageenan.

Figure 4

Effect of T0901317 on carrageenan-induced pro-inflammatory cytokine release in the lung. Lung sections taken from carrageenan-treated mice pre-treated with vehicle showed positive staining for TNF-α and IL-1β (b, d and f, h). There was a marked reduction in the immunostaining for TNF-α and IL-1β in the lungs of carrageenan-treated mice pre-treated with 20 mg/kg T0901317 (c, d and g, h). No staining for either TNF-α (a, d) or IL-1β (e, h) in lung tissues obtained from the sham group of mice. The figure is representative of at least 3 experiments performed on different experimental days. ND: not detectable. Data are expressed as mean ± s.e.m. from n = 10 mice for each group. *P < 0.01 versus sham group. °P < 0.01 versus carrageenan.

Figure 5

Representative Western blots showing the effects of T0901317 on IκB-α degradation and nuclear NF-κ Bp65 expression after carrageenan (CAR) injection. Basal expression of IκB-α was detected in lung samples from sham-treated animals, whereas IκB-α levels were substantially reduced in lung tissues obtained from vehicle-treated animals at 4 h after carrageenan injection (a, a1). T0901317 (20 mg/kg) treatment prevented carrageenan-induced IκB-α degradation (a, a1). NF-κB p65 levels in the lung nuclear fractions were also significantly increased at 4 h after carrageenan injection compared to the sham-treated mice (b, b1). T0901317 treatment significantly reduced the levels of NF-κB p65 (b, b1). A representative blot of lysates obtained from 5 animals per group is shown and densitometry analysis of all animals is reported. The results in panel a1 and b1 are expressed as mean ± s.e.m. from n = 5/6 lung tissues for each group. ND: not detectable. *P < 0.01 versus sham group. °P < 0.01 versus carrageenan.

Figure 6

Effect of T0901317 on carrageenan-induced Fas ligand expression and on apoptosis as measured by TUNEL like staining. Positive staining for Fas ligand was observed in lung sections taken from carrageenan-treated mice pre-treated with vehicle (b, d) compared to sham-operated mice (a, d). In contrast, T0901317 (20 mg/kg) treatment reduced the degree of positive staining for FAS Ligand in the lung tissues (c, d). Positive TUNEL staining was observed in lung sections taken from carrageenan-treated mice pre-treated with vehicle (f, h). In contrast, tissue obtained from carrageenan treated mice pre-treated with T0901317 (20 mg/kg) demonstrated no apoptotic cells or fragments (g, h). Almost no apoptotic cells were observed in lungs of sham mice (e, h). The figure is representative of at least 3 experiments performed on different experimental days. ND: not detectable. Data are expressed as mean ± s.e.m. from n = 10 mice for each group. *P < 0.01 versus sham group. °P < 0.01 versus carrageenan.

Figure 7

Effect of T0901317 on carrageenan-induced Bax and Bcl-2 expression in the lung. Representative Western blots showing no Bax expression in lung tissues obtained from sham-treated animals (Ae, Ae1). Bax levels were increased in the lung tissues from carrageenan-treated mice (Ae, Ae1). T0901317 (20 mg/kg) treatment prevented the carrageenan-induced Bax expression (Ae, Ae1). A basal level of Bcl-2 expression was detected in lung tissues from sham-treated mice (Be, Be1). At 4 hours after carrageenan administration, Bcl-2 expression was significantly reduced (Be, Be1). Treatment of mice with T0901317 (20 mg/kg) significantly attenuated carrageenan-induced inhibition of Bcl-2 expression (Be, Be1). A representative blot of lysates obtained from 5 animals per group is shown and densitometry analysis of all animals is reported. The results in panel Ae1 and Be1 are expressed as mean ± s.e.m. from n = 5/6 lung tissues for each group. *P < 0.01 versus sham group. °P < 0.01 versus carrageenan. Lung sections taken from carrageenan-treated mice pre-treated with vehicle showed positive staining for Bax (Ab, Ad) localized mainly in the inflammatory cells. The degree of positive staining for Bax was markedly reduced in lung sections obtained from mice pre-treated with 20 mg/kg T0901317 mice (Ac, Ad). Positive staining for Bcl-2 was observed in lung sections taken from sham mice (Ba, Bd). The degree of positive staining for Bcl-2 was markedly reduced in lung sections obtained from carrageenan-mice treated with vehicle (Bb, Bd). Pre-treatment with T0901317 20 mg/kg significantly attenuated the reduction in Bcl-2 expression caused by carrageenan (Bc, Bd). The figure is representative of at least 3 experiments performed on different experimental days. ND: not detectable Data are expressed as mean ± s.e.m. from n = 10 mice for each group. *P < 0.01 versus sham group. °P < 0.01 versus carrageena.