Inhibition of glycogen synthase kinase-3beta attenuates the development of carrageenan-induced lung injury in mice

Abstract

Background and purpose: Glycogen synthase kinase-3 (GSK-3) is a ubiquitous serine-threonine protein kinase that participates in a multitude of cellular processes and has recently been implicated in the pathophysiology of a number of diseases. The aim of this study was to investigate the effects of GSK-3beta inhibition in a model of acute inflammation. Here, we have investigated the effects of TDZD-8, a potent and selective GSK-3beta inhibitor, in a mouse model of carrageenan-induced pleurisy.

Experimental approach: 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), prostaglandin E2 (PGE2), tumour necrosis factor-alpha, (TNF-alpha) and interleukin-1beta (IL-1beta). Furthermore, carrageenan induced an upregulation of the adhesion molecules ICAM-1 and P-selectin, iNOS, COX-2 as well as nitrotyrosine as determined by immunohistochemical analysis of lung tissues.

Key results: Administration of TDZD-8 (1, 3 or 10 mg kg(-1), i.p.), 30 min prior to injection of carrageenan, caused a dose-dependent reduction in all the parameters of inflammation measured.

Conclusions and implications: Thus, based on these findings we propose that inhibitors of the activity of GSK-3beta, such as TDZD-8, may be useful in the treatment of various inflammatory diseases.

Figure 1

Effect of TDZD-8 on histological alterations of lung tissue 4 h after carrageenan-induced injury. No histological alterations were observed in lung sections taken from sham mice treated with TDZD-8 (10 mg kg⁻¹ a). Lung sections taken from carrageenan-treated mice pre-treated with vehicle demonstrated oedema, tissue injury (b) as well as infiltration of the tissue with neutrophils (see arrows b1). Carrageenan-treated animals pretreated with TDZD-8 (5 mg kg⁻¹ i.p.) (d) or at (10 mg kg⁻¹ i.p.) (e) demonstrated reduced lung injury and neutrophil infiltration. On the contrary, TDZD-8 (1 mg kg⁻¹) did not reduce the degree of lung injury (c). Original magnification: × 125. The figure is representative of at least three experiments performed on different experimental days.

Figure 2

Effect of TDZD-8 on the immunohistochemical localization of ICAM-1 expression in the lung after carrageenan injection. No positive staining for ICAM-1 was observed in lung sections taken from sham mice treated with TDZD-8 (10 mg kg⁻¹ a). Lung sections taken from carrageenan-treated mice pretreated with vehicle showed intense positive staining for ICAM-1 along the vessels (b) as well as the bronchial epithelium (b1). The degree of positive staining for ICAM-1 was markedly reduced in lung sections obtained from mice pretreated with 10 mg kg⁻¹ TDZD-8 mice (c). Original magnification: × 125. The figure is representative of at least three experiments performed on different experimental days.

Figure 3

Effect of TDZD-8 on carrageenan-induced P-selectin expression and PMN infiltration in the lung. No positive staining for P-selectin was observed in lung sections taken from sham mice treated with TDZD-8 (10 mg kg⁻¹ a). Lung sections taken from carrageenan-treated mice pre-treated with vehicle showed intense positive staining for P-selectin along the vessels (b). The degree of positive staining for P-selectin was markedly reduced in tissue sections obtained from mice pre-treated with 10 mg kg⁻¹ TDZD-8 (c). MPO activity, index of PMN infiltration, was significantly elevated at 4 h after carrageenan (CAR) administration in vehicle-treated mice (d). TDZD-8 (1, 3 and 10 mg kg⁻¹ i.p.) significantly reduced MPO activity in the lung in a dose-dependent fashion (d). The figure is representative of at least three experiments performed on different experimental days. 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 4

Effect of TDZD-8 on carrageenan-induced iNOS expression and NO formation in the lung. No positive staining for iNOS was observed in lung sections taken from sham mice treated with TDZD-8 (10 mg kg⁻¹ a). Lung sections taken from carrageenan-treated mice pretreated with vehicle showed positive staining for iNOS, localized mainly in inflammatory cells (b). The degree of positive staining for iNOS was markedly reduced in tissue sections obtained from mice pretreated with 10 mg kg⁻¹ TDZD-8 (c). Nitrite and nitrate levels, stable NO metabolites, were significantly increased in the pleural exudates at 4 h after carrageenan (CAR) administration (d). TDZD-8 (1, 3 and 10 mg kg⁻¹i.p.) significantly reduced the carrageenan-induced elevation of nitrite and nitrate exudates levels in a dose dependent manner. The figure is representative of at least three experiments performed on different experimental days. Data are expressed as meanc±s.e.m. from n=10 mice for each group. ^*P<0.01 versus sham group. °P<0.01 versus carrageenan.

Figure 5

A representative blot of iNOS (a) and COX-2 (b) expression in the lung after carrageenan (CAR) injection. A significant increase in iNOS (a, a1) and COX-2 (b, b1) expression, assayed by Western blot analysis, was detected in lungs obtained from mice subjected to carrageenan-induced pleurisy. Pretreatment with TDZD-8 10 mg kg⁻¹ significantly attenuated iNOS (a, a1) and COX-2 (b, b1) expression in the lung tissues. A representative blot of tissue lysates (a and b) obtained from five 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 lungs for each group. ^*P<0.01 versus carrageenan.

Figure 6

Effect of TDZD-8 on carrageenan-induced nitrotyrosine formation and lipid peroxidation in the lung. No positive staining for nitrotyrosine was observed in lung sections taken from sham mice treated with TDZD-8 (10 mg kg⁻¹ a). Lung sections taken from carrageenan-treated mice pre-treated with vehicle showed positive staining for nitrotyrosine, localized mainly in inflammatory cells (b). There was a marked reduction in the immunostaining for nitrotyrosine in the lungs of carrageenan-treated mice pretreated with 10 mg kg⁻¹ TDZD-8 (c). MDA levels, an index of lipid peroxidation, were significantly increased in lung tissues 4 h after carrageenan (CAR) administration (d). TDZD-8 (1, 3 and 10 mg kg⁻¹i.p.) significantly reduced the carrageenan-induced elevation of MDA tissues levels in a dose-dependent manner. The figure is representative of at least three experiments performed on different experimental days. 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 TDZD-8 on carrageenan-induced COX-2 expression and PGE₂ production in the lung. No positive staining for COX-2 was observed in lung sections taken from sham mice treated with TDZD-8 (10 mg kg⁻¹ a). Lung sections taken from carrageenan-treated mice pretreated with vehicle showed positive staining for COX-2, localized mainly in inflammatory cells (b). There was a marked reduction in the immunostaining for COX-2 in the lungs of carrageenan-treated mice pretreated with 10 mg kg⁻¹ TDZD-8 (c). PGE₂ levels were significantly increased in the pleural exudates at 4 h after carrageenan (CAR) administration (d). TDZD-8 (1, 3 and 10 mg kg⁻¹i.p.) significantly reduced the carrageenan-induced elevation of PGE₂ exudate levels in a dose-dependent manner. The figure is representative of at least three experiments performed on different experimental days. 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 8

Representative Western blots showing the effects of TDZD-8 on IκB-α degradation (a, a1) and phosphorylation of Ser536 on NF-κB subunit p65 (b, b1) after carrageenan (CAR) injection. A representative blot of lysates (a, b) obtained from five animals per group is shown and densitometry analysis of all animals is reported. The results in panel a1, b1 are expressed as mean±s.e.m. from n=5/6 lung tissues for each group. ^*P<0.01 versus carrageenan.

Figure 9

Effect of TDZD-8 on carrageenan-induced apoptosis as measured by TUNEL-like staining. Positive staining was observed in lung sections taken from carrageenan-treated mice pretreated with vehicle (a, a1, a2, a3). In contrast, tissues obtained from carrageenan-treated mice pretreated with 10 mg kg⁻¹ TDZD-8 demonstrated no apoptotic cells or fragments (b). Almost no apoptotic cells were observed in lungs of sham mice treated with TDZD-8 (10 mg kg⁻¹ c). A positive control is also included (d). The figure is representative of at least three experiments performed on different experimental days.

Figure 10

Representative Western blots showing the effects of TDZD-8 on Bax (a, a1) and Bcl-2 (b, b1) expression in lung tissue after carrageenan (CAR) injection. A representative blot of lysates (a, b) obtained from five animals per group is shown and densitometry analysis of all animals is reported. The results in panel a1, b1 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.

Figure 11

Effect of TDZD-8 on carrageenan-induced Bax expression in the lung. No positive staining for Bax was observed in lung sections taken from sham mice treated with TDZD-8 (10 mg kg⁻¹ a). Lung sections taken from carrageenan-treated mice pretreated with vehicle showed positive staining for Bax (b) localized mainly in the inflammatory cells (b1). The degree of positive staining for Bax was markedly reduced in lung sections obtained from mice pretreated with 10 mg kg⁻¹ TDZD-8 mice (c). The figure is representative of at least three experiments performed on different experimental days.

Figure 12

Effect of carrageenan and TDZD-8 on Bcl-2 expression in the lung. Positive staining for Bcl-2 was observed in lung sections taken from sham mice treated with TDZD-8 (10 mg kg⁻¹ a, a1). The degree of positive staining for Bcl-2 was markedly reduced in lung sections obtained from carrageenan-mice treated with vehicle (c). Pretreatment with TDZD-8 10 mg kg⁻¹ significantly attenuated the reduction in Bcl-2 expression caused by carrageenan (b, b1).