Glycogen Synthase Kinase-3 inhibition with the small molecule ATP-competitive inhibitor SB216763 [3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione] prevents bleomycyn-induced lung inflammation and fibrosis.

Objectives: Glycogen synthase kinase-3 (GSK-3) modulates inflammatory cytokines production. Since bleomycin (BLM)-induced lung injury in the mouse is char- acterized by an inflammatory response followed by fibrosis, we postulated that blocking GSK-3 activity could affect the BLM-induced damage in the lung. We inves- tigated the effects of the selective, ATP-competitive GSK-3 inhibitor SB216763 in this experimental setting. Methods: We randomised C57BL6 mice to receive intratracheal instillation of saline, saline plus SB216763, BLM plus vehicle, or BLM plus SB216763. Bronchoalve- olar lavage (BAL) and histopathological analysis of the lungs, flow-cytometry studies and cell sorting of BAL pulmonary leucocytes were performed on days 2, 7, and 28. By real time PCR, we examined cytokine gene expression levels in lung monocytes. In situ TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) assay was performed to score apoptotic lung cells. Results: SB216763, when co-administered with BLM, prevented lung inflammation and the subsequent fibrosis. BAL cell analysis of BLM + SB216763 treated mice showed a significant reduction in BLM-induced alveolitis (BLM+SB216763 X 0,5 × 106 ± 0,1 sd cells/ml compared with BLM-treated mice = 5,7 × 106 ± 2,1 sd cells/ml on day 7 and 2 × 106 ± 0,8 sd cells/ml on day 28). SB216763 treatment was associated with a significantly lower production of inflammatory cytokines by macrophages, as evaluated by qRT-PCR. TNF-a: BLM 2,04 ± 0,12 sd vs BLM+SB216763 0,98 ± 0,17 sd; CCL2/MCP1: BLM 2,4 ± 0,08 sd vs BLM+SB216763 0,79 ± 0,08 sd (arbitrary units). BLM-treated mice that received SB216763 developed pulmonary fibrosis to a much lesser extent as compared to bleomycin-treated controls ( BLM+SB216763 3,1% ± 2,1 sd vs BLM 21% ± 15 sd). Next, an in situ TUNEL assay on the lung sections showed that BLM-treated mice displayed a higher degree of alveolar epithelial damage as compared to the BLM + SB216763-treated mice. Conclusions: Altogether, these findings suggest that GSK-3 inhibition has therapeutic efficacy on bleomycin-induced lung injury by acting at multiple levels: 1. buffering the inflammatory cytokine production; 2. protection of alveolar cells from apoptosis. 3. reduction of fibrogenesis. GSK-3 may represent a potential therapeutic target for preventing inflammation-induced pulmonary damage.