GHK Peptide Inhibits Bleomycin-Induced Pulmonary Fibrosis in Mice by Suppressing TGF β1/Smad-Mediated Epithelial-to-Mesenchymal Transition

Objective: Idiopathic pulmonary fibrosis is an irreversible and progressive fibrotic lung disease that leads to declines in pulmonary function and, eventually, respiratory failure and has no effective treatment. Gly-His-Lys (GHK) is a tripeptide involved in the processes of tissue regeneration and wound healing and has significant inhibitory effects on transforming growth factor (TGF)-beta 1 secretion. The effect of GHK on fibrogenesis in pulmonary fibrosis and the exact underlying mechanism have not been studied previously. Thus, this study investigated the effects of GHK on bleomycin (BLM)-induced fibrosis and identified the pathway that is potentially responsible for these effects. Methods: Intratracheal injections of 3 mg/kg BLM were administered to induce pulmonary fibrosis in C57BL/6 mice. GHK was administered intraperitoneally at doses of 2.6, 26, and 260 mu g/ml/day every other day from the 4th to the 21st day after BLM instillation. Three weeks after BLM instillation, pulmonary injury and pulmonary fibrosis was evaluated by the hematoxylin-eosin (HE) and Masson's trichrome (MT) staining. Chronic inflammation index was used for the histological assessments by two pathologists blindly to each other. Tumor necrosis factor (TNF)-alpha and IL-6 levels in BALF and myeloperoxidase (MPO) activity in lung extracts were measured. For the pulmonary fibrosis evaluation, the fibrosis index calculated based on MT staining, collagen deposition and active TGF-beta 1 expression detected by ELISA, and the expression of TGF-beta 1, alpha-smooth muscle actin (SMA), fibronectin, MMP-9, and TIMP-1 by western blotting. The epithelial mesenchymal transition index, E-cadherin, and vimentin was also detected by western blot. The statistical analysis was performed by one-way ANOVA and the comparison between different groups were performed. Results: Treatment with GHK at all three doses reduced inflammatory cell infiltration and interstitial thickness and attenuated BLM-induced pulmonary fibrosis in mice. GHK treatment significantly improved collagen deposition, and MMP-9/TIMP-1 imbalances in lung tissue and also reduced TNF-alpha, IL-6 expression in bronchoalveolar lavage fluid (BALF) and MPO in lung extracts. Furthermore, GHK reversed BLM-induced increases in TGF-beta 1, p-Smad2, p-Smad-3 and insulin-like growth factor-1 (IGF-1) expression. Conclusion: GHK inhibits BLM-induced fibrosis progression, the inflammatory response and EMT via the TGF-beta 1/Smad2/3 and IGF-1 pathway. Thus, GHK may be a potential treatment for pulmonary fibrosis.