Protective effects of GHK-Cu in bleomycin-induced pulmonary fibrosis via anti-oxidative stress and anti-inflammation pathways

Abstract

Background

Idiopathic pulmonary fibrosis (IPF) is a serious lung problem with advancing and diffusive pulmonary fibrosis as the pathologic basis, and with oxidative stress and inflammation as the key pathogenesis. Glycyl-L-histidyl-l-lysine (GHK) is a tripeptide participating into wound healing and regeneration. GHK-Cu complexes improve GHK bioavailability. Thus, the current study aimed to explore the therapeutic role of GHK-Cu on bleomycin (BLM)-induced pulmonary fibrosis in a mouse model.

Methods

BLM (3 mg/kg) was administered via tracheal instillation (TI) to induce a pulmonary fibrosis model in C57BL/6j mice 21 days after the challenge of BLM. GHK-Cu was injected intraperitoneally (i.p.) at different dosage of 0.2, 2 and 20 μg/g/day in 0.5 ml PBS on alternate day. The histological changes, inflammation response, the collagen deposition and epithelial-mesenchymal transition (EMT) was evaluated in the lung tissue. EMT was evaluated by ɑ-SMA and fibronectin expression in the lung tissue. NF-κB p65, Nrf2 and TGFβ1/Smad2/3 signalling pathways were detected by immunoblotting analysis.

Results

GHK-Cu complex inhibited BLM-induced inflammatory and fibrotic pathological changes, alleviated the inflammatory response in the BALF by reducing the levels of the inflammatory cytokines, TNF-ɑ and IL-6 and the activity of MPO as well as reduced collagen deposition. In addition, the GHK-Cu treatment significantly reversed the MMP-9/TIMP-1 imbalance and partially prevented EMT via Nrf2, NF-κB and TGFβ1 pathways, as well as Smad2/3 phosphorylation.

Conclusions

GHK-Cu presented a protective effect in BLM-induced inflammation and oxidative stress by inhibiting EMT progression and suppressing TGFβ1/Smad2/3 signalling in pulmonary fibrosis.

Introduction

Pulmonary fibrosis is characterized by diffusive and rapidly developing pulmonary fibrosis resulting in gas exchange impairment and restrictive ventilatory problem, eventually respiratory failure [1]. Thus, the mortality associated with pulmonary fibrosis is considerably high [1,2]. Many factors participate in pulmonary fibrosis pathogenesis including inflammation, epithelial-mesenchymal transition (EMT), imbalanced extracellular matrix degradation and collagen deposition, and oxidative stress [3,4]. Oxidative stress and the inflammatory response may be two of the essential factors that initiating this disease and making it progress [[5], [6], [7], [8]]. Before now, the treatment options for pulmonary fibrosis were quite limited. In the past few decades, corticosteroid and immunosuppressant agents and cytokine antagonists were used to treat pulmonary fibrosis but had an unsatisfactory benefit/adverse effect ratio. As essential pathogenic factors, oxidative stress and the inflammatory response may serve as potential targets in pulmonary fibrosis treatment. The search for new anti-oxidative and/or anti-inflammatory agents that play protective, therapeutic roles in pulmonary fibrosis prevention and treatment continues.

Glycyl-L-histidyl-l-lysine (GHK), a tripeptide with glycyl-histidyl-lysine as the amino acid sequence, was first used in wound healing and anti-ageing skin care [9,10]. This peptide has a long history and has been studied in many systems, such as skin and wound healing, the digestive system and the respiratory system [11]. GHK is nontoxic, and produced naturally. The complex of GHK-Cu has been proved to improve GHK bioavailability, especially for antioxidant and anti-inflammatory functions. The GHK-Cu complex exerts antioxidant functions through several mechanisms [[12], [13], [14]]. GHK, the peptide alone, has been found to be involved in degenerative neuropathies and disorders by quenching peroxidation process [15]. The copper component, as the metal ion, activates the superoxide dismutase (SOD) which depends on Cu and Zn, which works as an endogenous antioxidant. The complex also reduces oxidative damage by haltering inflammation with decrease of ferritin iron release in injury tissue [16]. The complex of GHK-Cu was found to diminish the production of reactive oxygen species (ROS) production, increase SOD activity while decrease proinflammatory cytokine release in acute lung injury [17]. In addition, in wound healing tissue and skin repair, GHK-Cu decreases the release of proinflammatory cytokines, including transforming growth factor beta1 (TGFβ1), tumour necrosis factor (TNF)-α, et al. [10,[18], [19], [20]], which are overexpressed in pulmonary fibrosis lung tissue due to abnormal repair and regeneration following certain stimuli. Whether the complex of GHK-Cu could play the antioxidant and anti-inflammatory effect on the development of pulmonary fibrosis is still unknown now. Thus, we designed this study to verify the effect of GHK-Cu complex on bleomycin (BLM) -induced pulmonary fibrosis and to explore the potential underlying mechanism.