Glycyl-L-histidyl-L-lysine-Cu2+ attenuates cigarette smoke-induced pulmonary emphysema and inflammation by reducing oxidative stress pathway

Abstract

Background: Chronic obstructive pulmonary disease (COPD) is a common respiratory disorder manifested as chronic airway inflammation and persistent airflow limitation with the essential mechanism as inflammatory response and oxidative stress induced by toxic exposures such as cigarette smoke (CS). Glycyl-L-histidyl-L-lysine (GHK) is a nontoxic tripeptide involved in the process of healing and regeneration as a natural product. With the combination of Cu(II), glycyl-L-histidyl-L-lysine-Cu²⁺ (GHK-Cu) improves antioxidative and anti-inflammatory bioavailability, and they might offer potential therapeutic properties for COPD. Thus, the present study aimed to identify the potential effects of GHK-Cu on emphysema induced by cigarette smoke. Methods: In the in vivo experiment, C57BL/6J mice were exposed to CS for 12 weeks to induce pulmonary emphysema. GHK-Cu was injected intraperitoneally at doses of 0.2, 2 and 20 μg/g/day in 100 µl of saline on alternative days from the 1st day after CS exposure. The effects of GHK-Cu on the morphology of CS-induced emphysema, the inflammatory response and oxidative stress were evaluated. The antioxidative effect of GHK-Cu on human alveolar epithelial A549 cells was assessed in vitro. Results: GHK-Cu treatment attenuated the CS-induced emphysematous changes and partially reversed the matrix metalloprotein -9 (MMP-9)/tissue inhibitor of metalloproteinases-1 (TIMP-1) imbalance in the lung tissue. GHK-Cu reduced the inflammation and oxidation by decreasing the expression of inflammatory cytokines (IL-1β and TNF-α) in the bronchoalveolar lavage and the enzymatic activity of MPO and MDA in the lung homogenate while restoring the T-AOC and GSH content. Furthermore, administration of GHK-Cu reversed the increase in NF-κB expression induced by CS and increased the Nrf2 level, as an antioxidant defense component, in mice with chronic CS exposure. In CSE-exposed human alveolar epithelial A549 cells, GHK-Cu also inhibited oxidative stress by suppressing MDA levels and restoring T-AOC and GSH levels, which were modulated by upregulating Nrf2 expression. Conclusion: GHK-Cu treatment attenuated CS-induced emphysema by anti-inflammation by downregulating NF-κB and antioxidation via upregulation of the Nrf2/Keap1 in lung tissues.

Keywords: GHK-Cu; NF-κB; Nrf2; cigarette smoke; emphysema; inflammation; oxidative stress.

FIGURE 1

GHK-Cu alleviates cigarette smoke-induced emphysematous changes. (A) Photomicrograph of HE-stained slides for histological analysis of mice after chronic exposure to cigarette smoke or room air. Representative images of micrographs at ×20 and ×100 magnification. (B,C) Histological assessment of the sections was determined using Lm (B) and MAN (C) (n = 6 mice/group). (D) The body weight was measured (n = 10–12 mice/group). **p < 0.01, ***p < 0.001, and NS means not significant (p > 0.05).

FIGURE 2

GHK-Cu restored the MMP-9/TIMP-1 balance in mice with cigarette smoke exposure. (A) Photomicrograph of immunohistochemical stained slides for MMP-9 from mice with chronic cigarette smoke or room air exposure. Representative images of micrographs at ×400 magnification. (B) Expression of MMP-9 and TIMP-1 in lung tissue from different groups was assessed by western blot. (C,D) Western blotting analysis of MMP-9 (C) and TIMP-1 (D) protein expression in the lung tissue of mice in different groups. *p < 0.05, **p < 0.01, ***p < 0.001, and NS means not significant (p > 0.05).

FIGURE 3

Treatment with GHK-Cu ameliorated lung inflammation and oxidative stress induced by cigarette smoke. (A) MPO activity in lung tissue was assessed following chronic exposure to cigarette smoke or room air. (B,C) Proinflammatory cytokines, including TNF-α (B) and IL-1β (C), in BAL fluid supernatant were measured by ELISA (n = 6 mice/group). (D–F) The levels of T-AOC (D), GSH (E) and MDA (F) in lung homogenate were detected in mice of different groups (n = 3 mice/group). *p < 0.05, **p < 0.01, ***p < 0.001, and NS means not significant (p > 0.05).

FIGURE 4

GHK-Cu upregulated the Nrf2/Keap1 pathway in mice with chronic cigarette smoke exposure. (A,B) The protein expression of Nrf2/Keap1 pathway in the cytoplasm and nuclei of lung tissue in mice from different groups was evaluated by western blotting. (C–E) Western blotting analysis of Nrf2 (C), Keap1 (D) and HO-1 (E) expression in the cytoplasm of the lung tissue from mice in different groups. (F) Western blotting analysis of Nrf2 in the nuclei of lung tissue cells in mice from different groups was assessed. *p < 0.05, **p < 0.01 and ***p < 0.001, and NS means not significant (p > 0.05).

FIGURE 5

GHK-Cu prevented the activation of NF-κB and i-NOS induced by cigarette smoke. (A,B) The expression of NF-κB p65, p-IκBα and i-NOS in lung tissue in mice from different groups was assessed by western blotting. (C–E) Western blotting analysis of NF-κB p65 (C), p-IκBα (D) and i-NOS (E) expression in the lung tissue of mice from different groups. *p < 0.05, **p < 0.01 and ***p < 0.001.

FIGURE 6

GHK-Cu inhibits oxidative stress in human alveolar epithelial cells by upregulating Nrf2 levels in vitro. (A–C) The levels of T-AOC (A), GSH (B) and MDA (C) in A549 cells were detected. (D) The protein expression of Nrf2/Keap1 pathway and NF-κB p65 in the cytoplasm and nuclei of A549 cells were measured. (E–G) Western blotting analysis of cytoplasmic Nrf2 (E), nuclear Nrf2 (F), Keap1 (G), HO-1 (H) and NF-κB p65 (I) protein expression in the cytoplasm of the lung tissue from mice in different groups. *p < 0.05, **p < 0.01 and ***p < 0.001, and NS means not significant (p > 0.05).