A novel macrolide derivative ameliorates smoke-induced inflammation and emphysema by inhibiting NF-κB activation

Abstract

Although inflammation and emphysema in patients with chronic obstructive pulmonary disease (COPD) can be ameliorated by antibiotics such as erythromycin, the impact of drug resistance is still controversial. We aimed to evaluate the role of F528, a new macrolide derivative without antibacterial effect, in cigarette smoke (CS)-induced pulmonary inflammation and emphysema in a mouse model, as well as in a macrophage cell line. The inflammatory cell number and cell type in the BALF were counted, and the levels of cytokines in the BALF and cultured cell medium were measured by ELISA. The degree of emphysema and apoptosis was evaluated by H&E and immunohistochemical staining, respectively. The lung function of the mice was evaluated by a small animal lung function meter. Furthermore, the expression levels of MMP-2, MMP-9, and phospho-NF-κB in the cells and lung tissue were measured by Western blot and qRT-PCR. In the BALF of the CS-induced pulmonary inflammation and emphysema model, the numbers of inflammatory cells and cytokines were significantly decreased after F528 intervention. F528 intervention also significantly protected lung function from CS-induced emphysema, while the mean lining interception (MLI) of the F528-treated CS group was significantly lower than that of the vehicle-treated CS group. In addition, F528 treatment reduced the phosphorylation of NF-κB induced by smoke, and the expression of MMP-2 and MMP-9 was also obviously decreased by F528 treatment. We therefore conclude that F528 reduces cigarette smoke-induced inflammation and emphysema in vivo and in vitro through inhibition of the activation of NF-κB.

Keywords: Emphysema; cigarette smoke; inflammation; macrolide.

Figure 1

F528 treatment reduced the development of emphysema in mice. A. The different treatment for mice in four groups were performed at indicated time points. B. Representative HE photomicrographs of lung sections from mice treated with F528 and erythromycin. The circles represent the part of the enlarged and broken alveoli. Scale bar: 100 μm. C. Morphometric measurements of mean linear intercept (MLI) (μm). D, E. Total lung capacity and lung resistance assessment. n=6 mice in each group. Data are represented as mean ± SEM. NS6m: Non-smoke group; CS6m: Cigarette smoke group for 6 months; F528 100 mg/kg: Cigarette smoke 6 months + F528 100 mg/kg group; EM 100 mg/kg: Cigarette smoke 6 months + Erythromycin 100 mg/kg. *P<0.05, **P<0.01, ***P<0.001, n.s., not significant.

Figure 2

F528 treatment attenuated smoke-induced lung inflammation in mice. A-C. Total number, the number of neutrophils and macrophages in BALF were counted under the microscope. D, E. The levels of IL-6 and TNF-α in BALF were determined by ELISA. F, G. The levels of IL-6 and TNF-α in serum were measured by ELISA. n=6 mice in each group. Data are represented as mean ± SEM. NS6m: Non-smoke group; CS6m: Cigarette smoke group for 6 months; F528 100 mg/kg: Cigarette smoke 6 months + F528 100 mg/kg group; EM 100 mg/kg: Cigarette smoke 6 months + Erythromycin 100 mg/kg. *P<0.05, **P<0.01, ***P<0.001, n.s., not significant.

Figure 3

F528 treatment decreased the levels of IL-6 and TNF-α in RAW264.7 cells after CSE stimulation. F528 50 µM, 100 µM and erythromycin 100 µM were respectively given to RAW264.7 cells 1 h prior to 5% CSE stimulation for 6 h. The supernatant was taken and the content of (A) IL-6 and (B) TNF-α was measured by ELISA. Data are represented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, n.s., not significant.

Figure 4

F528 attenuated the expression of Bax and Bcl-2 protein in mice. (A) Representative immunostaining of Bax and Bcl-2 proteins in the lung of mice in the Non-smoke group (a), CS6m group: Cigarette smoke group for 6 months (b); F528 100 mg/kg: Cigarette smoke 6 months + F528 100 mg/kg group (c); EM 100 mg/kg: Cigarette smoke 6 months + Erythromycin 100 mg/kg (d). Original magnifications, (a-d), 200X. Scale bar: 100 μm. (B, C) Semi-quantitative analysis of the optical density of Bax and Bcl-2. *P<0.05, **P<0.01, ***P<0.001, n.s., not significant.

Figure 5

F528 reduced the expression of phospho-NFκB, MMPs in RAW 264.7 cells. (A) The protein expression of phospho-NF-κB and total NF-κB in RAW264.7 cells after the CSE stimulation. (B) The protein expression of MMP-2 and MMP-9 in RAW264.7 cells after the CSE stimulation. (C-E) Quantification of the bands shown in (A and B). Data are represented as mean ± SEM. a: Vehicle; b: Vehicle/CS; c: F528 100 μM/CS. *P<0.05, **P<0.01, ***P<0.001, n.s., not significant.

Figure 6

F528 treatment reduced the expression of phospho-NF-κB, MMPs in lung tissue. (A) The protein expression of phospho-NF-κB in lung tissue after 6 months of cigarette smoke. (B) The protein expression of MMP-2 and MMP-9 in lung tissue after 6 months of cigarette smoke. (C-E) Quantification of the bands shown in (A and B). (F, G) The mRNA level of MMP-2 and MMP-9 in lung tissue after 6 months of cigarette smoke. Data are represented as mean ± SEM. a: NS6m (Non-smoke group); b: CS6m (Cigarette smoke 6 months group); c: F528 100 mg/kg (Cigarette smoke 6 months + F528 100 mg/kg group). *P<0.05, **P<0.01, ***P<0.001, n.s., not significant.

Figure 7

F528 treatment ameliorated smoke-induced inflammation and emphysema through NF-κB signaling pathway. Cigarette smoke can induce the inflammation by the phosphorylation of the NF-κB signaling pathway of macrophages and the expression of pro-inflammatory cytokines and matrix metallopeptidases, leading to the destruction of the alveolar structure and the development of lung emphysema. F528 ameliorates the inflammation and emphysema by inhibition of the activation of the NF-κB signaling pathway in macrophages and attenuates CS-induced recruitment of pro-inflammatory cytokines and proteinases, such IL-6, TNF-α and MMPs.