Erythromycin attenuates metalloprotease/anti-metalloprotease imbalance in cigarette smoke-induced emphysema in rats via the mitogen-activated protein kinase/nuclear factor-κB activation pathway

Abstract

The present study investigated whether erythromycin (ERY) reduces cigarette smoke (CS)-induced emphysema in rats and aimed to determine the anti-inflammatory effect of ERY, which may identify potential treatments for chronic obstructive pulmonary disease. Furthermore, the current study focused on the potential effects on the imbalance between matrix metalloprotease (MMP) and anti-MMP activity, the phosphorylation of mitogen-activated protein kinases (MAPKs) and the nuclear factor‑κB (NF‑κB) signaling pathway. Wistar rats were divided into the following three groups (n=12 each): control (ERY vehicle only, without any CS exposure), CS (animals were exposed to CS for 12 weeks) and CS + ERY (animals were exposed to CS for 12 weeks and received 100 mg/kg/day ERY). The recruitment of inflammatory cells into the bronchoalveolar lavage fluid (BALF) and the histopathology of lung tissue from all groups was evaluated to grade the severity of the emphysema. The expression of MMP‑2, MMP‑9 and tissue inhibitor of metalloproteinase‑1 was evaluated by immunohistochemistry and western blotting. The activation of MAPKs, NF‑κB and inhibitor of NF‑κB (IκBα), in lung tissues was examined by western blotting. Treatment with ERY resulted in fewer inflammatory cells and cytokines in the BALF, and fewer emphysema‑associated changes in the lungs compared with control. The stimulus of CS promoted the phosphorylation of extracellular signal‑regulated kinase (ERK)1/2 and p38, but not c‑Jun NH2‑terminal kinase, thereby inducing the activation of the ERK/MAPK signaling pathway in rats. Furthermore, CS exposure increased the expression of NF-κB and decreased the expression of IκBα. The levels of phosphorylated ERK1/2 and p38 were significantly reduced in rats with CS‑induced emphysema when treated with ERY compared with the CS group. The results of the present study therefore indicate that oral administration of ERY may suppress CS‑induced emphysema by regulating inflammatory cytokines and the MMP/anti-MMP imbalance via the MAPK/NF-κB pathway.

Figure 1.

ERY has a protective effect on the histological changes observed in CS-induced emphysema in rats. (A) Lung tissue sections from different groups of rats were subjected to hematoxylin and eosin staining (original magnification, ×400). (B) Quantitative analysis of alveolar airspace by MLI and MAN. *P<0.05 and **P<0.01, comparisons indicated by brackets; n=12 per group. Control rats were exposed to normal air and received daily saline injections. CS rats were exposed to CS for 12 weeks. CS + ERY rats were exposed to CS for 12 weeks and received daily injections of 100 mg/kg ERY. CS, cigarette smoke; ERY, erythromycin; MLI, mean linear intercept; MAN, mean alveolar number.

Figure 2.

ERY has a protective role in the changes to inflammatory cytokinesIL-8 and LTB4 in the BALF from rats with CS-induced emphysema. The levels of IL-8 and LTB4 in BALF of individual rats were analyzed by ELISA. Data are presented as the mean + standard deviation of individual samples from three separate experiments. **P<0.01, comparisons indicated by brackets; n=12 per group. Control rats were exposed to normal air and received daily saline injections. CS rats were exposed to CS for 12 weeks. CS + ERY rats were exposed to CS for 12 weeks and received daily injections of 100 mg/kg ERY. BALF, bronchoalveolar lavage fluid; IL, interleukin; CS, cigarette smoke; ERY, erythromycin; LTB4, leukotriene B4.

Figure 3.

ERY has a protective role in the disturbance of MMP and anti-MMP activity in CS-induced emphysema in rats. (A) The protein expression of MMP-2, MMP-9 and TIMP-1 as measured by immunohistochemical staining (original magnification, ×400). (B) Protein expression levels of MMP-2, MMP-9 and TIMP-1 as measured by western blotting, with quantification relative to β-actin. For TIMP-1, the upper band was considered to be non-specific binding. Data are presented normalized to the control and are the mean ± standard deviation. Control rats were exposed to normal air and received daily saline injections. CS rats were exposed to CS for 12 weeks. CS + ERY rats were exposed to CS for 12 weeks and received daily injections of 100 mg/kg ERY. *P<0.05 vs. control group and ^#P<0.05 vs. CS group; n=12 per group. CS, cigarette smoke; ERY, erythromycin; MMP, metalloprotease; TIMP-1, tissue inhibitor of metalloproteinases.

Figure 4.

Effects of ERY on the expression of NF-κB, p65 and IκBα in CS-induced emphysema in rats by western blotting. Protein expression levels of NF-κB, p65 and IκBα as measured by western blotting, with quantification relative to β-actin. The upper band on the blot for NF-κB p65 was quantified and the lower band was considered to be non-specific binding and was not quantified. Data are presented as the mean ± standard deviation. Control rats were exposed to normal air and received daily saline injections. CS rats were exposed to CS for 12 weeks. CS + ERY rats were exposed to CS for 12 weeks and received daily injections of 100 mg/kg ERY. *P<0.05 vs. control group and ^#P<0.05 vs. CS group; n=12 per group. CS, cigarette smoke; ERY, erythromycin; IκBα, inhibitor of NF-κB; NF-κB, nuclear factor-κB.

Figure 5.

Effects of ERY on the expression of MAPK phosphorylation in CS-induced emphysema in rats by western blotting. (A) The protein expression of MAPKs and p-MAPKs as measured by western blotting. (B) For p-p38, p-ERK1/2, ERK1/2, p-JNK and JNK, both bands were quantified in the densitometry. Densitometrically analyzed target p-MAPKs bands normalized to MAPKs. Data are presented as the mean ± standard deviation. Control rats were exposed to normal air and received daily saline injections. CS rats were exposed to CS for 12 weeks. CS + ERY rats were exposed to CS for 12 weeks and received daily injections of 100 mg/kg ERY. *P<0.05 vs. control group and ^ΔP<0.05 vs. CS group; n=12 per group. MAPK, mitogen-activated protein kinase; p-, phospho-; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; CS, cigarette smoke; ERY, erythromycin.