(-)-Epigallocatechin-3-gallate Reduces Cigarette Smoke-Induced Airway Neutrophilic Inflammation and Mucin Hypersecretion in Rats

Abstract

Background: Cigarette smoking is the leading cause of chronic obstructive pulmonary disease. (-)-Epigallocatechin-3-gallate (EGCG), the major catechins in Chinese green tea, has been studied for its anti-oxidative and anti-inflammatory properties in cell and animal models. In this study, we aimed to analyze the effects of EGCG on cigarette smoke (CS)-induced airway inflammation and mucus secretion in the CS-exposed rat model. Methods: Male Sprague-Dawley rats were randomly divided into either sham air (SA) or CS exposure. EGCG (50 mg/kg b.wt.) was given by oral gavage every other day in both SA and CS-exposed animals. Oxidative stress and inflammatory markers were determined in serum and/or bronchoalveolar lavage fluid by biochemical assays or ELISA. Lung morphological changes were examined by Periodic Acid-Schiff, Masson's Trichrome staining and immunohistochemical analysis. Western blot analysis was performed to explore the effects of EGCG on epidermal growth factor receptor (EGFR)-mediated signaling pathway. Results: (-)-Epigallocatechin-3-gallate treatment attenuated CS-induced oxidative stress, lung cytokine-induced neutrophil chemoattractant-1 release and neutrophil recruitment. CS exposure caused an increase in the number of goblet cells in line with MUC5AC upregulation, and increased lung collagen deposition, which were alleviated in the presence of EGCG. In addition, CS-induced phosphorylation of EGFR in rat lung was abrogated by EGCG treatment. Conclusion: (-)-Epigallocatechin-3-gallate treatment ameliorated CS-induced oxidative stress and neutrophilic inflammation, as well as airway mucus production and collagen deposition in rats. The present findings suggest that EGCG has a therapeutic effect on chronic airway inflammation and abnormal airway mucus production probably via inhibition of EGFR signaling pathway.

Keywords: (-)-epigallocatechin-3-gallate; cigarette smoke; epidermal growth factor receptor; inflammation; mucus secretion; neutrophil infiltration.

FIGURE 1

Effect of EGCG on serum oxidative stress markers in rats. 8-isoprostane level (A) and advanced oxidation protein products (AOPP) level (B) were significantly increased in CS-exposed group of rats compared to SA group. Oral administration of EGCG significantly decreased 8-isoprostane and AOPP levels compared to CS group. Total antioxidant capacity (T-AOC) (C), total superoxide dismutase (SOD) activity (D), and catalase (CAT) activity (E) were inhibited in CS-exposed rats while EGCG treatment reversed these enzymes activities. CS exposure upregulated the activity of glutathione s-transferase (GST) and EGCG treatment reversed GST activity (F). The results are expressed as means ± SEM; n = 7 each group. SA group, water/sham air; CS group, water/cigarette smoke; EGCG/SA group, EGCG (50 mg/kg)/sham air; EGCG/CS, EGCG (50 mg/kg)/cigarette smoke. ^∗p < 0.05, ^∗∗p < 0.01 and ^∗∗∗p < 0.001 for the comparison between CS and SA group, ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001 for the comparison between EGCG/CS and CS group.

FIGURE 2

Effects of EGCG on CINC-1 and MCP-1 levels in serum and BALF of rats. Both CINC-1 and MCP-1 levels in serum and BALF were significantly elevated in the rats exposed to CS for 56 days (A–D). EGCG attenuated CS-induced CINC-1 elevation in serum (A) and BALF (C) and reduced serum MCP-1 level (B) but not BALF MCP-1 level (D). The results are expressed as means ± SEM; n = 7 each group. SA group, water/sham air; CS group, water/cigarette smoke; EGCG/SA group, EGCG (50 mg/kg)/sham air; EGCG/CS, EGCG (50 mg/kg)/cigarette smoke. ^∗p < 0.05 and ^∗∗∗p < 0.001 for the comparison between CS and SA group, ^#p < 0.05 for the comparison between EGCG/CS and CS group.

FIGURE 3

Effect of EGCG on neutrophil infiltration in rat lung. Rats treated with EGCG or control vehicle were killed on day 57 and the left lung was formalin-fixed and sectioned for immunofluorescence (magnification ×400). (A) Representative immunofluorescence images of tissue neutrophils as neutrophil elastase (NE)-positive cells with green fluorescence in lung sections. Nuclei were stained with DAPI in blue fluorescence. Scale bar, 50 μm. Arrows indicate representative cells with positive staining. (B) Quantification of NE-positive cells in the lung sections. The results are expressed as means ± SEM; n = 6 each group. SA group, water/sham air; CS group, water/cigarette smoke; EGCG/SA group, EGCG (50 mg/kg)/sham air; EGCG/CS, EGCG (50 mg/kg)/cigarette smoke. ^∗p < 0.05 and ^∗∗∗p < 0.001 for the comparison to SA group, ^###p < 0.001 for the comparison between EGCG/CS and CS group.

FIGURE 4

Effect of EGCG on airway mucus secretion in rats. Rats treated with EGCG or control vehicle were killed on day 57 and the left lung was formalin-fixed and sectioned for histology/immunohistochemistry (magnification ×200). (A) Representative photomicrographs of lung sections stained with periodic acid Schiff (PAS). Goblet cells appear as purple staining (arrows) over epithelium. PAS staining revealed increased goblet cell metaplasia after CS exposure and EGCG reduced the CS-induced goblet cell metaplasia. (B) Immunohistochemistry for MUC5AC was performed using an anti-MUC5AC peptide mouse polyclonal antibody, and detected with an anti-mouse/rabbit IgG peroxidase antibody and diaminobenzidine (DAB). MUC5AC-positive staining showed similar phenomenon of mucus secretion of PAS-staining in different groups. Scale bar, 100 μm. Arrows indicate representative cells with positive staining. (C) Quantification of PAS-positive cells per length of epithelium for goblet cells of different groups. (D) Quantification of MUC5AC-positive cells per length of epithelium for mucin of different groups. The results are expressed as means ± SEM; n = 5–6 each group. SA group, water/sham air; CS group, water/cigarette smoke; EGCG/SA group, EGCG (50 mg/kg)/sham air; EGCG/CS, EGCG (50 mg/kg)/cigarette smoke. ^∗∗∗p < 0.001 for the comparison to SA group, ^###p < 0.001 for the comparison between EGCG/CS and CS group.

FIGURE 5

Effect of EGCG on airway fibrosis in rats. Rats treated with EGCG or control vehicle were killed on day 57 and the left lung was formalin-fixed and sectioned for histology (magnification ×200). (A) Representative photomicrographs of lung sections stained with Masson’s Trichrome staining system. More collagen deposition/fibrosis in or near the airway wall was found in CS group compared to SA group. Compared with the CS group, collagen deposition in the EGCG/CS group was significantly reduced. Scale bar, 100 μm. (B) Quantification of staining-positive area (%) of the lung sections from different group. The results are expressed as means ± SEM; n = 5–6 each group. SA group, water/sham air; CS group, water/cigarette smoke; EGCG/SA group, EGCG (50 mg/kg)/sham air; EGCG/CS, EGCG (50 mg/kg)/cigarette smoke. ^∗∗∗p < 0.001 for the comparison between CS and SA group, ^###p < 0.001 for the comparison between EGCG/CS and CS group.

FIGURE 6

Effect of EGCG on the activation of EGFR in rats. (A) Representative photographs of Western blot for p-EGFR and EGFR proteins from homogenized rat lung tissue were shown. (B) Protein levels were normalized to the corresponding β-actin and the ratios of p-EGFR/EGFR were calculated. Western blot analysis showed that EGCG suppressed CS-induced elevated phosphorylation of EGFR, total EGFR and the ratio of p-EGFR/EGFR. The results are expressed as means ± SEM; n = 7 each group. SA group, water/sham air; CS group, water/cigarette smoke; EGCG/SA group, EGCG (50 mg/kg)/sham air; EGCG/CS, EGCG (50 mg/kg)/cigarette smoke. ^∗∗∗p < 0.001 for the comparison between CS and SA group, ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 for the comparison between EGCG/CS and CS group. EGFR, epidermal growth factor receptor.

FIGURE 7

Schematic diagram. Cigarette smoke (CS) exposure causes oxidative stress, inflammation, and airway mucus production. (-)-Epigallocatechin-3-gallate (EGCG) attenuated CS-induced oxidative stress, neutrophilic airway inflammation, and airway mucus production in rat lungs probably via the inhibition of epidermal growth factor receptor (EGFR) signaling pathway, leading to amelioration of airway remodeling.