Pulmonary, Gastrointestinal and Urogenital PharmacologyDoxycycline attenuates acrolein-induced mucin production, in part by inhibiting MMP-9
Introduction
Mucus is an important protective factor against environmental agents and pathogens, but excessive airway mucus production, termed mucus hypersecretion, may impair mucociliary clearance (Nadel, 2000). Many chronic airway diseases, including chronic obstructive pulmonary disease (COPD), cystic fibrosis, bronchiectasis, and asthma are associated with airway mucus hypersecretion (Perez-Vilar et al., 2003).
Airway mucus is mostly water (95% by weight), and the balance comprises mainly mucous glycoprotein (mucins), other secretory proteins, serum proteins, lipids, and salts. Mucin, the major macromolecular constituent of mucus, is secreted by goblet cells located in the epithelium and by mucus cells in the submucosal glands (Leikauf et al., 2002). In airway tissues from healthy individuals, goblet cells typically express MUC5AC mRNA, while mucus cells express MUC5B mRNA. MUC5AC is a highly inducible gene, whereas MUC5B is expressed constitutively (Leikauf et al., 1989, Williams et al., 2001, Rose and Voynow, 2006, Young et al., 2007, Henke et al., 2007).
Up-regulation of MUC genes and goblet cell hyperplasia (GCH) contribute to and sustain mucin overproduction in the airways of patients with chronic lung diseases. Cigarette smoking, air pollutants and bacterial infection are believed to be common causes of lung inflammation and are all associated with mucus overproduction. Acrolein (CH2═CHCHO) can be found in tobacco smoke at concentrations up to 50 parts per million (ppm), as well as in wood smoke (Altshuler and McPherson, 1963, Ayer and Yeager, 1982). Animals repeatedly exposed to acrolein develop histological changes, including epithelial damage, mucus hypersecretion, and bronchiolitis (Costa et al., 1986, Borchers et al., 1998, Wang et al., 2009a).
Matrix metalloproteinases (MMPs), a family of many structurally related enzymes, degrade components of the extracellular matrix in both physiological and pathophysiological states, and they may play an important role in chronic airway diseases (Murray et al., 1998, Greenlee et al., 2007). MMPs also process, activate, and deactivate a variety of non-matrix soluble factors, including several growth factors and chemokines. Thus, they are considered both proteinases of matrix catalysis and extracellular processing enzymes (Parks and Shapiro, 2001).
Epidermal growth factor (EGF) and its receptor may be involved in mucin secretion, and MMPs may mediate their involvement. Previous studies showed that activation of EGF receptor was responsible for airway mucus production induced by cigarette smoke (Nadel, 2000, Takeyama, 2001). Deshmukh et al. (2008) demonstrated that the EGF receptor and MAPK signaling pathway play an important role in the accumulation of MMP-9 transcripts and protein after acrolein exposure. Indeed, studies have shown that MMP-9, regulated by epidermal growth factor (EGF) receptor, plays a major role in the pathogenesis of inflammatory airway diseases such as COPD, asthma and cystic fibrosis (Wallace and Sandford, 2002, Deshmukh et al., 2008).
The tetracyclines are potent inhibitors of the MMP enzyme family and have been used to reduce tissue degradation in periodontal disease and arthritis (Bokor, 1995, Tamimi et al., 2008, Smieja et al., 2001). Doxycycline is one of the classic tetracycline derivatives that have been studied extensively in various diseases. Doxycycline exerts diverse inhibitory effects on MMP production and activity (Hidalgo and Eckhardt, 2001). It was reported to decrease serum MMP-9 levels by 50% after 6 months of oral administration following myocardial infarction (Brown et al., 2004). Doxycycline has also been reported to decrease the activity of MMP-9 in vitro (Kim et al., 2005), but the effects and mechanisms of doxycycline on acrolein-induced MMP-9 production are not known.
In the present study, levels of MMP-9 mRNA and protein, levels of MUC5AC mRNA, and mucus production were measured in rats exposed to acrolein. Rats were treated with doxycycline before and during the exposure, in order to determine whether the MMP inhibitor can affect acrolein-induced mucin overproduction.
Section snippets
Laboratory animals and experimental design
The Animal Care and Use Committee of West China Hospital of Sichuan University approved the animal protocol. Forty-eight male Sprague–Dawley rats weighing 200–250 g were used for experiments and randomly divided into four groups that received acrolein (A group, n = 18), acrolein + doxycycline (AD group, n = 18), saline (C group, n = 6), and doxycycline (D group, n = 6). Rats in groups A and AD were placed in chambers and exposed to acrolein aerosol made by an electric nebulizer as previously described (
Immunohistochemical staining for MMP-9
MMP-9 positive staining (dark brown color) was barely detectable in the control group, with an integrated optical density (IOD) of only 11.17 ± 1.08. After exposure to acrolein for 12 days, the IOD in airway epithelium of the acrolein group (46.75 ± 2.20) was significantly higher than in the control (P < 0.05), indicating that acrolein inhalation increased the expression of MMP-9 (Fig. 1). In the acrolein + doxycycline group, the IOD (20.13 ± 1.14) was significantly lower than that of the control group (P <
Discussion
The present study showed that acrolein exposure increased both MMP-9 and MUC5AC expression, as well as mucus production, in rat airways. These increases could be prevented with the MMP inhibitor doxycycline. Thus, we have demonstrated a prophylactic role for doxycycline in acrolein-induced mucous hypersecretion, and our results suggest that doxycycline exerts these effects, in part, by inhibiting MMP-9.
The pathogenesis of mucus overproduction involves several complex processes, including mucin
Conclusion
Doxycycline can attenuate acrolein-induced increases in MUC5AC mRNA expression and mucin synthesis in a rat model, in addition to decreasing airway inflammation and mucus production. It exerts these effects, in part, by inhibiting MMP-9. Doxycycline shows potential as a prophylactic agent in the treatment of chronic airway diseases.
Acknowledgements
This study was supported by grants from the National Natural Science Foundation of China (30971327, 30670921) and from the China Medical Board of New York (00-722, 06-834) to Dr. F.Q. Wen.
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