Glycopyrronium Bromide By Inhalation Inhibits Cigarette Smoke-induced Lung Inflammation Via JNK/NF-κB Pathways

Objictive:Cigarette smoke (CS) is the major risk factor for the development of chronic obstructive pulmonary disease (COPD) via inducing an abnormal and persistent inflammatory response with an increase of inflammatory cells, such as neutrophils, macrophages and T cells in the lungs. Glycopyrronium Bromide (Gly), a well-known quaternary ammonium compound, is a potential muscarinic receptor antagonist that has been used typically as a long-acting bronchodilator in patients with COPD. The aim of the study was to investigate the anti-inflammatory activity of Gly in cigarette smoke-induced pulmonary inflammation models and the possible signaling pathway.Methods:In vivo:ICR mice were exposed to cigarette smoke (CS) for four days with7,7,9,11per day to elicit pulmonary inflammation. One hour before smoke exposure, animals were treated with Gly (150,300and600μg/ml) and tiotropium (300μg/ml) or dexamethasone (1mg/kg) by aerosol and gavage. BALF was prepared for determinations of inflammatory cell accumulation and enzyme-linked immunosorbent assay (ELISA). Lungs were harvested for measurement of oxidative activities, qRT-PCR, immunohistochemistry as well as histological examination.In vitro:Human type II alveolar epithelial cells (A549cells) were incubated with Gly or tiotropium followed by treatments with cigarette smoke extract (CSE,2.5%) for24h. The release of TGF-p, MMP-9, TIMP-1was assessed by qRT-PCR and ELISA. To elucidate the underlying mechanism, we studied the signaling pathways. Then the cells were incubated with Gly or tiotropium for1h, and then stimulated with CSE (2.5%) for0.5h. Levels of p-JNK, p-ERK, p-p38and NF-κB were detected by Western bloting.Results:In vivo study, it was showed that aerosol administration with Gly at150,300or600μg/ml significantly inhibited inflammatory cells, including neutrophils, macrophages and lymphocytes accumulation in bronchoalveolar lavage fluids (BALF).***P<0.001,**P<0.01represents significant differences compared with the control group,###P<0.001,#P<0.05represents significant differences compared with CS-exposed vehicle treated animals. n=9-11animals per group. Infiltration into lungs attenuates the CS-induced lung inflammation to improve histopathologic deterioration. It also increased CS-induced glutathione (GSH) activity and decreased CS-induced myeloperoxidase (MPO) activity in BALF. Moreover, it could reduce the mRNA and protein expression of Interleukin-1β(IL-1β), tumor necrosis factor alpha (TNF-a), Interleukin-8(IL-8), keratinocyte-derived chemokine (KC), transforming growth factor-β1(TGF-β1) both in BALF and lung, respectively.***p<0.001,*p<0.05represents significant differences compared with the control group,###p<0.001,##p<0.01,#p<0.05represents significant differences compared with CS-exposed vehicle treated animals. In vitro study, treatment with CSE for24h, expressions of TGF-β1and MMP-9in A549cells were up-regulated and expression of TIMP-1was down-regulated. These effects were completely blocked by pre-treatment with Gly. Additionally, Gly can recuce phosphorylation levels of JUK and NF-κB,**P<0.01,***P<0.001represents significant differences compared with the control group,#P<0.05,##P<0.01represents significant differences compared with the treated group.Conclusion:Our results showed that Gly can reduce the expression of inflammatory chemokines induced by cigarette smoke (CS) and cigarette smoke extract (CSE) through activation of JNK/NF-κB signal pathways.