**4. Discussion**

Eight major findings were extracted from this study. (1) Oral administration of ≥25 mg/kg YE markedly reduced leukocytosis of neutrophils and eosinophils as well as lymphocytes and monocytes in the BALF of CS-exposed mice. (2) YE reduced the induction of these inflammatory proteins of COX-2, iNOS, and ICAM-1 together with reduction of the pathological alterations and oxidative stress in small airways and alveoli of CS-challenged mice. (3) The treatment with 25–100 mg/kg YE blunted the MMP-12 expression and emphysematous damage of airways through diminution of bax/bcl-2 ratio and inactivation of p53 and caspases in CS-exposed mouse lungs. (4) In 5% CSE-loaded A549 cells supplemented with 10–100 μg/mL YE the apoptotic cell death was significantly attenuated. (5) The OVA inhalation highly elevated total leukocyte number in BALF, while the YE treatment diminished OVA-triggered neutrophilic and eosinophilic inflammation. (6) The tissue levels of COX-2, iNOS, and MMP-12 were enhanced in OVA-exposed mouse lungs, which was curtailed by the supply of 25–100 mg/kg YE. (7) Treatment of 10–100 g/mL YE abrogated the induction of COX-2, iNOS, and ICAM-1 and the secretion of TNF-α and MCP-1 with blockade of NF-κB signaling in LPS-loaded alveolar cells. (8) The MMP-12 induction by pro-inflammatory TNF-α was blunted in YE-treated alveolar cells. Accordingly, CS- and OVA inhalation-induced pulmonary oxidative stress and inflammation may contribute to airway tissue destruction and emphysema. YE may inhibit emphysema and alveolar cell loss in airways and alveoli encountered in the oxidative and inflammatory milieu. Therefore, anti-inflammatory and antioxidant YE had a potential benefit in treating pulmonary diseases of COPD and asthma.

COPD is a chronic inflammatory lung disease with bronchial airflow impairment and obstruction caused by long-term exposure to irritating risk factors such as cigarette smoking and biomass fuel dust exposure [1,2]. The most common disorders involved in COPD are chronic bronchitis inflamed in the lining of the bronchial tubes and emphysema liable to the alveoli [5,6]. As expected, this study showed that CS and allergic OVA enhanced the lung induction of COX-2, iNOS, and ICAM-1 responsible for lung inflammation along with marked leukocytosis in BALF. Chronic lung inflammation involves the infiltration of inflammatory cells such as neutrophils, macrophages, and lymphocytes into the small airways of COPD [1,6]. Similarly, the exposure to CS stimulates the recruitment of inflammatory cells into the airways and elicits immune responses [14]. This study revealed that the exposure of both CS and OVA to mice evoked eosinophilic allergic inflammation in airways. The challenge of LPS to alveolar cells induced lung inflammation entailing the secretion of MCP-1 and TNFα through activating NF-κB-responsive mechanism(s). Several studies highlight the role of NF-κB signaling in these two important inflammatory lung diseases of asthma and COPD [33,34]. The definite mechanisms underlying bronchiolar and alveolar inflammation are ye<sup>t</sup> unsolved in COPD. However, to alleviate airway inflammation would be a primary therapeutic option for chronic bronchitis and COPD [12,13].

Emphysema is characterized by alveolar destruction with a loss of alveolar integrity and marked airspace enlargement [14,16]. This study showed that CS enhanced the MMP-12 induction in bronchiolar airways and alveoli, indicating that CS resulted in pulmonary emphysematous injury. Several promising mechanisms including oxidative stress and protease/anti-protease imbalance have been suggested for the small airway/alveolar destruction and structural changes, which may provide major therapeutic targets in COPD [10,11]. An activation of aberrant inflammation in bronchioles and alveoli lead to pathological alterations including loss of alveolar integrity [4]. Consistently, the pro-inflammatory TNFα released by LPS promoted alveolar MMP-12 expression. Accordingly, airway inflammation due to OVA and CS may be a major contributor in the pathogenesis of emphysema. Oxidative stress contributes to cause alveolar emphysema through activating transcription of pro-inflammatory cytokines [13,17]. Several antioxidant natural compounds of epigallocatechin gallate and glycyrrhizin ameliorate oxidative renal injury and experimental acute pancreatitis in rats [35,36]. A growing body of evidence reports that CS as a risk factor in COPD is closely associated with increased oxidative stress [37,38]. Smoke may accelerate apoptosis of structural cells in the lung by means of oxidative stress, which might possibly be an important upstream event involved in the development of COPD and pulmonary emphysema [39,40]. In this study the exposure of CS to mice markedly enhanced apoptotic mediators of lung tissues as well as the ROS formation in alveoli. Another mechanism involved in the development of COPD is imbalance between proteolytic and anti-proteolytic activity [17,18,39]. This study found that inflammatory CS and OVA elicited proteolytic MMP-12 expression, resulting in the destruction of lung tissues. However, the emphasis on alveolar matrix destruction by the combination of inflammation, oxidative stress, and excessive proteolysis has failed to fully account for the COPD-specific mechanisms [40].

There is a need for safe and e ffective treatments that prevent these pulmonary diseases and have beneficial impacts on the course of COPD and asthma. Diverse molecular therapeutic targets for emphysema have been proposed from the identification of cellular and molecular mechanisms of the pathogenesis of COPD and asthma [12,16,39]. The pharmacological use of inflammatory mediator-targeted therapeutic agents in patients of COPD and asthma depends on clinical phenotypes and pathophysiological mechanisms [41]. Novel anti-inflammatory agents targeting lung inflammation include inhaled corticosteroids and β-adrenergic receptor agonists, phosphodiesterase-4 inhibitors, macrolides, and statins [14,19,20]. These agents can alter COPD-specific mechanisms involved in inflammation, mucin hypersecretion, and tissue destruction [14,19]. Asthmatics with eosinophilic inflammation but not with neutrophilic inflammation respond better to corticosteroids [41]. The choice of the optimal treatment In COPD and asthma should be based on the underlying immunopathology [15]. Although therapeutic approaches aim to target the chronic neutrophilic inflammation in COPD, targeting the underlying causes of the pulmonary neutrophilia such as smoking and oxidative stress might be more promising strategies. Due to the unique interplay between oxidative stress and pathogenesis of COPD, oxidative stress represents a novel target for the treatment of COPD and therapeutic opportunity with antioxidants arising [42].

Numerous therapeutic strategies with naturally occurring bioactive compounds that have mostly proven to be safe are currently under development for treating COPD and asthma [21]. Evidence

suggests that the anti-inflammatory and anti-oxidative roles of some of the existing natural agents have potential values in the treatment of inflammatory lung diseases [21,43]. The dietary natural polyphenolic compounds of kaempferol and astragalin antagonized airway epithelial apoptosis and fibrosis and airway thickening along with pulmonary inflammation in OVA-inhaled mice [27,28]. Flavonoids rich in fruits and vegetables show their beneficial effects in asthmatic animal models [44]. Several studies demonstrate potential roles of natural compounds for the treatment of COPD [22,26]. Dietary polyphenols of curcumin, resveratrol, green tea catechins, quercetin, sulforaphane, and lycopene that possess anti-oxidative activities can influence various COPD-specific mechanistic aspects for the treatment and managemen<sup>t</sup> of COPD [25]. Our recent study showed that dietary oleuropein rich in olive blunted pulmonary inflammation and alveolar destruction led to emphysema [29]. Oleuropein also blocked the infiltration of inflammatory and allergic cells into airways in CS-challenged mice. Our recent study found that YE inhibited bronchial epithelial eosinophilia and mucus-secreting goblet cell hyperplasia in OVA-exposed mice [30]. The present study revealed that YE blocked bronchiolar and alveolar inflammation and subsequent pulmonary emphysema in CS- and OVA-challenged COPD models, in which YE abrogated chronic inflammation, MMP-12 proteolytic activity, oxidative stress, and apoptosis of structural alveolar cells in the airways that might possibly be an important upstream event in the pathogenesis of COPD.
