**3. Results**

#### *3.1. Inhibition of Airway Inflammation of CS-Exposed Airways by YE*

This study examined how YE inhibited CS-evoked inflammation in mouse airways. Exposure of mice to CS increased total leukocyte number in the BALF by ≈1.5-fold (Figure 1A). Surprisingly, the challenge of CS promoted the influx of neutrophils and eosinophils in the BALF, indicating that CS resulted in neutrophilic and eosinophilic inflammation (Figure 1A). Oral administration of ≥25 mg/kg YE reduced CS-induced leukocytosis of neutrophils and eosinophils, which was incomparable to that of control mice (Figure 1A). In addition, YE curtailed the number of lymphocytes and monocytes in the BALF elevated in CS-exposed mice.

It was examined whether CS-stimulated airway inflammation was attenuated in YE-supplemented mice. The tissue level of COX-2 responsible for prostaglandin biosynthesis and inflammation was elevated in CS-exposed mouse lungs (Figure 1B). In addition, the airway tissue levels of iNOS and ICAM-1 directly involved in inflammatory responses were enhanced in CS-challenged mice (Figure 1C,D). However, orally-administrated YE reduced the induction of these inflammatory proteins promoted by exposure of airways to CS (Figure 1B–D).

**Figure 1.** Leukocytes in the bronchoalveolar lavage fluid (BALF; **A**) and inhibition of pulmonary inflammation (**B**–**D**) in cigarette smoke (CS)-challenged mouse lungs treated with dried yeas<sup>t</sup> extracts (YE). Mice were orally administrated with 25–100 mg/kg YE and exposed to CS for 30 min. Cells in BALF were counted using a Hemavet HV950 Multispecies Hematologic Analyzer (**A**). Tissue extracts were subject to Western blot analysis with a primary antibody against COX-2, iNOS, or ICAM-1. β-Actin protein was used as an internal control. The bottom bar graphs represent quantitative results of the upper bands obtained from a densitometer. Values (mean ± SEM, *n* = 3–4) in respective bar graphs not sharing a same small letter indicate a significant difference at *p* < 0.05.

#### *3.2. Blockade of Emphysematous Injury of CS-Challenged Airways by YE*

The histological examination was conducted in the lung tissues stained with H&E. The CS exposure induced the airway wall damage in mice (Figure 2). However, oral administration of 25–100 mg/kg YE highly attenuated the pathological alterations observed in bronchiolar and alveolar tissues of CS-challenged mice. In addition, MMP-12 was highly expressed in mouse airways exposed to CS, evidenced by FITC-immunostaining (Figure 3). In contrast, the treatment with 25–100 mg/kg YE reduced its induction in CS-exposed bronchioles (Figure 2). Moreover, the H&E histological staining revealed that the CS challenge to mice evoked destruction in pulmonary alveoli (Figure 2). Consistently, the MMP-12 induction was markedly enhanced. However, 25–100 mg/kg YE diminished the emphysematous damage in pulmonary alveoli and curtailed the FITC-staining of MMP-12 (Figure 3). Therefore, YE may inhibit emphysema and alveolar cell loss in CS-exposed small airways and alveoli.

**Figure 2.** Blockade of airway destruction by dried yeas<sup>t</sup> extracts (YE) in cigarette smoke (CS)-challenged mouse bronchioles and alveoli. Mice were orally administrated with 25–100 mg/kg YE and exposed to CS for 30 min. Airway tissue sections were stained by using a hematoxylin and eosin (H&E) reagent. Each photograph is representative of four mice. The arrows indicate damaged bronchioles and alveolar air sacs. Scale bars = 100 μm.

#### *3.3. Inhibitory E*ff*ects of YE on CS-Induced Pulmonary Apoptosis and Oxidative Stress*

This study attempted to examine whether YE inhibited emphysematous airway damage through blocking pulmonary apoptosis and oxidative stress induced by CS. Western blot analysis showed that CS diminished the lung tissue level of anti-apoptotic bcl-2 and increased the level of pro-apoptotic bax, consequently elevating the bax/bcl-2 ratio (Figure 4A). Oral treatment of YE reduced the bax/bcl-2 ratio in CS-exposed mouse alveolar tissues. The tumor suppressor p53 is known to directly activate bax and mediate mitochondrial membrane permeabilization and apoptosis [31]. As expected, the activation of p53 was enhanced in CS-loaded lung tissues, which was retarded by supplementing 25–100 mg/kg YE to mice (Figure 4B). In addition, caspase-9 and its downstream executioner caspase-3 responsible for executing cell death were upregulated in lung tissues by exposure to CS (Figure 4C). In contrast, YE highly attenuated the activation of these caspases in lung tissues.

**Figure 4.** Inhibition of apoptotic lung injury and reactive oxygen species (ROS) production by 25–100 mg/kg yeas<sup>t</sup> extracts (YE) in cigarette smoke (CS)-challenged mouse lungs. Tissue extracts were subject to Western blot with a primary antibody against bcl-2, bax, phospho-p53, cleaved caspase-9, or cleaved caspase-3 (**A**–**C**). β-Actin protein was used as an internal control. The bar graphs (mean ± SEM, *n* = 3) represent quantitative results of the upper bands obtained from a densitometer. Values in respective bar graphs not sharing a same small letter indicate a significant difference at *p* < 0.05. Dihydroethidium (DHE) staining showing pulmonary ROS production (**D**). Tissue sections of small airways and alveoli were stained with DHE, and nuclear staining was done with DAPI (blue). Each photograph is representative of four mice. Scale bars=100 μm.

The reciprocal interactions among ROS, airway inflammation, and alveolar cell death play crucial role in the pathogenesis of COPD [32]. This study introduced DHE staining for ROS production in airways exposed to CS. DHE exhibits blue-fluorescence in the cytosol until oxidized, where it intercalates within the cell DNA, with subsequent staining of nuclei as a bright fluorescent red. There was a strong DHE staining in CS-loaded bronchioles and alveoli, indicating marked superoxide

production by CS (Figure 4D). However, oral administration of 25–100 mg/kg YE highly attenuated ROS production in bronchiolar and alveolar tissues of CS-challenged mice.

#### *3.4. Suppressive E*ff*ects of YE on CSE-Loaded Alveolar Apoptotic Injury*

This study further explored how CS evoked alveolar damage in mice and how YE protected alveoli from CS. The treatment of A549 cells with 10–100 μg/mL YE did not cause cytotoxicity for 24 h (Figure 5A). When 5% CSE was applied to alveolar epithelial A549 cells for 24 h, the viability dropped to below 20% (Figure 5B). When 5% CSE-loaded A549 cells were supplemented with ≥10 μg/mL YE for 24 h, the viability was highly boosted (Figure 5B). On the other hand, Hoechst 33258 nuclear staining and TUNEL staining showed that 5% CSE resulted in nuclear condensation and DNA fragmentation of A549 cells in an apoptotic manner (Figure 5C). The apoptotic cell death by CSE was significantly curtailed in YE-added alveolar cells. Accordingly, CS-induced alveolar emphysema may be ascribed to its apoptotic death of alveolar cells.

**Figure 5.** Viability of alveolar epithelial A549 cells and effects of dried yeas<sup>t</sup> extracts (YE) on alveolar apoptosis. Alveolar epithelial cells were incubated in media containing 5% cigarette smoke extract (CSE) in the absence and presence of 10–100 μg/mL YE for up to 24 h. A549 cell viability (mean ± SEM, *n* = 5) was measured by using MTT assay and expressed as percent cell survival relative to untreated controls (**A**,**B**). Values in respective bar graphs not sharing a same small letter indicate a significant difference at *p* < 0.05. Nuclear staining was done with Hoechst 33258 dye for the detection of apoptotic cells (**C**). A transferase dUTP nick end labeling (TUNEL) assay was conducted to detect DNA fragmentation of apoptotic A549 cells and nuclear staining was accomplished with DAPI (**C**). Representative microphotographs were obtained by fluorescence microscopy. Scale bars = 50 μm.

#### *3.5. Inhibition of Airway Inflammation of OVA-Exposed Airways by YE*

This study investigated that YE inhibited allergic airway inflammation evoked by OVA in mouse airways. When mice underwent OVA inhalation, total leukocyte number in BALF was highly elevated by ≈2.5-fold (Figure 6A). The OVA inhalation prompted neutrophilic and eosinophilic inflammation in BALF, while oral administration of 25–100 mg/kg YE reduced OVA-induced leukocytosis of neutrophils and lymphocytes (Figure 6A). In addition, YE diminished eosinophilic inflammation elevated in OVA-exposed mice (Figure 6A).

The tissue levels of inflammatory COX-2 and iNOS were enhanced in OVA inhalation-experienced mouse lungs in a similar manner to the CS challenge (Figure 6B). However, the YE supply attenuated the lung tissue induction of these proteins promoted by OVA (Figure 6B). Accordingly, YE may alleviate OVA inhalation-induced allergic inflammation in airways. In addition, this study examined whether OVA induced pulmonary emphysema in mice. The Cy3-immunostaining revealed that OVA promoted the MMP-12 expression in mouse bronchioles and alveoli (Figure 6C). It should be noted that the MMP-12 induction by OVA inhalation was less than that of CS challenge. Nevertheless, YE curtailed the Cy3-MMP-12 staining in airways, indicating that YE abrogated pulmonary emphysema due to OVA (Figure 6C).

**Figure 6.** Suppressive effects of dried yeas<sup>t</sup> extract (YE) on airway inflammation and induction of airway target proteins in ovalbumin (OVA) inhalation-challenged mouse lungs. OVA-sensitized mice were orally administrated with 25–100 mg/kg YE. Cells in BALF were counted using a Hemavet HV950 Multispecies Hematologic Analyzer (**A**). Lung tissue extracts were subject to Western blot with a primary antibody against COX-2 and iNOS (**B**). β-Actin protein was used as an internal control. The bar graphs (mean ± SEM, *n* = 3) represent quantitative results of the left bands obtained from a densitometer. Values in respective bar graphs not sharing a same small letter indicate a significant difference at *p* < 0.05 Immunohistofluorescence analysis was done in tissues of small airways and alveoli of OA-challenged mice (**C**). The MMP-12 localization was identified as Cy3-red staining in mouse airways exposed to OVA. Nuclear staining was done with DAPI (blue). Each photograph is representative of four mice. Scale bars = 100 μm.

#### *3.6. Blockade of LPS-Triggered Airway Inflammation by YE*

The endotoxin LPS stimulated alveolar inflammation through the induction of COX-2, iNOS, and ICAM-1 in A549 cells (Figure 7A–C). In addition, LPS prompted the secretion of pro-inflammatory cytokines of TNF-α and MCP-1 from alveolar epithelial cells (Figure 7D,E). When LPS-loaded alveolar cells were treated with 10–100 μg/mL YE, such induction and secretion of these inflammatory proteins were reduced (Figure 7A–E).

This study further examined whether pro-inflammatory TNF-α produced by alveolar cells might be involved in evoking alveolar emphysema by pathological stimuli. When TNF-α was treated to A549 cells, the MMP-12 protein was highly induced (Figure 7F). In contrast, ≥10 μg/mL YE blunted its induction in TNF-α-experienced alveolar cells. Thus, one can speculate that airway inflammation may be a contributor to pulmonary emphysema.

**Figure 7.** Blockade of alveolar inflammation by dried yeas<sup>t</sup> extracts (YE) in lipopolysaccharide (LPS)-exposed A549 cells. Alveolar epithelial cells were incubated in media containing 2 μg/mL LPS or 10 ng/ml TNF-α in the absence and presence of 10–100 μg/mL YE for up to 24 h. Cell lysates were prepared for Western blot analysis with a primary antibody against COX-2, iNOS, ICAM-1, or MMP-12 (**A**–**C**,**F**). β-Actin protein was used as an internal control. The bar graphs (mean ± SEM, *n* = 3) represent quantitative results of the upper bands obtained from a densitometer. Alveolar secretion of TNF-α and MCP-1 was measured by using ELISA kits (**D**,**E**). Values in respective bar graphs not sharing a same small letter indicate a significant difference at *p* < 0.05.

It has been reported that sustained activation of NF-κB pathway links airway inflammation and COPD, which provides its potential as target for treatment of asthma and COPD [33]. LPS highly increased IκB phosphorylation of A549 cells, leading to induction of nuclear translocation of NF-κB (Figure 8A). The treatment of ≥10 μg/mL YE retarded its phosphorylation. Consistently, the Cy3-NF-κB staining supported the Western blot data showing nuclear translocation of NF-κB that was inhibited by YE (Figure 8B).

**Figure 8.** Involvement of NF-κB signaling in lipopolysaccharide (LPS)-induced alveolar inflammation and blockade by dried yeas<sup>t</sup> extracts (YE). Alveolar epithelial cells were incubated in media containing 2 μg/mL LPS in the absence and presence of 10–100 μg/mL YE for up to 24 h. Cell lysates were prepared for Western blot analysis with a primary antibody against IκB and phospho-Iκ<sup>B</sup> (**A**). β-Actin protein was used as an internal control. The bar graphs (mean ± SEM, *n* = 3) represent quantitative results of the left bands obtained from a densitometer. Values in respective bar graphs not sharing a same small letter indicate a significant difference at *P* < 0.05. Immunocytofluorescence analysis was done in LPS-treated A549 alveolar epithelial cells (**B**). The NF-κB localization was identified as Cy3-red staining in cells exposed to LPS. Nuclear staining was done with DAPI (blue). Each photograph is representative of stained cells (*n* = 4). Scale bar = 50 μm.
