The Fermented Soy Product ImmuBalanceTM Suppresses Airway Inflammation in a Murine Model of Asthma.

The fermented soy product ImmuBalance contains many active ingredients and its beneficial effects on some allergic diseases have been reported. We hypothesized that ImmuBalance could have potential effects on airway inflammation in a murine model of asthma. Mice sensitized and challenged with ovalbumin developed airway inflammation. Bronchoalveolar lavage fluid was assessed for inflammatory cell counts and levels of cytokines. Lung tissues were examined for cell infiltration and mucus hypersecretion. Oral administration of ImmuBalance significantly inhibited ovalbumin-induced eosinophilic inflammation and decreased Th2 cytokine levels in bronchoalveolar lavage fluid (p < 0.05). In addition, lung histological analysis showed that ImmuBalance inhibited inflammatory cell infiltration and airway mucus production. Our findings suggest that supplementation with ImmuBalance may provide a novel strategy for the prevention or treatment of allergic airway inflammation.

Ninjin'yoeito Ameliorates Skeletal Muscle Complications in COPD Model Mice by Upregulating Peroxisome Proliferator-Activated Receptor γ Coactivator-1α Expression.

Purpose: Sarcopenia, the loss of skeletal muscle mass and strength, is a common systemic consequence of chronic obstructive pulmonary disease (COPD) and is correlated with higher mortality. Ninjin'yoeito (NYT) is a Japanese herbal medicine used to treat athrepsia and anorexia and is reported to ameliorate weight loss and muscular dysfunction. Recent studies have shown that its crude components upregulate the peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α)-related pathway, which is involved in skeletal muscle functions. Here, we examined whether NYT improves skeletal muscle complications by upregulating PGC-1α in COPD model mice. Materials and Methods: Mice were divided into four groups: control, NYT, smoking, and smoking + NYT. The smoking and smoking + NYT groups were exposed to cigarette smoke for 60 min once daily. The mice in the NYT and smoking + NYT groups were fed an NYT-containing diet (3% w/w). We performed cellular analysis of bronchoalveolar lavage fluid, assessed pulmonary morphological changes, examined the expression of PGC-1α mRNA and protein in the gastrocnemius and soleus muscle, measured the hindlimb muscle volume with micro-computed tomography, and determined the myofiber proportion in soleus muscle after 12 weeks. Results: Cigarette smoke exposure resulted in reduced skeletal muscle volume and slow-twitch muscle fibers and development of pulmonary emphysema. NYT feeding induced partial recovery of the damaged alveolar wall; however, NYT did not ameliorate smoke-induced alveolar enlargement. These findings revealed that NYT did not have sufficient efficacy in suppressing pulmonary emphysema. On the other hand, PGC-1α expression in muscle tissue of the NYT-fed mice increased significantly, resulting in suppression of smoke-induced loss of muscle mass and alteration in the muscle fiber distribution. Conclusion: NYT increases PGC-1α expression in the muscle of COPD model mice and is involved in suppressing cigarette smoke-induced muscle complications. NYT may be a novel preventive and therapeutic medication for muscular dysfunctions in COPD.

Exercise Ameliorates Emphysema Of Cigarette Smoke-Induced COPD In Mice Through The Exercise-Irisin-Nrf2 Axis.

Background: Oxidative stress is one of the important mechanisms underlying the pathogenesis of chronic obstructive pulmonary disease (COPD). Irisin is a type of myokine secreted from the muscle during exercise and acts against oxidative stress via nuclear factor erythroid 2-related factor 2 (Nrf2), a transcription factor with antioxidant properties. Here, we examined the emphysema suppressive effects of the exercise-irisin-Nrf2 axis in mice. Methods: Mice were divided into three groups, namely, the control, smoking, and exercise + smoking groups. All mice from the smoking and exercise + smoking groups were exposed to cigarette smoke once a day. The mice from the exercise + smoking group were adapted to a treadmill once a day. To investigate the Nrf2 cascade, after 12 weeks, serum irisin concentration and Nrf2 and heme oxygenase-1 (HO-1) expression in the lung homogenate were determined. To evaluate cigarette smoke-induced COPD, the number of inflammatory cells in bronchoalveolar lavage fluid (BALF), mean linear intercept (MLI), and destructive index in the lung tissue were examined. Results: Serum irisin concentration and the expression levels of Nrf2 and HO-1 in the lung homogenate were significantly higher in mice from the exercise + smoking group than in those from the control and smoking groups. The proportion of neutrophils in the BALF was significantly lower in the exercise + smoking group than in the smoking group. The MLI and destructive index were also significantly smaller in mice from the exercise + smoking group than mice from the smoking group. Conclusion: Irisin secreted from the muscle during exercise may exert protective effects against oxidative stress via Nrf2 and HO-1, and ameliorate emphysema of cigarette smoke-induced COPD. The exercise-irisin-Nrf2 axis may serve as a novel target for COPD treatment.

Astaxanthin Suppresses Cigarette Smoke-Induced Emphysema through Nrf2 Activation in Mice.

Oxidative stress plays an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD). The activation of nuclear factor erythroid 2-related factor 2 (Nrf2) is a key cellular defense mechanism against oxidative stress. Recent studies have shown that astaxanthin protects against oxidative stress via Nrf2. In this study, we investigated the emphysema suppression effect of astaxanthin via Nrf2 in mice. Mice were divided into four groups: control, smoking, astaxanthin, and astaxanthin + smoking. The mice in the smoking and astaxanthin + smoking groups were exposed to cigarette smoke for 12 weeks, and the mice in the astaxanthin and astaxanthin + smoking groups were fed a diet containing astaxanthin. Significantly increased expression levels of Nrf2 and its target gene, heme oxygenase-1 (HO-1), were found in the lung homogenates of astaxanthin-fed mice. The number of inflammatory cells in the bronchoalveolar lavage fluid (BALF) was significantly decreased, and emphysema was significantly suppressed. In conclusion, astaxanthin protects against oxidative stress via Nrf2 and ameliorates cigarette smoke-induced emphysema. Therapy with astaxanthin directed toward activating the Nrf2 pathway has the potential to be a novel preventive and therapeutic strategy for COPD.

Isoflavone Aglycones Attenuate Cigarette Smoke-Induced Emphysema via Suppression of Neutrophilic Inflammation in a COPD Murine Model.

Chronic obstructive pulmonary disease (COPD), a lung disease caused by chronic exposure to cigarette smoke, increases the number of inflammatory cells such as macrophages and neutrophils and emphysema. Isoflavone is a polyphenolic compound that exists in soybeans. Daidzein and genistein, two types of isoflavones, have been reported to have anti-inflammatory effects in various organs. We hypothesized that the daidzein-rich soy isoflavone aglycones (DRIAs) attenuate cigarette smoke-induced emphysema in mice. Mice were divided into four groups: the (i) control group, (ii) isoflavone group, (iii) smoking group, and (iv) isoflavone + smoking group. The number of inflammatory cells in the bronchoalveolar lavage fluid (BALF) and the airspace enlargement using the mean linear intercept (MLI) were determined 12 weeks after smoking exposure. Expressions of neutrophilic inflammatory cytokines and chemokines were also examined. In the isoflavone + smoking group, the number of neutrophils in BALF and MLI was significantly less than that in the smoking group. Furthermore, the gene-expressions of TNF-α and CXCL2 (MIP-2) in the isoflavone + smoking group were significantly less than those in the smoking group. Supplementation of the COPD murine model with DRIAs significantly attenuates pathological changes of COPD via suppression of neutrophilic inflammation.

Decreased levels of irisin, a skeletal muscle cell-derived myokine, are related to emphysema associated with chronic obstructive pulmonary disease.

BACKGROUND: Cigarette smoking-induced oxidant-antioxidant imbalance is a factor that contributes to the pathogenesis of COPD through epithelial cell apoptosis. Irisin is a skeletal muscle cell-derived myokine associated with physical activity. Irisin is also known to decrease oxidant-induced apoptosis in patients with diabetes mellitus. However, the correlation between irisin and emphysema in COPD and its role in epithelial cell apoptosis remains unknown. SUBJECTS AND METHODS: Forty patients with COPD were enrolled in this study. Pulmonary function tests and measurements of the percentage of low-attenuation area on high-resolution computed tomography images were performed, and the results were evaluated for correlation with serum irisin levels. The effect of irisin on cigarette-smoke extract-induced A549 cell apoptosis and the expression of Nrf2, a transcription factor for antioxidants, was also examined in vitro. RESULTS: Serum irisin levels were significantly correlated with lung diffusing capacity for carbon monoxide divided by alveolar volume (r=0.56, P<0.01) and percentage of low-attenuation area (r=-0.79, P<0.01). Moreover, irisin significantly enhanced Nrf2 expression (P<0.05) and reduced cigarette-smoke extract-induced A549 cell apoptosis (P<0.05). CONCLUSION: Decreased serum irisin levels are related to emphysema in patients with COPD and involved in epithelial apoptosis, resulting in emphysema. Irisin could be a novel treatment for emphysema in patients with COPD.

Down-Regulation of Soluble α-Klotho is Associated with Reduction in Serum Irisin Levels in Chronic Obstructive Pulmonary Disease.

PURPOSE: The klotho gene was originally identified as a putative aging-suppressor gene. Klotho-depleted mice display a shortened life span and exhibit a variety of premature aging-related phenotypes such as pulmonary emphysema and sarcopenia. This study was designed to determine the roles of secreted-type klotho protein on lung and skeletal muscle in chronic obstructive pulmonary disease (COPD). METHODS: Serum α-klotho and irisin levels were assayed in 16 non-smokers, 13 smokers without COPD, and 24 smokers with COPD. Moreover, we examined correlations between soluble α-klotho levels and the results of lung function test, cardiopulmonary exercise test (CPET), and skeletal muscle function in smokers with COPD. RESULTS: Soluble α-klotho levels were significantly lower in smokers with COPD compared to non-smokers and smokers without COPD. In smokers with COPD, those levels did not significantly correlate with any parameters of lung function test. In CPET, peak VO2 significantly correlated with FEV1 (% predicted) (r = 0.76, p = 0.0003) and DLCO (% predicted) (r = 0.62, p = 0.003). In contrast, soluble α-klotho levels did not significantly correlate with peak VO2. Irisin levels were also significantly lower in smokers with COPD. Moreover, there was a significant correlation between soluble α-klotho and serum irisin levels (r = 0.61, p = 0.004). CONCLUSIONS: Our findings could provide a critical first step to understanding the impacts of soluble α-klotho on skeletal muscle in COPD and may lead to the identification of new molecular targets for the treatment of COPD.

Impaired nuclear factor erythroid 2-related factor 2 expression increases apoptosis of airway epithelial cells in patients with chronic obstructive pulmonary disease due to cigarette smoking.

BACKGROUND: Cigarette smoking-induced oxidative stress is known to be a key mechanism in COPD pathogenesis. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a central transcription factor that regulates the antioxidant defense system. The aim of this study was to compare Nrf2 expression in COPD subjects and control subjects, and to determine the role of Nrf2 in protecting against oxidative stress-induced apoptosis. METHODS: We enrolled 8 COPD subjects and 7 control subjects in this study. We performed bronchial brushing by bronchoscopy and obtained bronchial epithelial cells from the airways. Nrf2 expression in bronchial epithelial cells was evaluated by real-time PCR and Western blotting. We examined the effect of 10 or 15 % cigarette smoke extract (CSE) induced A549 cells apoptosis using a time-lapse cell imaging assay with caspase-3/7 activation detecting reagent and performed Terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling assay for confirming A549 cells apoptosis. We also examined the effects of Nrf2 knockdown and, 0.1, 0.5, and 1.0 mM N-acetyl cysteine on CSE-induced apoptosis. Statistical analyses were performed using t-test, paired t-test or an analysis of variance followed by the Tukey-Kramer method. RESULTS: Nrf2 mRNA expression in COPD subjects was significantly lower than that in control subjects and Nrf2 mRNA were negatively correlated with pack year. Nrf2 protein in COPD subjects was significantly lower than that in control subjects. CSE-induced A549 cells apoptosis was increased in a time-, concentration-dependent manner, and was significantly increased by Nrf2 knockdown. N-acetyl cysteine significantly ameliorated CSE-induced apoptosis. CONCLUSIONS: Nrf2 expression was lower in COPD patients than in control subjects. Nrf2 might have a protective role against apoptosis caused by CSE-induced oxidative stress. These results suggest an involvement of Nrf2 in COPD and administration of antioxidants to patients with COPD might be a basic therapeutic option.

Up-regulation of the MicroRNA miR-30c Induced by High Mobility Group Box 1 in A549 Cells Used as an Alveolar Epithelial Model.

Background: MicroRNAs (miRNAs) have been reported to be involved in multiple diseases, including chronic obstructive pulmonary disease (COPD), a progressive disease in which alveolar apoptosis may play a role. We hypothesized that miRNAs are associated with the response to injury. induced by high mobility group box 1 (HMGB1), a cytokine crucial for the development of COPD, and studied the potential link between HMGB1 and miRNAs. Materials and Methods: A549 cells were stimulated with recombinant HMGB1. RNA and protein were extracted and culture supernatants were collected. Molecules downstream of HMGB1 signaling were analyzed by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Expression levels of miRNA were analyzed by quantitative RT-PCR. Cellular injury was evaluated by western blotting of relevant proteins. Apoptosis was evaluated by in situ terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL). Results: HMGB1 treatment of A549 cells resulted in the up-regulation of tumor necrosis factor (TNF)-α and macrophage inflammatory protein (MIP)-2 mRNAs and over expression of matrix metalloprotease (MMP)-7 protein in the supernatant. The miRNA miR-30c was also up-regulated in response to HMGB1 treatment. Cellular injury and apoptosis were observed following HMGB1 treatment, as demonstrated by the oyerexpression of cyclin A2 (CCNA2) and phosphatase and tensin homolog (PTEN) proteins and-b'y decreased levels of pro-caspase-7 protein. The TUNEL assay showed that A549 cells with HMGB1 stimulation underwent apoptosis. Conclusions: Up-regulation of miR-30c and apoptosis of A549 cells were observed following HMGB1 stimulation. Our model demonstrates the potential for utilization of HMGB1 and miR-30c in further studies of alveolar apoptosis in COPD.

Pentraxin-3 as a Biomarker for Febrile Neutropenia in Patients with Lung Cancer.

BACKGROUND: Pentraxin-3 (PTX3) is a newly discovered biomarker for various inflammatory conditions. We measured plasma PTX3 levels in patients with febrile neutropenic lung cancer and examined the utility of PTX3 levels as a biomarker for febrile neutropenia. METHODS: Fourteen patients with febrile neutropenic lung cancer were enrolled in the study. In addition, 10 untreated lung cancer patients and 12 healthy adults were enrolled as a disease control group and a healthy control group, respectively. On the day of onset of febrile neutropenia (day 1) and days 3 and 7, PTX3 and C-reactive protein (CRP) levels were measured. In the control groups, PTX3 and CRP levels were measured once. RESULTS: On day 1, plasma CRP levels in febrile neutropenia during chemotherapy or chemoradiotherapy for lung cancer (FN/LC) patients (8.11 ± 6.42 mg/dL) were significantly higher than those in healthy controls (HC) and chemotherapy/chemoradiotherapy-naïve lung cancer (CN/LC) patients (p < 0.05). However, CRP levels of the CN/LC group (0.33 ± 0.02 mg/dL) were also significantly higher than those of the HC group (0.07 ± 0.09 mg/dL) (p < 0.05). In contrast, plasma PTX3 levels of the FN/LC group (6.14 ± 5.28 ng/mL) were significantly higher than those of the HC and CN/LC groups on day 1 (p < 0.05), but PTX3 levels of the CN/LC group (1.60 ± 0.64 ng/mL) were not significantly higher than those of the HC group (1.05 ± 0.25 ng/mL). In the FN/LC group, PTX3 levels peaked immediately on day 1. CONCLUSIONS: PTX3 may be a useful biomarker for diagnosis of FN in patients with LC.

Irisin, a newly discovered myokine, is a novel biomarker associated with physical activity in patients with chronic obstructive pulmonary disease.

BACKGROUND AND OBJECTIVE: Irisin is a recently identified hormone secreted by skeletal myocytes, which has been proposed to mediate the beneficial effects of exercise. Physical activity has been emphasized as one of the principal targets of the treatment for chronic obstructive pulmonary disease (COPD). This study was designed to evaluate the possibility of using serum irisin level as a novel biomarker associated with physical activity in patients with COPD. METHODS: We measured the serum irisin level in 72 COPD patients and 27 control subjects, and investigated its correlation to pulmonary function parameters, exercise capacity and physical activity level. In addition, we analysed the effects of acute and chronic exercise on serum irisin level. RESULTS: Fat-free mass index was not significantly different between the two study groups. However, lower serum irisin level was observed in COPD patients than in the control subjects (COPD patients: median (interquartile range) 31.6 (22.7-40.4) ng/mL; control subjects: 50.7 (39.3-65.8) ng/mL; P < 0.001). The serum irisin level did not significantly correlate with any pulmonary function parameters and 6-min walk distance. However, serum irisin level was associated with the physical activity level in all subjects. In COPD patients, acute exercise did not affect serum irisin level, but an 8-week exercise training was linked to the significant increase in its level. CONCLUSIONS: Circulating irisin could be used to evaluate physical activity in COPD patients and increased after an 8-week exercise training. Serum irisin level may prove to be a valuable biomarker in clinical follow up of COPD.

Application of a new parameter in the 6-minute walk test for manifold analysis of exercise capacity in patients with COPD.

BACKGROUND: New parameters in the 6-minute walk test (6 MWT) are required for comprehensive analysis of exercise capacity in patients with chronic obstructive pulmonary disease (COPD). The aim of the present study was to apply a novel index, the desaturation distance ratio (DDR), to clinical research on COPD as an estimate of exercise capacity and to examine whether DDR is a potential parameter for manifold analysis of exercise capacity in patients with COPD. METHODS: A total of 41 patients with COPD (median age [interquartile range] =75 [68-79] years; and body mass index [BMI] =22.3 [19.4-23.8] kg/m(2)) participated in the study. The 6 MWT was performed along with anthropometric measurements and a pulmonary function test. The "desaturation area" was measured as the total area above the curve created using peripheral oxygen saturation (SpO2) values observed at each minute during the 6 MWT. Then the DDR was calculated as the ratio of the desaturation area to the 6-minute walk distance (6 MWD). RESULTS: The 6 MWD was 370 (328-445) m, and the decline in SpO2 values (ΔSpO2) was -5.0% (-8.0% to -1.5%). The DDR correlated modestly with baseline pulmonary function in patients with COPD (forced expiratory volume in 1 second [% of predicted value]: r=-0.658, P<0.001; and diffusing capacity of the lung for carbon monoxide [DL(CO)]: r=-0.470, P=0.002), comparable with the findings of the 6 MWD. The DDR correlated well with ΔSpO2 (r=-0.656, P<0.001) and with the increase in subjective sense of dyspnea during the 6 MWT, as assessed by Borg scale scores (ΔBorg) (r=0.486, P=0.001), in contrast with the 6 MWD, which was not significantly correlated with ΔSpO2 and ΔBorg scale scores. CONCLUSION: The DDR is more informative for manifold analysis of exercise capacity associated with oxygen desaturation and subsequent sense of dyspnea by exercise in patients with COPD.

Analysis of the contributing factors to airway hyperresponsiveness by a forced oscillation technique in patients with asthma.

BACKGROUND: Forced oscillation technique (FOT) is increasingly used to obtain much information on the state of the respiratory system. However, there are little data about FOT parameters on methacholine provocation test in adult asthma. This study was designed to determine the physiological implications of FOT parameters during methacholine provocation and analyze the major contributing factors to airway hyperresponsiveness (AHR) in asthma. METHODS: Spirometry and FOT were performed in 22 asthmatic patients and 21 normal control subjects before and after provocation with a maximal dose of methacholine. RESULTS: In asthmatic patients, the percent increase in resistance at 5 Hz (R5) and resistance at 20 Hz (R20) after the methacholine provocation was 70 [45-93] % and 16 [5-23] %. The percent change in R20 was not significantly correlated with the percent change in FVC or FEV1. Similarly, the percent change in R5 was not significantly correlated with the percent change in FEV1, but was significantly correlated with the percent change in FVC. Moreover, the percent change in R5 was significantly correlated with the closing index (r = 0.55, p = 0.01). In addition, AHR to methacholine was closely correlated with the percent change in R5 (r = -0.71, p = 0.001). CONCLUSIONS: Simultaneous measurement of FOT and bronchial challenge test provide meaningful information, and greater change in R5 may represent exaggerated response of small airways in asthmatic patients. This study will provide new insights into the physiological implications of each FOT parameter in asthmatic patients.