Life Sciences 108(2014)109-115 Contents lists available at ScienceDirect Life Sciences ELSEVIER journal homepage:www.elsevier.com/locate/lifescie Lavender essential oil inhalation suppresses allergic airway inflammation CrossMark and mucous cell hyperplasia in a murine model of asthma Tomoe Ueno-lio a,Misako Shibakuraa.*,Kanayo Yokota2,Michinori Aoe a,Tomoko Hyoda Ryoko Shinohata3, Arihiko Kanehiro b,Mitsune Tanimoto b,Mikio Kataoka a Field of Medical Technology.Okayama University Graduate School of Health Sciences,Okayama.Japan PDepartment of Hematology,Oncology,Allergy,and Respiratory Medicine,Okayama University Graduate School of Medicine,Dentistry.Pharmaceutical Sciences,Okayama,Japan ARTICLE INFO ABSTRACT Article history: Aims:Lavender essential oil (Lvn)has been reported to have anti-inflammatory effects.Bronchial asthma is Received 14 March 2014 characterized by bronchial allergic inflammation with airway remodeling.Therefore,we evaluated the anti- Accepted 25 May 2014 inflammatory effect of Lvn on experimentally induced bronchial asthma in a murine model. Available online 5 June 2014 Main methods:BALB/c mice were sensitized by an intraperitoneal injection of ovalbumin (OVA)at days 0 and 14. Keywords: and subsequently challenged with nebulized OVA on days 28-30(Control-Asthma group).Mice in the treatment Lavender group inhaled Lvn on days 14-31 (Lvn-Asthma group).The allergic inflammatory response was determined on Asthma days 32 and 33. Alternative medicine Key findings:An increase in airway resistance was inhibited in the Lvn-Asthma group than in the Control-Asthma Cytokines group.The Lvn-Asthma group showed lower total cell numbers and eosinophils in bronchoalveolar lavage(BAL) Mucin fluids and peribronchial and perivascular tissues when compared with the Control-Asthma group.The Lvn- Asthma group also had less mucin hyperplasia than the Control-Asthma group.Furthermore,the Lvn-Asthma group showed lower interleukin (IL)-5 and IL-13 cytokine levels in BAL fluids,as well as reduced IL-4 and IL-5 mRNA expression in lung tissue,compared with the Control-Asthma group and determined by FlowCytomix and reverse transcriptase-polymerase chain reaction(RT-PCR).respectively.In addition,Lvn inhalation reduced Muc5b mRNA expression in the lungs without significantly changing the expression of Muc5ac mRNA. Significance:Lvn inhibits allergic inflammation and mucous cell hyperplasia with suppression of T-helper-2 cell cytokines and Muc5b expression in a murine model of asthma.Consequently.Lvn may be useful as an alternative medicine for bronchial asthma. 2014 Elsevier Inc.All rights reserved. Introduction Hypersecretion of mucus contributes to mucous plugging and air- way obstruction in bronchial asthma.MUC5AC and MUC5B are the Atopic asthma is the most prevalent form of asthma which is initiated most prominent gel-forming mucins secreted in the respiratory tract by the exposure to allergens,and resultant of allergen-specific immune (Curran and Cohn,2010).Muc5ac and Muc5b are produced by mucous responses,such as airflow obstruction,airway hyperresponsiveness cells,such as goblet cells,submucosal glands,and Clara cells (Evans (AHR),and airway inflammation(Busse and Lemanske,2001).The air- et al,2004;Zhu et al.,2008).Muc5ac and Muc5b are upregulated in mu- way dysfunction by inflammation is the major feature in murine asthma cous cells during asthmatic inflammation in murine models(Yu et al. model,and causes development of AHR(Takeda and Gelfand,2009).The 2006:Zuhdi Alimam et al,2000).Th2 cytokines are also known to in- development of AHR and airway eosinophilic inflammation depends on duce mucus production(Whittaker et al,2002) T-helper-2(Th2)cells producing several cytokines,including interleukin Aromatherapy uses volatile aromatic compounds found in essential (IL)-4,IL-5 and IL-13 (Foster et al.,1996:Perkins et al.,2006). oils extracted from many types of plants.Essential oils help to improve health and alleviate symptoms,including anxiety,pain,insomnia,and bacterial infections.Lavender essential oil (Lvn)has been reported to have sedative and antibacterial effects (Cavanagh and Wilkinson, Abbreviations:Lvn,lavender essential oil:BAL,bronchoalveolar lavage:AHR.airway 2002).Several biological studies using animal models have clarified Corresponding author at:Field of Medical Technology.Okayama University Graduate that Lvn inhibited the mast cell-dependent ear swelling response in- School of Health Sciences,2-5-1 Shikata-cho Kita-ku,Okayama 700-8558.Japan duced by injection of compound 48/80 in mice.Lvn also inhibited pas- E-mail address:m_shiba@mdokayama-uac.jp (M.Shibakura). sive cutaneous anaphylaxis induced by anti-dinitrophenyl(DNP)IgE http:/ddoi.org/10.1016/j1fs.2014.05.018 0024-3205 2014 Elsevier Inc.All rights reserved
Lavender essential oil inhalation suppresses allergic airway inflammation and mucous cell hyperplasia in a murine model of asthma Tomoe Ueno-Iio a , Misako Shibakura a, ⁎, Kanayo Yokota a , Michinori Aoe a , Tomoko Hyoda a , Ryoko Shinohata a , Arihiko Kanehiro b , Mitsune Tanimoto b , Mikio Kataoka a a Field of Medical Technology, Okayama University Graduate School of Health Sciences, Okayama, Japan b Department of Hematology, Oncology, Allergy, and Respiratory Medicine, Okayama University Graduate School of Medicine, Dentistry, Pharmaceutical Sciences, Okayama, Japan article info abstract Article history: Received 14 March 2014 Accepted 25 May 2014 Available online 5 June 2014 Keywords: Lavender Asthma Alternative medicine Cytokines Mucin Aims: Lavender essential oil (Lvn) has been reported to have anti-inflammatory effects. Bronchial asthma is characterized by bronchial allergic inflammation with airway remodeling. Therefore, we evaluated the antiinflammatory effect of Lvn on experimentally induced bronchial asthma in a murine model. Main methods: BALB/c mice were sensitized by an intraperitoneal injection of ovalbumin (OVA) at days 0 and 14, and subsequently challenged with nebulized OVA on days 28–30 (Control-Asthma group). Mice in the treatment group inhaled Lvn on days 14–31 (Lvn-Asthma group). The allergic inflammatory response was determined on days 32 and 33. Key findings: An increase in airway resistance was inhibited in the Lvn-Asthma group than in the Control-Asthma group. The Lvn-Asthma group showed lower total cell numbers and eosinophils in bronchoalveolar lavage (BAL) fluids and peribronchial and perivascular tissues when compared with the Control-Asthma group. The LvnAsthma group also had less mucin hyperplasia than the Control-Asthma group. Furthermore, the Lvn-Asthma group showed lower interleukin (IL)-5 and IL-13 cytokine levels in BAL fluids, as well as reduced IL-4 and IL-5 mRNA expression in lung tissue, compared with the Control-Asthma group and determined by FlowCytomix and reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. In addition, Lvn inhalation reduced Muc5b mRNA expression in the lungs without significantly changing the expression of Muc5ac mRNA. Significance: Lvn inhibits allergic inflammation and mucous cell hyperplasia with suppression of T-helper-2 cell cytokines and Muc5b expression in a murine model of asthma. Consequently, Lvn may be useful as an alternative medicine for bronchial asthma. © 2014 Elsevier Inc. All rights reserved. Introduction Atopic asthma is the most prevalent form of asthma which is initiated by the exposure to allergens, and resultant of allergen-specific immune responses, such as airflow obstruction, airway hyperresponsiveness (AHR), and airway inflammation (Busse and Lemanske, 2001). The airway dysfunction by inflammation is the major feature in murine asthma model, and causes development of AHR (Takeda and Gelfand, 2009). The development of AHR and airway eosinophilic inflammation depends on T-helper-2 (Th2) cells producing several cytokines, including interleukin (IL)-4, IL-5 and IL-13 (Foster et al., 1996; Perkins et al., 2006). Hypersecretion of mucus contributes to mucous plugging and airway obstruction in bronchial asthma. MUC5AC and MUC5B are the most prominent gel-forming mucins secreted in the respiratory tract (Curran and Cohn, 2010). Muc5ac and Muc5b are produced by mucous cells, such as goblet cells, submucosal glands, and Clara cells (Evans et al., 2004; Zhu et al., 2008). Muc5ac and Muc5b are upregulated in mucous cells during asthmatic inflammation in murine models (Yu et al., 2006; Zuhdi Alimam et al., 2000). Th2 cytokines are also known to induce mucus production (Whittaker et al., 2002). Aromatherapy uses volatile aromatic compounds found in essential oils extracted from many types of plants. Essential oils help to improve health and alleviate symptoms, including anxiety, pain, insomnia, and bacterial infections. Lavender essential oil (Lvn) has been reported to have sedative and antibacterial effects (Cavanagh and Wilkinson, 2002). Several biological studies using animal models have clarified that Lvn inhibited the mast cell-dependent ear swelling response induced by injection of compound 48/80 in mice. Lvn also inhibited passive cutaneous anaphylaxis induced by anti-dinitrophenyl (DNP) IgE Life Sciences 108 (2014) 109–115 Abbreviations: Lvn, lavender essential oil; BAL, bronchoalveolar lavage; AHR, airway hyperresponsiveness; RL, lung resistance; OVA, ovalbumin. ⁎ Corresponding author at: Field of Medical Technology, Okayama University Graduate School of Health Sciences, 2-5-1 Shikata-cho Kita-ku, Okayama 700-8558, Japan. E-mail address: m_shiba@md.okayama-u.ac.jp (M. Shibakura). http://dx.doi.org/10.1016/j.lfs.2014.05.018 0024-3205/© 2014 Elsevier Inc. All rights reserved. Contents lists available at ScienceDirect Life Sciences journal homepage: www.elsevier.com/locate/lifescie
110 T.Ueno-lio et al Life Sciences 108 (2014)109-115 in rats (Kim and Cho,1999).Degranulating mast cells secrete several lungs and bronchoalveolar lavage (BAL)fluids were obtained for further chemical mediators and accelerate the accumulation of inflammatory assays. cells in the bronchial wall,causing multiple features of asthma such as increased AHR,inflammatory cell accumulation in the lungs,and Inhalation of lavender essential oil mucous cell hyperplasia (Amin,2012:Yu et al.,2006).Based on these considerations,we hypothesized that Lvn could attenuate allergic in- Lavandula angustifolia (lavender)essential oil was purchased from flammation through suppression of inflammatory cell accumulation ROHTO Pharmaceutical Co.,Ltd (Osaka,Japan).The analysis sheet of and mucous cell hyperplasia.There are no reports evaluating Lvn's essential oil was supplied by ROHTO and analyzed by gas chromatogra- effect on allergic inflammation in asthma.Using a murine model of phy mass spectrometry.The main component of essential oil was as acute asthma,we present the previously unreported effect of Lvn on follows;linalyl acetate 31.78%,linalool 25.56%,cis-B-ocimene 4.89%, allergic inflammation and mucous cell hyperplasia. B-caryophyllene 4.78%,lavandulyl acetate 4.7%,terpinene-4-ol 4.03% cis-B-farnesene 3.7%,trans-B-ocimene 2.77%,and other component Materials and methods were less than 2%.The specific gravity is 0.882 and the refractive index is 1.459.Mice were transported into inhalation cages Animals (28.5 cmx 21.5 cm x10.0 cm)according to group assignment.Lavender essential oil (5 uL or 20 uL)was applied to a piece of 10 cm x 10 cm filter BALB/c mice (females,6-8 weeks of age)were purchased from paper and placed on the upper side of the inhalation cage.The Lvn- Charles River Japan,Inc.(Yokohama,Japan).The mice were maintained Asthma group was treated by inhalation of Lvn 5 days per week for on diets free of ovalbumin (OVA).All experimental animals used in this 20 min from day 14 to day 31(Fig.1).The Non-Asthma group and study were housed under constant temperature and light cycles and Control-Asthma group were treated by water in the same manner. under a protocol approved by the Institutional Animal Care and Use Committee of Okayama University. Determination of lung resistance Sensitization and airway challenge A flexiVent small animal ventilator (SCIREQ,Montreal,PO.Canada) was used to assess pulmonary function(SnapShot)in anesthetized(in- Mice were sensitized and challenged with OVA(Control-Asthma traperitoneal injection of sodium pentobarbital,70 mg/kg),mechani- group and Lvn-Asthma group).Sensitization was carried out by intra- cally ventilated mice,measuring changes in lung resistance (RL)in peritoneal injection of 20 ug OVA(albumin from chicken egg.Grade V: response to increasing doses of inhaled methacholine (Wako Pure Sigma Chemical Co.,St.Louis,IL,USA)emulsified in 2.25 mg of alumi- Chemical Industries,Ltd.,Osaka,Japan)as previously described (Koga num hydroxide (Imject Alum,Thermo Fisher Scientific Inc.,Rockford, et al.,2013).Briefly,RL was assessed (150 breaths/min,tidal volume: IL,USA)in a total volume of 0.1 mL on days 0 and 14.Mice were chal- 10 mL/kg)as a change in airway resistance after a challenge with aero- lenged via the airways with aerosolized 1%OVA administered by ultra- solized methacholine administered for 10 s (60 breaths/min,tidal sonic nebulizer(Omron Healthcare,Kyoto.Japan)for 20 min on days 28. volume:20 mL/kg)in increasing concentrations(0.3.125,6.25,12.5. 29,and 30(Fig.1).Mice were divided into 4 groups:Non-Asthma group, and 25 mg/mL).Maximum values of RL were recorded and expressed Control-Asthma group,and 5 uL or 20 uL of Lvn-treated asthma group as percent change from baseline after saline aerosol. (Lvn-Asthma group).For mice in the Non-Asthma group,saline (0.1 mL)was intraperitoneally administered on days 0 and 14.Lvn treat- Bronchoalveolar lavage fluid and blood collection and preparation of lung ment in the Lvn-Asthma group was performed as indicated below. tissue AHR was assessed from 48 to 72 h after the last challenge.and the After the assessment of airway responsiveness,the lungs were Non-Asthma group lavaged via the tracheal tube with saline (1 mL 37 C).The volume of Day 0 14 28.29,3032.33 collected BAL fluid was measured in each sample,and the number of 444 A▲ cells in the BAL fluid was counted.BAL fluid was centrifuged at 1500 rpm for 10 min at 4C.After centrifugation,the supernatant was 1.P .P. OVA Assay Saline Saline challenge stored at-80 C.The sediment was resuspended in 1.0 mL of phos- phate buffered saline (PBS),and smear preparations were prepared by an automatic cytocentrifuge system (1000 rpm,1 min).Preparations Control-Asthma group were stained with Giemsa,and cell differentiation was performed in a blinded fashion by counting at least 200 cells under light microscopy. Blood samples were collected from the abdominal space by excising L.P. IP OVA Assay the vena cava.Blood was centrifuged at 2500 rpm for 15 min.After cen- OVA OVA challenge trifugation,serum was stored at-80 C.The lungs were removed and fixed in 10%formalin for histological studies. Lvn-Asthma group 5 or 20 ul of Lvn 5days/week ■。■。。0”0■■■■”■■■■■■ Histological studies I I.P. 1.P OVA Formalin-fixed blocks of lung tissue were cut around the main bron- Assay OVA OVA challenge chus and embedded in paraffin blocks.Tissue sections(4 um thick)were affixed to microscope slides and deparaffinized.After staining with he- Fig.1.Experimental protocols.Mice were divided into 4 groups:non-sensitized and ovalbu- matoxylin-eosin(HE)and periodic acid-Schiff (PAS)for the identifica- min (OVA)-challenged mice (Non-Asthma group):OVA-sensitized and OVA-challenged tion of mucus-containing cells,the slides were examined under light mice (Control-Asthma group):5 or 20 uL of lavender essential oil (Lvn)-treated,OVA- microscopy.In HE-stained lung sections,the number of inflammatory sensitized,and OVA-challenged mice (Lvn-Asthma group).In the Control-Asthma group, cells (eosinophils,lymphocytes,and neutrophils)was counted in areas mice were sensitized by two intraperitoneal injections of OVA/alum and subsequently received 3 consecutive days of an aerosolized OVA challenge on days 28-30.In the Lvn- of peribronchial and perivascular tissues using the NIH Image Analysis Asthma group.OVA-sensitized and OVA-challenged mice inhaled 5 or 20 ul of Lvn for system (National Institutes of Health,Bethesda,MD,USA).More than 20 min,5 consecutive days per week,from day 14 to day 31. 10 bronchioles in a minimum of 10 high-power fields per lung tissue
in rats (Kim and Cho, 1999). Degranulating mast cells secrete several chemical mediators and accelerate the accumulation of inflammatory cells in the bronchial wall, causing multiple features of asthma such as increased AHR, inflammatory cell accumulation in the lungs, and mucous cell hyperplasia (Amin, 2012; Yu et al., 2006). Based on these considerations, we hypothesized that Lvn could attenuate allergic in- flammation through suppression of inflammatory cell accumulation and mucous cell hyperplasia. There are no reports evaluating Lvn's effect on allergic inflammation in asthma. Using a murine model of acute asthma, we present the previously unreported effect of Lvn on allergic inflammation and mucous cell hyperplasia. Materials and methods Animals BALB/c mice (females, 6–8 weeks of age) were purchased from Charles River Japan, Inc. (Yokohama, Japan). The mice were maintained on diets free of ovalbumin (OVA). All experimental animals used in this study were housed under constant temperature and light cycles and under a protocol approved by the Institutional Animal Care and Use Committee of Okayama University. Sensitization and airway challenge Mice were sensitized and challenged with OVA (Control-Asthma group and Lvn-Asthma group). Sensitization was carried out by intraperitoneal injection of 20 μg OVA (albumin from chicken egg, Grade V; Sigma Chemical Co., St. Louis, IL, USA) emulsified in 2.25 mg of aluminum hydroxide (Imject Alum, Thermo Fisher Scientific Inc., Rockford, IL, USA) in a total volume of 0.1 mL on days 0 and 14. Mice were challenged via the airways with aerosolized 1% OVA administered by ultrasonic nebulizer (Omron Healthcare, Kyoto, Japan) for 20 min on days 28, 29, and 30 (Fig. 1). Mice were divided into 4 groups: Non-Asthma group, Control-Asthma group, and 5 μL or 20 μL of Lvn-treated asthma group (Lvn-Asthma group). For mice in the Non-Asthma group, saline (0.1 mL) was intraperitoneally administered on days 0 and 14. Lvn treatment in the Lvn-Asthma group was performed as indicated below. AHR was assessed from 48 to 72 h after the last challenge, and the lungs and bronchoalveolar lavage (BAL) fluids were obtained for further assays. Inhalation of lavender essential oil Lavandula angustifolia (lavender) essential oil was purchased from ROHTO Pharmaceutical Co., Ltd (Osaka, Japan). The analysis sheet of essential oil was supplied by ROHTO and analyzed by gas chromatography mass spectrometry. The main component of essential oil was as follows; linalyl acetate 31.78%, linalool 25.56%, cis-β-ocimene 4.89%, β-caryophyllene 4.78%, lavandulyl acetate 4.7%, terpinene-4-ol 4.03%, cis-β-farnesene 3.7%, trans-β-ocimene 2.77%, and other component were less than 2%. The specific gravity is 0.882 and the refractive index is 1.459. Mice were transported into inhalation cages (28.5 cm × 21.5 cm ×10.0 cm) according to group assignment. Lavender essential oil (5 μL or 20 μL) was applied to a piece of 10 cm × 10 cm filter paper and placed on the upper side of the inhalation cage. The LvnAsthma group was treated by inhalation of Lvn 5 days per week for 20 min from day 14 to day 31 (Fig. 1). The Non-Asthma group and Control-Asthma group were treated by water in the same manner. Determination of lung resistance A flexiVent small animal ventilator (SCIREQ, Montreal, PQ, Canada) was used to assess pulmonary function (SnapShot) in anesthetized (intraperitoneal injection of sodium pentobarbital, 70 mg/kg), mechanically ventilated mice, measuring changes in lung resistance (RL) in response to increasing doses of inhaled methacholine (Wako Pure Chemical Industries, Ltd., Osaka, Japan) as previously described (Koga et al., 2013). Briefly, RL was assessed (150 breaths/min, tidal volume: 10 mL/kg) as a change in airway resistance after a challenge with aerosolized methacholine administered for 10 s (60 breaths/min, tidal volume: 20 mL/kg) in increasing concentrations (0, 3.125, 6.25, 12.5, and 25 mg/mL). Maximum values of RL were recorded and expressed as percent change from baseline after saline aerosol. Bronchoalveolar lavage fluid and blood collection and preparation of lung tissue After the assessment of airway responsiveness, the lungs were lavaged via the tracheal tube with saline (1 mL, 37 °C). The volume of collected BAL fluid was measured in each sample, and the number of cells in the BAL fluid was counted. BAL fluid was centrifuged at 1500 rpm for 10 min at 4 °C. After centrifugation, the supernatant was stored at −80 °C. The sediment was resuspended in 1.0 mL of phosphate buffered saline (PBS), and smear preparations were prepared by an automatic cytocentrifuge system (1000 rpm, 1 min). Preparations were stained with Giemsa, and cell differentiation was performed in a blinded fashion by counting at least 200 cells under light microscopy. Blood samples were collected from the abdominal space by excising the vena cava. Blood was centrifuged at 2500 rpm for 15 min. After centrifugation, serum was stored at −80 °C. The lungs were removed and fixed in 10% formalin for histological studies. Histological studies Formalin-fixed blocks of lung tissue were cut around the main bronchus and embedded in paraffin blocks. Tissue sections (4 μm thick) were affixed to microscope slides and deparaffinized. After staining with hematoxylin–eosin (HE) and periodic acid-Schiff (PAS) for the identification of mucus-containing cells, the slides were examined under light microscopy. In HE-stained lung sections, the number of inflammatory cells (eosinophils, lymphocytes, and neutrophils) was counted in areas of peribronchial and perivascular tissues using the NIH Image Analysis system (National Institutes of Health, Bethesda, MD, USA). More than 10 bronchioles in a minimum of 10 high-power fields per lung tissue Fig. 1. Experimental protocols. Mice were divided into 4 groups: non-sensitized and ovalbumin (OVA)-challenged mice (Non-Asthma group); OVA-sensitized and OVA-challenged mice (Control-Asthma group); 5 or 20 μL of lavender essential oil (Lvn)-treated, OVAsensitized, and OVA-challenged mice (Lvn-Asthma group). In the Control-Asthma group, mice were sensitized by two intraperitoneal injections of OVA/alum and subsequently received 3 consecutive days of an aerosolized OVA challenge on days 28–30. In the LvnAsthma group, OVA-sensitized and OVA-challenged mice inhaled 5 or 20 μL of Lvn for 20 min, 5 consecutive days per week, from day 14 to day 31. 110 T. Ueno-Iio et al. / Life Sciences 108 (2014) 109–115
T.Ueno-lio et al Life Sciences 108 (2014)109-115 111 were randomly examined in a blinded fashion.The number of mucus- Software,La Jolla,CA,USA).Probability values of less than 0.05 were containing cells was counted in more than 10 bronchioles in 10 high- considered significant.All results were expressed as the mean+SEM. power fields per animal by measuring the length of the epithelium de- fined along the basement membrane and luminal area using the NIH Results Image Analysis system. Inflammatory cell accumulation in bronchoalveolar lavage fluid Measurement of serum anti-ovalbumin antibody The numbers and types of inflammatory cells in the airway were Anti-OVA IgE antibody levels in the serum were measured by determined from the BAL fluid after the last of 3 consecutive OVA enzyme-linked immunosorbent assay (ELISA)according to the challenges(Fig.2).Macrophages were mainly detected in the Non- manufacturer's instructions.The limit of detection was 2.7 ng/mL (DS Asthma group.Total cell numbers,lymphocytes,and eosinophils Pharma Biomedical,Osaka,lapan). were higher in the Control-Asthma group than in the Non-Asthma group(9.36±1.98×104/mLvs4.80±0.54×10/mL.p<0.05: Measurement of bronchoalveolar lavage fluid cytokines 1.56±0.31×10/mLvs0.07±0.02×10°/mL,p<0.01:and4.05± 1.52×10/mLvs0±0×10/mL:p<0.01,respectively).Mice in the The cytokine concentrations in the BAL fluid supernatants were Lvn-Asthma group treated with 20 uL of Lvn showed significantly small- measured by FlowCytomix according to the manufacturer's instruc- er numbers of total cells (4.47+0.56 x 10/mL,p<0.05)and eosino- tions (Affymetrix,Santa Clara,CA,USA).The limits of detection phils (0.650.27 x 10/mL,p<0.01)in the BAL fluid than the mice were 0.7 pg/mL for IL-4,4.0 pg/mL for IL-5,and 9.3 pg/mL for IL-13. in the Control-Asthma group;however,there were no significant differ- ences in total cells and eosinophils between the Lvn-Asthma group treated with 5 uL of Lvn and the Control-Asthma group.Therefore,we Measurement of messenger RNA expression of cytokines and mucin in lung administered 20 uL of Lvn for inhalation in further experiments. tissue by reverse transcriptase-polymerase chain reaction Total cellular RNA was isolated from lung tissues using Trizol Effect of lavender essential oil on airway hyperresponsiveness reagent(Life Technologies,Carlsbad,CA,USA).Reverse transcriptase- polymerase chain reaction (RT-PCR)analysis was performed in a final Lung resistance was applied as an index of AHR.Fig.3 shows the volume of 20 uL using a Transcriptor First Strand cDNA Synthesis Kit ratio of change from baseline for RL The increase in RL in the Control- (Roche Applied Science,Basel,Switzerland)in a Veriti thermal cycler Asthma group was greater than the increase in the Non-Asthma group (Life Technologies).The sequences of each primer for cytokines and at12.5and25mg/mL of methacholine(7.93±0.59vs4.79±0.84，p mucins are shown in Table 1.PCR assays were performed for 35 cycles. <0.05;13.1±153s4.68±0.53，p<0.0001,respectively).RL did PCR products were analyzed using 2.5%agarose gel containing ethidium not increase as much in mice treated with Lvn (Lvn-Asthma group)as bromide.Finally,the intensity of the PCR product bands and glyceralde- it did in the Control-Asthma group at 25 mg/mL of methacholine hyde 3-phosphate dehydrogenase (Gapdh)was determined using (8.83+0.87.p 0.001).Inhalation of Lvn reduced the increase of Image Lab(BIO-RAD,Hercules,CA,USA). AHR in the Lvn-Asthma group. Statistical analysis Effects of lavender essential oil on inflammatory cell accumulation in peribronchial and perivascular tissues A two-way analysis of variance with repeated measures followed by Bonferroni correction was used to compare RL responses to different Staining with HE detected very few eosinophils and lymphocytes in methacholine concentrations among the Non-Asthma,Control-Asthma the peribronchial and perivascular tissues in the Non-Asthma group and Lvn-Asthma groups.For comparison of cytokine levels,cell counts, (2.0+1.0 and 122.3+22.1 cells/mm2,respectively)(Fig.4b,d).The and mucin levels among the three groups,one-way analysis of variance total cell numbers and eosinophils in the lung tissue sections of the followed by Bonferroni correction was used to compare each group. Control-Asthma group (1194+102.1 and 1011+91.09 cells/mm, Data analysis was conducted using GraphPad Prism 5.04 (GraphPad 15 20 Lyn Ashm Table 1 Primer sequence for T-helper-2(Th2)cytokines and mucins 10 Primers Annealing L-4 Forward 5'-AGGAGAAGGGACGCCATGCACGGA-3 65 Reverse 5-ATCGAAAAGCCCGAAAGAGTCTCTG-3 L-5 Forward 5-GAGCACAGTGGTGAAAGAGACCTT-3 62 Reverse 5'-ATGACAGGTTTTGGAATAGCATTT-3 L-13 Forward 5-TCTTGCTTGCCTTGGIGGICTCGC-3' 子 Reverse 5'-GATGGCATTGCAATTGGAGATGTTG-3 Muc5ac Forward 5-AAAGACACCAGTAGTCACTCAGCAA-3 55 Reverse 5'-CTGGGAAGTCAGTGTCAAACC-3 Muc5b Forward 5'-GCACCTGCTGTCAGGAGAGCA-3 58 Total Mac Lym Nt Eo Reverse 5-TGGTCAGTTGTGCAGGTTCTG-3 Gapdh Forward 5'-TTGGCATTGTGGAAGGGCTCAT-3 65 Fig 2.Cellular composition of bronchoalveolar lavage (BAL)fluids.The BAL fluids were Reverse 5'-GATGACCTTGCCCACAGCCTT-3' obtained from each group described in Fig.1.Non-Asthma group.non-sensitized and ov- The primer sequences were selected from the references.The number of amplification albumin (OVA)-challenged mice (n =7):Control-Asthma group,OVA-sensitized and cycles and annealing temperature for each set was determined experimentally.The OVA-challenged mice (n 7):5 uL Lvn-Asthma group.5 uL of lavender essential oil sequences of IL-4.IL-5.and IL-13 primers are referenced from Krzesicki et al.(1997) (Lvn)-treated,OVA-sensitized,and OVA-challenged mice (n 4):20 ulL Lvn-Asthma The sequences of Muc5ac and Muc5b primers are referenced from Kim et al (2011)and group,20 uL of Lvn-treated.OVA-sensitized,and OVA-challenged mice (n 7).Total, Wu et al.(2008).respectively.The sequences of Gapdh are referenced from Nacher et aL total cell:Mac,macrophage:Lym,lymphocyte:Nt,neutrophil:Eo,eosinophil 'p<0.05 (2011). and "p<0.01.The results for each group are represented as mean+SEM
were randomly examined in a blinded fashion. The number of mucuscontaining cells was counted in more than 10 bronchioles in 10 highpower fields per animal by measuring the length of the epithelium de- fined along the basement membrane and luminal area using the NIH Image Analysis system. Measurement of serum anti-ovalbumin antibody Anti-OVA IgE antibody levels in the serum were measured by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions. The limit of detection was 2.7 ng/mL (DS Pharma Biomedical, Osaka, Japan). Measurement of bronchoalveolar lavage fluid cytokines The cytokine concentrations in the BAL fluid supernatants were measured by FlowCytomix according to the manufacturer's instructions (Affymetrix, Santa Clara, CA, USA). The limits of detection were 0.7 pg/mL for IL-4, 4.0 pg/mL for IL-5, and 9.3 pg/mL for IL-13. Measurement of messenger RNA expression of cytokines and mucin in lung tissue by reverse transcriptase-polymerase chain reaction Total cellular RNA was isolated from lung tissues using Trizol reagent (Life Technologies, Carlsbad, CA, USA). Reverse transcriptasepolymerase chain reaction (RT-PCR) analysis was performed in a final volume of 20 μL using a Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Science, Basel, Switzerland) in a Veriti® thermal cycler (Life Technologies). The sequences of each primer for cytokines and mucins are shown in Table 1. PCR assays were performed for 35 cycles. PCR products were analyzed using 2.5% agarose gel containing ethidium bromide. Finally, the intensity of the PCR product bands and glyceraldehyde 3-phosphate dehydrogenase (Gapdh) was determined using Image Lab (BIO-RAD, Hercules, CA, USA). Statistical analysis A two-way analysis of variance with repeated measures followed by Bonferroni correction was used to compare RL responses to different methacholine concentrations among the Non-Asthma, Control-Asthma and Lvn-Asthma groups. For comparison of cytokine levels, cell counts, and mucin levels among the three groups, one-way analysis of variance followed by Bonferroni correction was used to compare each group. Data analysis was conducted using GraphPad Prism 5.04 (GraphPad Software, La Jolla, CA, USA). Probability values of less than 0.05 were considered significant. All results were expressed as the mean ± SEM. Results Inflammatory cell accumulation in bronchoalveolar lavage fluid The numbers and types of inflammatory cells in the airway were determined from the BAL fluid after the last of 3 consecutive OVA challenges (Fig. 2). Macrophages were mainly detected in the NonAsthma group. Total cell numbers, lymphocytes, and eosinophils were higher in the Control-Asthma group than in the Non-Asthma group (9.36 ± 1.98 × 104 /mL vs 4.80 ± 0.54 × 104 /mL, p b 0.05; 1.56 ± 0.31 × 104 /mL vs 0.07 ± 0.02 × 104 /mL, p b 0.01; and 4.05 ± 1.52 × 104 /mL vs 0 ± 0 × 104 /mL; p b 0.01, respectively). Mice in the Lvn-Asthma group treated with 20 μL of Lvn showed significantly smaller numbers of total cells (4.47 ± 0.56 × 104 /mL, p b 0.05) and eosinophils (0.65 ± 0.27 × 104 /mL, p b 0.01) in the BAL fluid than the mice in the Control-Asthma group; however, there were no significant differences in total cells and eosinophils between the Lvn-Asthma group treated with 5 μL of Lvn and the Control-Asthma group. Therefore, we administered 20 μL of Lvn for inhalation in further experiments. Effect of lavender essential oil on airway hyperresponsiveness Lung resistance was applied as an index of AHR. Fig. 3 shows the ratio of change from baseline for RL. The increase in RL in the ControlAsthma group was greater than the increase in the Non-Asthma group at 12.5 and 25 mg/mL of methacholine (7.93 ± 0.59 vs 4.79 ± 0.84, p b 0.05; 13.1 ± 1.53 vs 4.68 ± 0.53, p b 0.0001, respectively). RL did not increase as much in mice treated with Lvn (Lvn-Asthma group) as it did in the Control-Asthma group at 25 mg/mL of methacholine (8.83 ± 0.87, p b 0.001). Inhalation of Lvn reduced the increase of AHR in the Lvn-Asthma group. Effects of lavender essential oil on inflammatory cell accumulation in peribronchial and perivascular tissues Staining with HE detected very few eosinophils and lymphocytes in the peribronchial and perivascular tissues in the Non-Asthma group (2.0 ± 1.0 and 122.3 ± 22.1 cells/mm2 , respectively) (Fig. 4b, d). The total cell numbers and eosinophils in the lung tissue sections of the Control-Asthma group (1194 ± 102.1 and 1011 ± 91.09 cells/mm2 , Table 1 Primer sequence for T-helper-2 (Th2) cytokines and mucins. Primers Annealing IL-4 Forward 5′-AGGAGAAGGGACGCCATGCACGGA-3′ 65 Reverse 5′-ATCGAAAAGCCCGAAAGAGTCTCTG-3′ IL-5 Forward 5′-GAGCACAGTGGTGAAAGAGACCTT-3′ 62 Reverse 5′-ATGACAGGTTTTGGAATAGCATTT-3′ IL-13 Forward 5′-TCTTGCTTGCCTTGGTGGTCTCGC-3′ 65 Reverse 5′-GATGGCATTGCAATTGGAGATGTTG-3′ Muc5ac Forward 5′-AAAGACACCAGTAGTCACTCAGCAA-3′ 56 Reverse 5′-CTGGGAAGTCAGTGTCAAACC-3′ Muc5b Forward 5′-GCACCTGCTGTCAGGAGAGCA-3′ 58 Reverse 5′-TGGTCAGTTGTGCAGGTTCTG-3′ Gapdh Forward 5′-TTGGCATTGTGGAAGGGCTCAT-3′ 65 Reverse 5′-GATGACCTTGCCCACAGCCTT-3′ The primer sequences were selected from the references. The number of amplification cycles and annealing temperature for each set was determined experimentally. The sequences of IL-4, IL-5, and IL-13 primers are referenced from Krzesicki et al. (1997). The sequences of Muc5ac and Muc5b primers are referenced from Kim et al. (2011) and Wu et al. (2008), respectively. The sequences of Gapdh are referenced from Nacher et al. (2011). Fig. 2. Cellular composition of bronchoalveolar lavage (BAL) fluids. The BAL fluids were obtained from each group described in Fig. 1. Non-Asthma group, non-sensitized and ovalbumin (OVA)-challenged mice (n = 7); Control-Asthma group, OVA-sensitized and OVA-challenged mice (n = 7); 5 μL Lvn-Asthma group, 5 μL of lavender essential oil (Lvn)-treated, OVA-sensitized, and OVA-challenged mice (n = 4); 20 μL Lvn-Asthma group, 20 μL of Lvn-treated, OVA-sensitized, and OVA-challenged mice (n = 7). Total, total cell; Mac, macrophage; Lym, lymphocyte; Nt, neutrophil; Eo, eosinophil. *p b 0.05 and **p b 0.01. The results for each group are represented as mean ± SEM. T. Ueno-Iio et al. / Life Sciences 108 (2014) 109–115 111
112 T.Ueno-lio et al Life Sciences 108 (2014)109-115 201 three groups (Fig.4c,d).Results show that treatment with Lvn -●Ncn-Asthma group decreased inflammatory cell accumulation in peribronchial and Control-Asthma group perivascular tissues. 合20LlLm-Asthma group 15 Effect of lavender essential oil on IgE levels in serum Serum IgE levels were significantly higher in the Control-Asthma group and the Lvn-Asthma group than in the Non-Asthma group. 10- There were no significant differences in serum IgE levels between the Control-Asthma group and the Lvn-Asthma group (Table 2).Treatment with Lvn did not decrease serum IgE levels. 5 Effects of lavender essential oil on mucous cell hyperplasia Periodic acid-Schiff(PAS)-positive cells and areas were not detected in the Non-Asthma group (Fig.5a,d,e).PAS-positive cells and areas were detected in the Control-Asthma group(65.71+3.31 cells/mm 0 10 20 30 BM and 2475+288.7 um2/mm BM,respectively;p<0.0001)(Fig.5b. Methacholine(mg/mL) d,e).The Lvn-Asthma group showed significantly lower PAS-positive cell numbers(37.4+5.5 cells/mm BM,p<0.001)and PAS-positive Fig.3.Effect of lavender essential oil (Lvn)on airway hyperresponsiveness(AHR).Lung areas (1262+244.3 um2/mm BM,p<0.05)compared to the Control- resistance(RL)values were obtained in response to increasing concentrations of inhaled Asthma group (Fig.5c,d,e).These results suggest that treatment with methacholine(MCh).as indicated in Materials and methods.Values are mean+SEM.'p <0.05 compared with Non-Asthma group (n 10)and Control-Asthma group (n Lvn inhibited mucous cell hyperplasia 10)."*p <0.001 compared with Non-Asthma group and 20 ulL Lvn-Asthma group (n 10).and Control-Asthma group and 20 uL Lvn-Asthma group.""p 0.0001 compared Effects of lavender essential oil on cytokine expression in bronchoalveolar with Non-Asthma group and Control-Asthma group. lavage fluids and lung tissue respectively;p<0.0001)were higher than those in the Non-Asthma FlowCytomix measurements showed that the expression of IL-5 and group (158.9+30.1 and 1.95+0.95 cells/mm2,respectively)(Fig.4a IL-13 cytokines in BAL fluids was significantly higher in the Control- b).The Lvn-Asthma group showed significantly lower total cell num- Asthma group compared with the Non-Asthma group (Table 3).The bers(834.9+75.9 cells/mm2,p<0.0001)and eosinophils(668.4 Lvn-Asthma group showed significantly lower IL-5 and IL-13 cytokine 70.88 cells/mm2.p<0.01)in lung tissue sections when compared levels in BAL fluids when compared with the Control-Asthma group. with the Control-Asthma group(Fig.4a.b).There were no significant The IL-4,IL-5,and IL-13 mRNA expression levels in lung tissue were differences in numbers of neutrophils and lymphocytes among the significantly higher in the Control-Asthma group compared with the a 1500 500- 400 1000 300 200 500 100 0 b d 1500 500 有有有 1000 300 200 500 100 0 ☐Nonm-Asthma group Control-Asthma group 目20uLLn-Asthma group Fig.4.Inflammatory cell accumulation in peribronchial and perivascular tissues.The specimens were obtained from the same groups described in Fig.2.Total inflammatory cell (a).eosinophil (b).neutrophil(c)and lymphocyte (d)numbers in peribronchial and perivascular tissues."p<0.01 and""p<0.0001.The results for each group are represented as mean±SEM
respectively; p b 0.0001) were higher than those in the Non-Asthma group (158.9 ± 30.1 and 1.95 ± 0.95 cells/mm2 , respectively) (Fig. 4a, b). The Lvn-Asthma group showed significantly lower total cell numbers (834.9 ± 75.9 cells/mm2 , p b 0.0001) and eosinophils (668.4 ± 70.88 cells/mm2 , p b 0.01) in lung tissue sections when compared with the Control-Asthma group (Fig. 4a, b). There were no significant differences in numbers of neutrophils and lymphocytes among the three groups (Fig. 4c, d). Results show that treatment with Lvn decreased inflammatory cell accumulation in peribronchial and perivascular tissues. Effect of lavender essential oil on IgE levels in serum Serum IgE levels were significantly higher in the Control-Asthma group and the Lvn-Asthma group than in the Non-Asthma group. There were no significant differences in serum IgE levels between the Control-Asthma group and the Lvn-Asthma group (Table 2). Treatment with Lvn did not decrease serum IgE levels. Effects of lavender essential oil on mucous cell hyperplasia Periodic acid-Schiff (PAS)-positive cells and areas were not detected in the Non-Asthma group (Fig. 5a, d, e). PAS-positive cells and areas were detected in the Control-Asthma group (65.71 ± 3.31 cells/mm BM and 2475 ± 288.7 μm2 /mm BM, respectively; p b 0.0001) (Fig. 5b, d, e). The Lvn-Asthma group showed significantly lower PAS-positive cell numbers (37.4 ± 5.5 cells/mm BM, p b 0.001) and PAS-positive areas (1262 ± 244.3 μm2 /mm BM, p b 0.05) compared to the ControlAsthma group (Fig. 5c, d, e). These results suggest that treatment with Lvn inhibited mucous cell hyperplasia. Effects of lavender essential oil on cytokine expression in bronchoalveolar lavage fluids and lung tissue FlowCytomix measurements showed that the expression of IL-5 and IL-13 cytokines in BAL fluids was significantly higher in the ControlAsthma group compared with the Non-Asthma group (Table 3). The Lvn-Asthma group showed significantly lower IL-5 and IL-13 cytokine levels in BAL fluids when compared with the Control-Asthma group. The IL-4, IL-5, and IL-13 mRNA expression levels in lung tissue were significantly higher in the Control-Asthma group compared with the Fig. 3. Effect of lavender essential oil (Lvn) on airway hyperresponsiveness (AHR). Lung resistance (RL) values were obtained in response to increasing concentrations of inhaled methacholine (MCh), as indicated in Materials and methods. Values are mean ± SEM. *p b 0.05 compared with Non-Asthma group (n = 10) and Control-Asthma group (n = 10). ***p b 0.001 compared with Non-Asthma group and 20 μL Lvn-Asthma group (n = 10), and Control-Asthma group and 20 μL Lvn-Asthma group. ****p b 0.0001 compared with Non-Asthma group and Control-Asthma group. Fig. 4. Inflammatory cell accumulation in peribronchial and perivascular tissues. The specimens were obtained from the same groups described in Fig. 2. Total inflammatory cell (a), eosinophil (b), neutrophil (c) and lymphocyte (d) numbers in peribronchial and perivascular tissues. **p b 0.01 and ****p b 0.0001. The results for each group are represented as mean ± SEM. 112 T. Ueno-Iio et al. / Life Sciences 108 (2014) 109–115
T.Ueno-lio et aL Life Sciences 108 (2014)109-115 113 Table 2 Table 3 gE levels in serum Cytokine levels in bronchoalveolar lavage fluids Groups IgE (ng/mL) Groups IL-4 L-5 L-13 Non-Asthma 9.66±3.01 (pg/mL) (pg/mL) (pg/mL) Control-Asthma 4545±1611* Non-Asthma 5.4±3.4 16.6±4.8 51.0±1.7 Lvn-Asthma 4397±853.9* Control-Asthma 117±82 65.1±4.7* 1448±35.7* Lvn-Asthma Mean values+SEM are given.Non-sensitized and ovalbumin 0 283±6.8+ 646±3.1* (OVA)-challenged group.Non-Asthma:OVA-sensitized and OVA- Mean values+SEM are given.Non-sensitized and ovalbumin (OVA)-challenged group. challenged group.Control-Asthma:20 uL of Lvn treated,OVA- Non-Asthma:OVA-sensitized and OVA-challenged group,Control-Asthma:20 ul of Lvn- sensitized,and OVA-challenged group,Lvn-Asthma. treated,OVA-sensitized,and OVA-challenged group.Lvn-Asthma. p 0.05 compared with Non-Asthma (n 5)and Control- p<0.05 compared with Non-Asthma (n =5)and Control-Asthma(n =5). Asthma (n 5)or Lvn-Asthma (n =5). compared with Control-Asthma and Lvn-Asthm p<0.01 compared with Control-Asthma and Lvn-Asthma. Non-Asthma group (p<0.01,p<0.001,and p<0.01,respectively) (Fig.6).Significantly lower IL-4 and IL-5 mRNA expression levels significantly lower Muc5b mRNA expression compared with the in lung tissue were observed in the Lvn-Asthma group compared to Control-Asthma group(p <0.05).Treatment with Lvn decreased the Control-Asthma group(p<0.05 and p<0.01.respectively).The Muc5b mRNA expression in lung tissue. Lvn-Asthma group tended to show lower IL-13 mRNA expression in lung tissue compared to the Control-Asthma group(no significance) Therefore,treatment with Lvn decreased the production of Th2 cyto Discussion kines in BAL fluids and reduced mRNA expression in lung tissue. A significant increase in mRNA expression of Muc5ac and Muc5b The results of this study revealed that Lvn suppressed AHR and accu- was detected in the Control-Asthma group compared with the mulation of inflammatory cells in BAL fluids as well as in bronchial and Non-Asthma group (p<0.001).There was no significant difference perivascular tissues.In addition,Lvn attenuated the increase in Th2 cy- in Muc5ac mRNA expression between the Lvn-Asthma group and tokines in BAL fluids and their mRNA expression in lung tissue.Further- the Control-Asthma group.The Lvn-Asthma group showed more,Lvn suppressed mucous cell hyperplasia.Our findings indicate d 来安光我 行 60 40 30 e 4000- 3000 2000: 1000 ▣Nom-Asthma group Control-Asthma group ☐20ulLn-Asthma group Fig.5.Detection of mucus-producing cells by periodic acid-Schiff(PAS)staining.The lung sections were stained by PAS staining and analyzed as indicated in Materials and methods (a)Non-Asthma group (n =7).(b)Control-Asthma group (n =7).and (c)20 uL Lvn-Asthma group (n =7).The number of mucus-positive cells per mm of basement membrane (d)and PAS-stained area per mm ofbasement membrane(e)were measured in PAS-stained sections as described in Materials and methods.BM:basement membrane.Scale bar indicates 100 um."p<0.05,"p<0.01,*"p 0.001,and*p<0.0001.Data are represented as mean+SEM
Non-Asthma group (p b 0.01, p b 0.001, and p b 0.01, respectively) (Fig. 6). Significantly lower IL-4 and IL-5 mRNA expression levels in lung tissue were observed in the Lvn-Asthma group compared to the Control-Asthma group (p b 0.05 and p b 0.01, respectively). The Lvn-Asthma group tended to show lower IL-13 mRNA expression in lung tissue compared to the Control-Asthma group (no significance). Therefore, treatment with Lvn decreased the production of Th2 cytokines in BAL fluids and reduced mRNA expression in lung tissue. A significant increase in mRNA expression of Muc5ac and Muc5b was detected in the Control-Asthma group compared with the Non-Asthma group (p b 0.001). There was no significant difference in Muc5ac mRNA expression between the Lvn-Asthma group and the Control-Asthma group. The Lvn-Asthma group showed significantly lower Muc5b mRNA expression compared with the Control-Asthma group (p b 0.05). Treatment with Lvn decreased Muc5b mRNA expression in lung tissue. Discussion The results of this study revealed that Lvn suppressed AHR and accumulation of inflammatory cells in BAL fluids as well as in bronchial and perivascular tissues. In addition, Lvn attenuated the increase in Th2 cytokines in BAL fluids and their mRNA expression in lung tissue. Furthermore, Lvn suppressed mucous cell hyperplasia. Our findings indicate Table 2 IgE levels in serum. Groups IgE (ng/mL) Non-Asthma 9.66 ± 3.01 Control-Asthma 4545 ± 1611⁎ Lvn-Asthma 4397 ± 853.9⁎ Mean values ± SEM are given. Non-sensitized and ovalbumin (OVA)-challenged group, Non-Asthma; OVA-sensitized and OVAchallenged group, Control-Asthma; 20 μL of Lvn treated, OVAsensitized, and OVA-challenged group, Lvn-Asthma. ⁎ p b 0.05 compared with Non-Asthma (n = 5) and ControlAsthma (n = 5) or Lvn-Asthma (n = 5). Fig. 5. Detection of mucus-producing cells by periodic acid-Schiff (PAS) staining. The lung sections were stained by PAS staining and analyzed as indicated in Materials and methods. (a) Non-Asthma group (n = 7), (b) Control-Asthma group (n = 7), and (c) 20 μL Lvn-Asthma group (n = 7). The number of mucus-positive cells per mm of basement membrane (d) and PAS-stained area per mm of basement membrane (e) were measured in PAS-stained sections as described in Materials and methods. BM; basement membrane. Scale bar indicates 100 μm. *p b 0.05, **p b 0.01, ***p b 0.001, and ****p b 0.0001. Data are represented as mean ± SEM. Table 3 Cytokine levels in bronchoalveolar lavage fluids. Groups IL-4 (pg/mL) IL-5 (pg/mL) IL-13 (pg/mL) Non-Asthma 5.4 ± 3.4 16.6 ± 4.8 51.0 ± 1.7 Control-Asthma 11.7 ± 8.2 65.1 ± 4.7⁎ 144.8 ± 35.7⁎ Lvn-Asthma 0 28.3 ± 6.8## 64.6 ± 3.1# Mean values ± SEM are given. Non-sensitized and ovalbumin (OVA)-challenged group, Non-Asthma; OVA-sensitized and OVA-challenged group, Control-Asthma; 20 μL of Lvntreated, OVA-sensitized, and OVA-challenged group, Lvn-Asthma. ⁎ p b 0.05 compared with Non-Asthma (n = 5) and Control-Asthma (n = 5). # p b 0.05 compared with Control-Asthma and Lvn-Asthma (n = 6). ## p b 0.01 compared with Control-Asthma and Lvn-Asthma. T. Ueno-Iio et al. / Life Sciences 108 (2014) 109–115 113