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Baicalein, wogonin, and Scutellaria baicalensis ethanol extract alleviate ovalbumin-induced allergic airway inflammation and mast cell-mediated anaphylactic shock by regulation of Th1/Th2 imbalance and histamine release

Baicalein, wogonin, and Scutellaria baicalensis ethanol extract alleviate ovalbumin-induced... Original Article https://doi.org/10.5115/acb.2017.50.2.124 pISSN 2093-3665 eISSN 2093-3673 Baicalein, wogonin, and Scutellaria baicalensis ethanol extract alleviate ovalbumin-induced allergic airway inflammation and mast cell-mediated anaphylactic shock by regulation of Th1/Th2 imbalance and histamine release 1 1 1,2 3 4,5 1,2 Thi Tho Bui , Chun Hua Piao , Chang Ho Song , Chang-Hyun Lee , Hee Soon Shin , Ok Hee Chai 1 2 3 Department of Anatomy, Institute for Medical Sciences, Chonbuk National University Medical School, Jeonju, Department of Anatomy, College of 4 5 Korean Medicine, Woosuk University, Samrye, Food Biotechnology Program, Korea University of Science and Technology, Daejeon, Division of Nutrition and Metabolism Research, Korea Food Research Institute, Seongnam, Korea Abstract: Asthma is characterized by chronic inflammation, goblet cell hyperplasia, the aberrant production of the Th2 cytokines, and eosinophil infiltration into the lungs. In this study, we examined the effects of baicalein, wogonin, and Scutellaria baicalensis ethanol extract on ovalbumin (OVA)-induced asthma by evaluating Th1/Th2 cytokine levels, histopathologic analysis, and compound 48/80-induced systemic anaphylaxis and mast cell activation, focusing on the histamine release from rat peritoneal mast cells. Baicalein, wogonin, and S. baicalensis ethanol extract also decreased the number of inflammatory cells especially eosinophils and downregulated peribronchial and perivascular inflammation in the lungs of mice challenged by OVA. Baicalein, wogonin, and S. baicalensis ethanol extract significantly reduced the levels of tumor necrosis factor α, interleukin (IL)-1β, IL-4, IL-5 and the production of OVA-specific IgE and IgG1, and upregulated the level of interferon-γ and OVA-specific IgG2a. In addition, oral administration of baicalein, wogonin, and S. baicalensis ethanol extract inhibited compound 48/80-induced systemic anaphylaxis and plasma histamine release in mice. Moreover, baicalein, wogonin, and S. baicalensis ethanol extract suppressed compound 48/80-induced mast cell degranulation and histamine release from rat peritoneal mast cells. Conclusively, baicalein and wogonin as major flavonoids of S. baicalensis may have therapeutic potential for allergic asthma through modulation of Th1/Th2 cytokine imbalance and histamine release from mast cells. Key words: Baicalein, Wogonin, Scutellaria baicalensis, Th1/Th2 cytokine imbalance, OVA-specific IgE/IgG1/IgG2a Received February 6, 2017; Revised March 9, 2017; Accepted March 18, 2017 Introduction Bronchial asthma is an increasingly prevalent and often severe disease characterized by variable airway obstruction Corresponding author: in response to allergen, airway eosinophilic inflammation, Ok Hee Chai and airway hyperresponsiveness [1, 2]. Even though, allergic Department of Anatomy, Chonbuk National University Medical asthma is one of the most common chronic inflammatory School, 567 Baekje-daero, Deokjin-gu, Jeonju 54896, Korea Tel: +82-63-270-3109, Fax: +82-63-274-9880, E-mail: okchai1004@jbnu. disorders of the airways in children and adults. However, it is ac.kr not completely curable yet. Airway inflammation in asthma Copyright © 2017. Anatomy & Cell Biolog y This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Anat Cell Biol 2017;50:124-134 Anti-asthmatic effects of baicalein, wogonin, and Scutellaria baicalensis is associated with T-cell immune response in which Th2 cell Materials and Methods derived cytokines including interleukins (IL)-1 , IL-4, IL- 5, IL-6 and tumor necrosis factor (TNF- ) are thought to α α Materials contribute to eosinophil recruitment, mucus hypersecretion, OVA (grade VI), baicalein, and wogonin were purchase and airway hyperresponsiveness [3-5] by controlling the key from Sigma-Aldrich (St, Louis, MO, USA). S. baicalensis process of IgE production, the growth of mast cells and the ethanol extract was provided from the Korea Food Research differentiation and activation of mast cells and eosinophil [6- Institute (KFRI-SL-101). The ethanol extract was obtained 8]. Mast cells are widely known to contribute to the develop- by microwave extraction in 70% ethanol for 5 minutes, and ment of allergic airway disease. Mast cells are tissue cells that concentrated under vacuum in a rotary evaporator. The con- are located preferentially at the host-environment interface centrated extract was lyophilized and dissolved in saline prior and in proximity to blood vessels [9]. Mast cells are known to use. mainly for their being involved in mediating various harmful inflammatory reactions and can be activated to release potent Animals mediators by antibody-dependent mechanisms, and can re- Six-week-old male BALB/c mice for a murine asthma spond to very low dose of specific antigen [10-12]. model and 8-week-old male Sprague-Dawley rats for ac- Scutellaria baicalensis Georgi is one of the most popular quirement of RPMC were purchased from Damool Science and multi-purpose herbal medicines or medicinal plants used (Daejeon, Korea). These animals were housed in an air-con- in oriental countries including China, Japan, and Korea to ditioned room with a 12-hour light/dark cycle. All animal ex- treat inflammation, allergy, and bacterial and viral infections periments were performed in accordance with the guidelines [13-15]. Recently, investigations have shown that S. baicalen- for Animal Care and Use of Chonbuk National University sis has beneficial properties such as anti-oxidative effect [16], Laboratory Animal Center. and inhibits anti-dinitrophenyl IgE-medicated anaphylactic reactions and compound 48/80-induced histamine release Induction of murine asthma model and calcium uptake into rat peritoneal mast cells (RPMC) [15, Mice were divided into 6 groups according to treatment: (1) 17]. The flavonoids such as baicalein and wogonin isolated saline as a vehicle control, (2) OVA-induced asthma mice, (3) from S. baicalensis have also various biological activities such 1.25 mg/kg/day dexamethasone as reference drug for posi- as anti-oxidative, anti-inflammatory and anti-allergic effects tive control, (4) 10 mg/kg/day baicalein, (5) 10 mg/kg/day [18-23]. However, despite studies on anti-allergic effects of S. wogonin, and (6) 200 mg/kg/day S. baicalensis ethanol extract baicalensis have progressed extensively, little is known about in OVA-induced asthma mice. Mice of groups 2, 3, 4, 5, and the detailed effects of baicalein, wogonin, and S. baicalensis 6 were immunized by intraperitoneal injection of 50 g OVA ethanol extract on ovalbumin (OVA)-induced Th2 cytokine- with 1 mg Imject Alum (Thermo Scientific, Rockford, IL, dependent murine asthma model and compound 48/80-in- USA) in a total volume of 200 l on days 1 and by intraperi- duced systemic anaphylaxis in vivo and compound 48/80-in- toneal injection of 50 g OVA without Alum on 14. On days duced mast cell activation in vitro. 27, 28, and 29 after the beginning of the sensitization period, In this study, we evaluated the levels of IgE, OVA-specific these mice were challenged for 30 minutes with an aerosol IgE and IgG1, IgG2a and balance of Th1/Th2 cytokines of of 5% (wt/vol) OVA in saline using ultrasonic nebulization OVA-induced murine asthma model in vivo to reveal the (NE-U12, Omron Crop., Tokyo, Japan). On days 15 to 26, the anti-asthmatic effect of S. baicalensis ethanol extract and its treatment groups were also orally treated once daily with ba- major flavonoids, baicalein, and wogonin. To further prove icalein, wogonin, S. baicalensis ethanol extract or dexametha- the effects of them on mast cell-mediated immune responses, sone. Saline group and OVA-induced asthma group were the inhibitory effect of them on compound 48/80-induced received only saline. Animals were sacrificed 24 hours after systemic anaphylaxis and mast cell activation was evaluated the last challenge on day 30 to investigate the inhibitory effect focusing on the histamine release from mast cells. of baicalein, wogonin, and S. baicalensis ethanol extract. Collection and analysis of bronchoalveolar lavage fluid Twenty-four hours after the final OVA challenge, bron- www.acbjournal.org https://doi.org/10.5115/acb.2017.50.2.124 Anat Cell Biol 2017;50:124-134 Thi Tho Bui, et al choalveolar lavage fluid (BALF) was collected by cannulat- density gradient, as described in detail elsewhere [25]. RPMC ing the upper part of the trachea and lavaging, as described preparation was approximately 95% pure as assessed by tolu- previously [24]. The total number of viable cells in BALF was idine blue staining and at least 98% of these cells were viable determined by trypan blue exclusion using a hemocytometer. as assessed by trypan blue exclusion. Differential cell counts were determined with cytospin (Cen- trifuge 5403, Eppendorf, Hamburg, Germany) preparation, Observation of mast cell degranulation followed by Diff Quik staining (Sysmex Co., Kobe, Japan). Purified RPMC (1×10 cells/ml) were resuspended in HEPES-Tyrode buffer. The RPMC were pretreated with vari- ous concentrations of baicalein, wogonin, or S. baicalensis Histopathologic analysis Histopathologic analysis of lung was performed as previ- ethanol extract for 10 minutes at 37°C and observed for 10 ously described [24]. Lung were fixed in 10% formalin and minutes after addition of compound 48/80 (5 g/ml) un- embedded in paraffin. Serial 5 m thickness sections were der phase contrast microscopy and photographed [25]. The stained with congo red for eosinophils and inflammatory mast cells were classified (×1,000) as follows: (1) extensively cells, periodic acid-Schiff (PAS) for goblet cells and mucus degranulated (>50% of the cytoplasmic granules exhibiting and Masson trichrome for collagen fiber deposits. fusion, staining alterations and extrusion from the cell), (2) slight to moderately degranulated (10%–50% of the granules exhibiting fusion or discharge), or (3) normal. Measurement of Th1, Th2 cytokines and OVA-specific IgE, IgG1, and IgG2a The levels of Th1 cytokines such as interferon (IFN- ) γ γ Histamine assay and Th2-related cytokines such as TNF- , IL-4, and IL-6 RPMC (2×10 cells/well) were pre-incubated with various levels in the BALF from each mouse using the appropriate concentrations of baicalein, wogonin, or S. baicalensis etha- enzyme-linked immunosorbent assay (ELISA; BioSource In- nol extract at 37°C for 10 minutes and then incubated with ternational, Camarillo, CA, USA) were measured as described compound 48/80 (5 g/ml) for 30 minutes. The cells were earlier [24]. Also OVA-specific IgE, IgG1, and IgG2a were separated from the released histamine by centrifugation at measured according to the manufacturer’s instructions. 150 ×g for 10 minutes at 4°C. Residual histamine in the cells was released by boiling cells. After centrifugation, histamine content was measured by using ELISA. Induction of systemic anaphylaxis Mice intraperitoneally received 8 mg/kg body weight (BW) of mast cell degranulator Compound 48/80 or saline as previ- Statistical analysis ously described [17]. Baicalein (10 mg/kg BW), wogonin (10 Results were expressed as mean±SD for the number of mg/kg BW), or S. baicalensis ethanol extract (100, 200, and experiments. Student’s t test and ANOVA with Dunnett’s test 400 mg/kg BW) were dissolved in saline and administered were used for statistical comparison among the groups. Re- orally at 24, 12, and 1 hour prior to injection of compound sults with P<0.05 were considered statistically significant. 48/80 (n=20/group). Mortality was monitored for 1 hour af- ter induction of anaphylactic shock. After the mortality test, Results blood was obtained from the heart of each mouse. After cen- trifugation of blood from mouse heart, the plasma was with- Effect of baicalein, wogonin, and S. baicalensis drawn and histamine content was measured by ELISA kit. ethanol extract on infiltration of eosinophils and other inflammatory cells in BALF As shown in Fig. 1, the numbers of eosinophils, lympho- Preparation of RPMC RPMC were isolated as previously described [25]. Briefly, cytes, macrophages and total cells in BALF of mice treated rats were anesthetized with ether and peritoneally injected 10 with OVA was significantly increased compared with those of 2+ ml of Ca -free HEPES-Tyrode buffer, following which the mice treated with saline alone. However, these increases in the abdomen was gently massaged for approximately 90 seconds. numbers of eosinophils, lymphocytes, macrophages, and total The peritoneal cavity was opened, and fluid was aspirated cells were significantly reduced by the administration of ba- using a Pasteur pipette. RPMC were purified using a Percoll icalein, wogonin, or S. baicalensis ethanol extract. Dexameth- https://doi.org/10.5115/acb.2017.50.2.124 www.acbjournal.org Anat Cell Biol 2017;50:124-134 Anti-asthmatic effects of baicalein, wogonin, and Scutellaria baicalensis AB C Fig. 1. Effect of baicalein, wogonin, and Scutellaria baicalensis ethanol extract on DE F infiltration of inflammatory cells (A– F) and differential cellular components and total cells (G) in bronchoalveolar lavage fluid (BALF) of mice induced by ovalbumin (OVA) sensitization and Dexamethasone OVA Control challenge. (A) Control. (B) Saline. (C) Baicalein Wogonin Scutellaria baicalensis ### Dexamethasone 1.5 mg/kg. (D) Bai- 15 calein 10 mg/kg. (E) Wogonin 10 mg/ kg. (F) S. baicalensis ethanol extract 200 mg/kg. Values are presented as the ### *** 10 mean±SD (n=6 per group). Data were analyzed using ANOVA followed by ### # Student’s t test. P<0.001, P<0.05, *** *** *** *** sig ni ficantly different from the value *** *** * * of saline group. ***P<0.001, *P<0.05, *** *** *** ### sig ni ficantly different from the value *** *** of OVA group. Results are presented as Eosinophil Neutrophil Lymphocyte Macrophage Total cell the mean±SD (n=6 per group). Control OVA Dexamethasone Baicalein Wogonin Scutellaria baicalensis Fig. 2. Effect of baicalein, wogonin, and Scutellaria baicalensis ethanol extract on ovalbumin (OVA)-induced histopathological changes. Mice were sensitized on days 1 and 14, and challenged on days 27, 28, and 29 by OVA. On days 15 to 26, the treatment groups were also orally treated once daily with saline, dexamethasone 1.5 mg/kg, baicalein 10 mg/kg, wogonin 10 mg/kg, and S. baicalensis ethanol extract 200 mg/kg. Lung tissues from each group were stained with congo red for eosinophils, periodic acid-Schiff (PAS) for goblet cells (white arrows) and mucus (white asterisk) and Masson trichrome for collagen fiber deposits (black arrows). Only a representative picture is shown for each group. Scale bars=100 m. www.acbjournal.org https://doi.org/10.5115/acb.2017.50.2.124 Cells in BALF (x10 /ml) Trichrome PAS Congo red Anat Cell Biol 2017;50:124-134 Thi Tho Bui, et al asone also significantly inhibited the levels of eosinophils and of bronchioles in OVA-challenged mice compared to control inflammatory cells in BALF. mice (Fig. 2). However, mice treated with baicalein, wogonin, or S. baicalensis ethanol extract showed significantly reduc- tions in the thickening of the airway epithelium, the infiltra- Effect of baicalein, wogonin, and S. baicalensis ethanol tion of eosinophils around bronchioles and blood vessels, the extract on histopatholocial changes Histopathological analyses revealed typical pathological amount of mucus in the airway lumen (Fig. 2). This increased features of asthma in OVA-sensitized and -challenged mice. peribronchial and perivascular lung inflammation was mark- There are increased in the thickness of airway epithelium, edly alleviated by the administration of baicalein, wogonin, the infiltration of eosinophils around bronchioles and blood or S. baicalensis ethanol extract. Dexamethasone also signifi- vessels, the accumulation of mucus and debris in the lumen cantly decreased peribronchial and perivascular lung inflam- A B 25 3 ### ### ** ** ** ** ** 10 ** ** 0 0 C D 150 200 ### *** ** ** *** *** ### *** 0 0 Fig. 3. Effect of baicalein, wogonin, and Scutellaria baicalensis ethanol extract on the serum levels of total IgE (A), ovalbumin (OVA)-specific IgE ### (B), OVA-specific IgG1 (C), and OVA-specific IgG2 (D). Data were analyzed using ANOVA followed by Student’s t test. P<0.001, significantly different from the value of saline group. P<0.001, P<0.01, P<0.05, significantly different from the value of OVA group. Results are presented *** ** * as the mean±SD (n=6 per group). https://doi.org/10.5115/acb.2017.50.2.124 www.acbjournal.org Control OVA Dexamethasone Baicalein Wogonin Scutellaria baicalensis Control Control OVA OVA Dexamethasone Dexamethasone Baicalein Baicalein Wogonin Wogonin Scutellaria baicalensis Scutellaria baicalensis Control OVA Dexamethasone Baicalein Wogonin Scutellaria baicalensis OVA-specific lgG1 (mU/ml) Total lgE (pg/ml) OVA-specific lgG2a (mU/ml) OVA-specific lgE (pg/ml) Anat Cell Biol 2017;50:124-134 Anti-asthmatic effects of baicalein, wogonin, and Scutellaria baicalensis mation (Fig. 2). increased compared to control mice. The numbers of goblet cells with stained with PAS in airway epithelium was mark- edly higher in OVA-challenged mice than in control mice Effect of baicalein, wogonin, and S. baicalensis (Fig. 2). However, mice treated with baicalein, wogonin, or S. ethanol extract on mucus accumulation and goblet cell baicalensis ethanol extract significantly inhibited the accumu- hyperplasia Mucus hypersecretion which contributes significantly to lation of mucus and debris in the lumen of bronchioles, and airflow restriction is accompanied by goblet cell hyperplasia hyperplasia of goblet cells in the epithelium of bronchioles (Fig. 2). In OVA-challenged mice, the accumulation of mu- (Fig. 2). Dexamethasone also significantly suppressed the ac- cus and debris in the lumen of bronchioles, and hyperplasia cumulation of mucus and debris in the lumen of bronchioles, of goblet cells in the epithelium of bronchioles significantly and hyperplasia of goblet cells in the epithelium of bronchi- A B C 60 250 60 ### ### ## 150 * ** * 30 30 ** ** ** ** *** 0 0 0 D E F 2.5 150 150 ** * * 2.0 100 100 1.5 1.0 50 50 0.5 0 0 0 Fig. 4. Effect of baicalein, wogonin, and Scutellaria baicalensis ethanol extract on the levels of Th1/Th2 cytokines in bronchoalveolar lavage fluid. (A) Tumor necrosis factor α (TNF-α). (B) Interleukin (IL)-1β. (C) IL-4. (D) IL-5. (E) IL-12. (F) Interferon γ (IFN-γ). Data were analyzed using ### ## # ANOVA followed by Student’s t test. P<0.001, P<0.01, P<0.05, significantly different from the value of saline group. P<0.001, P<0.01, *** ** *P<0.05, significantly different from the value of ovalbumin (OVA) group. Results are presented as the mean±SD (n=6 per group). www.acbjournal.org https://doi.org/10.5115/acb.2017.50.2.124 Control OVA Dexamethasone Baicalein Wogonin Scutellaria baicalensis Control OVA Dexamethasone Baicalein Control Wogonin OVA Scutellaria baicalensis Dexamethasone Baicalein Wogonin Scutellaria baicalensis Control OVA Dexamethasone Baicalein Control Wogonin OVA Scutellaria baicalensis Dexamethasone Baicalein Wogonin Scutellaria baicalensis Control OVA Dexamethasone Baicalein Wogonin Scutellaria baicalensis IL-5 (pg/ml) TNF- (pg/ml) IL-12 (pg/ml) IL-1 (pg/ml) INF- (pg/ml) IL-4 (pg/ml) Anat Cell Biol 2017;50:124-134 Thi Tho Bui, et al oles (Fig. 2). with control mice. The administration of baicalein, wogonin, or S. baicalensis ethanol extract reduced the levels of total IgE and both the OVA-specific IgE and IgG1 in serum. However, Effect of baicalein, wogonin, and S. baicalensis ethanol neither baicalein, wogonin nor S. baicalensis ethanol extract extract on collagen deposit around bronchiole Collagen deposit among inflammatory cells around inhibited the production of OVA-specific IgG2a (Fig. 3D). bronchiole was significantly increased in the lung of OVA- challenged mice compared to that of control mice (Fig. 2). Effect of baicalein, wogonin, and S. baicalensis ethanol The treatment of baicalein, wogonin, or S. baicalensis ethanol extract on the levels of Th1/Th2 cytokines in BALF extract reduced the collagen deposit around bronchiole by Next, to further delineat the anti-asthmatic effects of ba- OVA. Dexamethasone also inhibited the levels of peribron- icalein, wogonin S. baicalensis ethanol extract, we investigated chial fibrosis. the levels of Th1/Th2 cytokines including IFN- , TNF- , IL- γ α 4, IL-5, and IL-6 that known to regulate IgE-mediated allergy and asthma [26, 27] in the BALF using appropriate ELISA. Effect of baicalein, wogonin, and S. baicalensis ethanol The levels of Th1 cytokines such as IFN- and IL-12 in BALF extract on the serum levels of total IgE and γ were decreased and the levels of Th2 cytokines IL-1 , IL- OVA-specific IgE, IgG1, and IgG2a β To investigate the underlying immunoregulatory mecha- 4, IL-5, and TNF- in BALF were significantly increased in nism of baicalein, wogonin, and S. baicalensis ethanol extract OVA-challenged compared with control mice (Fig. 4). The de- on anti-asthma, we detected serum levels of total IgE and creased levels of IFN- and IL-12 were significantly increased OVA-specific IgE, IgG1 (Th2 related Ig), and IgG2a (Th1 re- and the increased levels of IL-1 , IL-4, IL-5, and TNF- were β α lated Ig). As shown in Fig. 3, the levels of total IgE (Fig. 3A), reduced by the administration of baicalein, wogonin or S. ba- OVA-specific IgE (Fig. 3B), and OVA-specific IgG1 (Fig. 3C) icalensis ethanol extract, which were similar to the increment were markedly increased in OVA-challenged mice compared of IFN- and IL-12 and the reduction of IL-1 , IL-4, IL-5, and γ β A B Control Compound 48/80 Baicalein Wogonin 100 200 400 Scutellaria baicalensis (mg/kg) ### ** ** *** *** 0 0 0 51015202530354045505560 Times after compound 48/80 injection (min) Scutellaria baicalensis (mg/kg) Fig. 5. Inhibitory effect of baicalein, wogonin, and Scutellaria baicalensis ethanol extract on compound 48/80-induced systemic anaphylaxis. Five groups of mice (n=7/group) were orally administered saline, baicalein (10 mg/kg body weight [BW ]), wogonin (10 mg/kg BW ) or S. baicalensis ethanol extract (100, 200, and 400 mg/kg BW), at 24, 12, and 1 hour prior to injection of compound 48/80. The compound 48/80 was intraperitoneally given to mice. (A) Survival rates (%) were monitored for 1 hour after induction of anaphylactic shock. (B) Histamine concentrations in serum were measured by enzyme-linked immunosorbent assay. Each data represents the mean±SD (n=7 per group). Data were analyzed using ANOVA followed by Student’s t test. P<0.01, P<0.001, significantly different from the value of saline treated group. Results ** *** ### are presented as the mean±SD. P<0.001, significantly different from the value of saline group. https://doi.org/10.5115/acb.2017.50.2.124 www.acbjournal.org Control Compound 48/80 Baicalein Wogonin Survival (%) Histamine (ng/ml) Anat Cell Biol 2017;50:124-134 Anti-asthmatic effects of baicalein, wogonin, and Scutellaria baicalensis TNF- in BALF by dexamethasone (Fig. 4). pound 48/80-induced plasma histamine release in mice was investigated. The content of plasma histamine in mice treated with compound 48/80 was 315.7±21.8 ng/ml. As shown in Effect of baicalein, wogonin, or S. baicalensis ethanol Fig. 5B, the inhibition effects on histamine release by oral extract on compound 48/80-induced systemic administration of S. baicalensis ethanol extract was significant anaphylaxis To investigate the effect of S. baicalensis ethanol extract at doses of 200 to 400 mg/kg BW. In addition, baicalein and in anaphylactic shock, we first used the in vivo model of wogonin significantly decreased the plasma histamine release systemic anaphylaxis using compound 48/80. As shown in induced by compound 48/80 (Fig. 5B). Fig. 5, the intraperitoneal injection of compound 48/80 (8 mg/kg BW) resulted in 93.33% death of mice. However, oral Effect of baicalein, wogonin, and S. baicalensis ethanol administration of S. baicalensis ethanol extract (200 to 400 extract on rat mast cell degranulation and histamine mg/kg BW) reduced compound 48/80-induced mortality in release from RPMC dose-dependent manner (Fig. 5A). Also, baicalein (10 mg/kg Mast cells in asthma have been known to exert their patho- BW) and wogonin (10 mg/kg BW) significantly suppressed physiological role through histamine release by degranulation the compound 48/80-induced mortality (Fig. 5A). The effect [28]. To investigate whether baicalein, wogonin, or S. ba- of baicalein, wogonin S. baicalensis ethanol extract on com- icalensis ethanol extract directly suppressed histamine release AB C Fig. 6. Effect of baicalein, wogonin, and Scutellaria baicalensis ethanol extract on rat mast cell degranulation and his- ta mine release from rat peritoneal mast DE F cells (RPMCs). RPMCs were prein- cubated with dexamethasone, baic alein, wogonin, or S. baicalensis ex tract at 37°C G H for 10 minutes before in cu ba tion with 80 100 ### compound 48/80. The RPMCs were ### micrographed within 10 minutes after the addition of saline or compound 48/80 by using inverted phase contrast microscopy. (A) Saline as a control. (B) Compound 48/80 5 μg/ml. (C) Dexamethasone 1.5 mg/ml. (D) Baicalein 10 mg/ml. (E) ** *** *** ** Wogonin 10 mg/ml. (F) S. baicalensis ethanol extract 200 mg/ml. Scale bars=10 20 μm (A–F). (G) Quantitation of mast cell deg ra nulation. (H) Histamine release (%). Data were analyzed using ANOVA 0 0 ### followed by Student’s t test. P<0.001, sig ni fi cantly different from the value of saline group. ***P<0.001, **P<0.01, *P<0.05, significantly different from the value of ovalbumin group. Results are pre- sented as the mean±SD (n=6 per group). www.acbjournal.org https://doi.org/10.5115/acb.2017.50.2.124 Control Compound 48/80 Dexamethasone Baicalein Wogonin Scutellaria baicalensis Control Compound 48/80 Dexamethasone Baicalein Wogonin Scutellaria baicalensis Mast cell degranulation (%) Histamine release (%) Anat Cell Biol 2017;50:124-134 Thi Tho Bui, et al from RPMC, we used compound 48/80 that has been known [22-24]. Eosinophils are markers of allergic airway inflam- as a stimulator the degranulation of RPMC through perturba- mation in asthma and play a critical role in the pathogenesis tion of the membrane [29]. of asthma [29, 32]. It has been known that there are a correla- Compound 48/80-treated RPMC showed cell swelling, cy- tion between pulmonary eosinophilia and asthma, as well as toplasmic vacuoles, irregular membrane, and extruded gran- a correlation with the level of eosinophils in the BALF [24, ules near the cell surface and in the surrounding media that 29, 32]. Similar to some studies [18, 23], baicalein, wogonin, is referred as mast cell degranulation (Fig. 6B). The normal and S. baicalensis ethanol extract significantly inhibited the RPMC were generally oval or round in shape and were full of airway inflammation around bronchi and blood vessels, the many fine granules surrounding a prominent nucleus in their hyperplasia of goblet cells, the deposit of collagen fibers, the cytoplasm (Fig. 6A). However, pretreatment with baicalein, infiltration of inflammatory cells especially eosinophils in the wogonin or S. baicalensis ethanol extract inhibited RPMC OVA-induced asthmatic lung tissues. Also, we observed ba- degranulation by compound 48/80 (Fig. 6D–F), were similar icalein, wogonin, and S. baicalensis ethanol extract resulted in to normal RPMC. Dexamethasone also inhibited the mast cell a significant decrease in the number of eosinophils and lym- degranulation (Fig. 6C). Baicalein, wogonin, and S. baicalensis phocytes in BALF, these cells could be the targets of baicalein, ethanol extract might protect the lipid membrane via the in- wogonin and S. baicalensis. hibition of the perturbation induced by compound 48/80 (Fig. In murine asthma model, OVA challenges induced a sig- 6G). Moreover, baicalein, wogonin, and S. baicalensis ethanol nificant increase in the total serum IgE and OVA-specific extract diminished compound 48/80-induced histamine re- IgG1 and BALF IgE [33, 34]. Our present study showed oral lease from RPMC at concentrations used in this study (Fig. administration of baicalein, wogonin, and S. baicalensis etha- 6H). These results suggested that baicalein, wogonin, and S. nol extract markedly reduced the OVA-induced total IgE and baicalensis ethanol extract may alleviate compound 48/80-in- OVA-specific IgE in serum, which was similar to the reduc- duced histamine release from mast cells by the suppression of tion in dexamethasone treated mice. These results suggest mast cell degranulation. that baicalein and wogonin have therapeutic potential on the allergic asthma that developed in an IgE-dependent manner. Airway inflammation in asthma is associated with T-cell Discussion immune response in which Th2 cell derived cytokines includ- The radix of S. baicalensis has been used as a remedy in the ing IL-1 , IL-4, IL-5, and TNF- are thought to contribute β α traditional medicine due to its powerful anti-inflammatory to eosinophil recruitment, mucus hypersecretion, and airway activity and low toxicity to humans. S. baicalensis contains hyperresponsiveness [3-5] by controlling the key process of various components, and more than 60 flavonoids have IgE production, the growth of mast cells and the differentia- been identified from different sources of S. baicalensis [30, tion and activation of mast cells and eosinophil [6-8]. Asthma 31]. Many reports show that baicalein and wogonin are the and inflammation are associated with enhanced production of principal active components of S. baicalensis and have potent IL-4 and decreased production of IFN- . Compared with Th2 anti-cancer and anti-inflammatory effects [18-23, 31]. Here, cytokines, Th1 cytokines such as IFN- and IL-12 are associ- compared with their anti-inflammatory effects, we dem- ated to antagonism of Th2 cell responses and IgE synthesis to onstrated the anti-allergic and anti-anaphylactic effects of restrain the progress of asthma. In physiological condition, these flavonoids using OVA-induced asthma and compound immune responses of Th1 and Th2 cytokines maintain dy- 48/80-induced systemic anaphylaxis mice models and studied namic balance. Whenever this balance is disturbed, diseases with mast cell-mediated responses in vitro. will occur [35]. Accordingly, examination of levels of Th1/ In this study, we demonstrated the inhibitory effect of ba- Th2 cytokines is important index in the evaluation of asthma. icalein, wogonin and S. baicalensis ethanol extract on airway To confirm effects of baicalein, wogonin, and S. baicalensis inflammation using a classical asthmatic murine model in- ethanol extract on Th1/Th2 cytokines further, we examined duced by OVA. OVA-induced allergic asthma is well known the production of Th2 cytokines such as TNF- , IL-1 , IL- α β as a disease that results from chronic airway inflammation 4, and IL-5, and Th1 cytokines such as IFN- and IL-12. We typically associated with the infiltration of eosinophils, showed here that administration of baicalein, wogonin, and lymphocytes, and macrophages into the bronchial lumen S. baicalensis ethanol extract markedly reduced the produc- https://doi.org/10.5115/acb.2017.50.2.124 www.acbjournal.org Anat Cell Biol 2017;50:124-134 Anti-asthmatic effects of baicalein, wogonin, and Scutellaria baicalensis tion of IL-4 and IL-5, and also counteracted the OVA-induced production. These data suggest that baicalein, wogonin and production of IL-1 and TNF- , pro-inflammatory cyto- S. baicalensis ethanol extract may have preventive and thera- β α kines, induced by OVA challenges. Consistent with reduction peutic activities for Th2 type or mast cell-mediated allergic of the levels of Th2 cytokines, the infiltration of eosinophils diseases. were significantly suppressed in the BALF and lung tissues after treatment of baicalein or wogonin. However, oral ad- Acknowledgements ministration of baicalein, wogonin, and S. baicalensis ethanol extract enhanced the production of Th1 cytokines including This study was supported by research grants (E0121304-05) IFN- and IL-12. From these results, the imbalanced Th1/ from the Korea Food Research Institute and by Basic Science Th2 cytokines are confirmed to be a key factor for asthma Research Program through the National Research Foundation that increases airway inflammation. These results suggest that of Korea (NRF) funded by the Ministry of Education, Science baicalein and wogonin could regulate the balance of Th1/ and Technology (2012R1A1A303857). Th2 cytokines by suppression of the development of airway inflammation via shifting from a Th2 to Th1 response in the References OVA-induced asthma. However, detailed molecular and cel- 1. Walford HH, Doherty TA. 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J Immunol 1993;151: in Th2 cytokine-dependent asthma model through production 3460-6. https://doi.org/10.5115/acb.2017.50.2.124 www.acbjournal.org http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Anatomy & Cell Biology Pubmed Central

Baicalein, wogonin, and Scutellaria baicalensis ethanol extract alleviate ovalbumin-induced allergic airway inflammation and mast cell-mediated anaphylactic shock by regulation of Th1/Th2 imbalance and histamine release

Anatomy & Cell Biology , Volume 50 (2) – Jun 27, 2017

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Abstract

Original Article https://doi.org/10.5115/acb.2017.50.2.124 pISSN 2093-3665 eISSN 2093-3673 Baicalein, wogonin, and Scutellaria baicalensis ethanol extract alleviate ovalbumin-induced allergic airway inflammation and mast cell-mediated anaphylactic shock by regulation of Th1/Th2 imbalance and histamine release 1 1 1,2 3 4,5 1,2 Thi Tho Bui , Chun Hua Piao , Chang Ho Song , Chang-Hyun Lee , Hee Soon Shin , Ok Hee Chai 1 2 3 Department of Anatomy, Institute for Medical Sciences, Chonbuk National University Medical School, Jeonju, Department of Anatomy, College of 4 5 Korean Medicine, Woosuk University, Samrye, Food Biotechnology Program, Korea University of Science and Technology, Daejeon, Division of Nutrition and Metabolism Research, Korea Food Research Institute, Seongnam, Korea Abstract: Asthma is characterized by chronic inflammation, goblet cell hyperplasia, the aberrant production of the Th2 cytokines, and eosinophil infiltration into the lungs. In this study, we examined the effects of baicalein, wogonin, and Scutellaria baicalensis ethanol extract on ovalbumin (OVA)-induced asthma by evaluating Th1/Th2 cytokine levels, histopathologic analysis, and compound 48/80-induced systemic anaphylaxis and mast cell activation, focusing on the histamine release from rat peritoneal mast cells. Baicalein, wogonin, and S. baicalensis ethanol extract also decreased the number of inflammatory cells especially eosinophils and downregulated peribronchial and perivascular inflammation in the lungs of mice challenged by OVA. Baicalein, wogonin, and S. baicalensis ethanol extract significantly reduced the levels of tumor necrosis factor α, interleukin (IL)-1β, IL-4, IL-5 and the production of OVA-specific IgE and IgG1, and upregulated the level of interferon-γ and OVA-specific IgG2a. In addition, oral administration of baicalein, wogonin, and S. baicalensis ethanol extract inhibited compound 48/80-induced systemic anaphylaxis and plasma histamine release in mice. Moreover, baicalein, wogonin, and S. baicalensis ethanol extract suppressed compound 48/80-induced mast cell degranulation and histamine release from rat peritoneal mast cells. Conclusively, baicalein and wogonin as major flavonoids of S. baicalensis may have therapeutic potential for allergic asthma through modulation of Th1/Th2 cytokine imbalance and histamine release from mast cells. Key words: Baicalein, Wogonin, Scutellaria baicalensis, Th1/Th2 cytokine imbalance, OVA-specific IgE/IgG1/IgG2a Received February 6, 2017; Revised March 9, 2017; Accepted March 18, 2017 Introduction Bronchial asthma is an increasingly prevalent and often severe disease characterized by variable airway obstruction Corresponding author: in response to allergen, airway eosinophilic inflammation, Ok Hee Chai and airway hyperresponsiveness [1, 2]. Even though, allergic Department of Anatomy, Chonbuk National University Medical asthma is one of the most common chronic inflammatory School, 567 Baekje-daero, Deokjin-gu, Jeonju 54896, Korea Tel: +82-63-270-3109, Fax: +82-63-274-9880, E-mail: okchai1004@jbnu. disorders of the airways in children and adults. However, it is ac.kr not completely curable yet. Airway inflammation in asthma Copyright © 2017. Anatomy & Cell Biolog y This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Anat Cell Biol 2017;50:124-134 Anti-asthmatic effects of baicalein, wogonin, and Scutellaria baicalensis is associated with T-cell immune response in which Th2 cell Materials and Methods derived cytokines including interleukins (IL)-1 , IL-4, IL- 5, IL-6 and tumor necrosis factor (TNF- ) are thought to α α Materials contribute to eosinophil recruitment, mucus hypersecretion, OVA (grade VI), baicalein, and wogonin were purchase and airway hyperresponsiveness [3-5] by controlling the key from Sigma-Aldrich (St, Louis, MO, USA). S. baicalensis process of IgE production, the growth of mast cells and the ethanol extract was provided from the Korea Food Research differentiation and activation of mast cells and eosinophil [6- Institute (KFRI-SL-101). The ethanol extract was obtained 8]. Mast cells are widely known to contribute to the develop- by microwave extraction in 70% ethanol for 5 minutes, and ment of allergic airway disease. Mast cells are tissue cells that concentrated under vacuum in a rotary evaporator. The con- are located preferentially at the host-environment interface centrated extract was lyophilized and dissolved in saline prior and in proximity to blood vessels [9]. Mast cells are known to use. mainly for their being involved in mediating various harmful inflammatory reactions and can be activated to release potent Animals mediators by antibody-dependent mechanisms, and can re- Six-week-old male BALB/c mice for a murine asthma spond to very low dose of specific antigen [10-12]. model and 8-week-old male Sprague-Dawley rats for ac- Scutellaria baicalensis Georgi is one of the most popular quirement of RPMC were purchased from Damool Science and multi-purpose herbal medicines or medicinal plants used (Daejeon, Korea). These animals were housed in an air-con- in oriental countries including China, Japan, and Korea to ditioned room with a 12-hour light/dark cycle. All animal ex- treat inflammation, allergy, and bacterial and viral infections periments were performed in accordance with the guidelines [13-15]. Recently, investigations have shown that S. baicalen- for Animal Care and Use of Chonbuk National University sis has beneficial properties such as anti-oxidative effect [16], Laboratory Animal Center. and inhibits anti-dinitrophenyl IgE-medicated anaphylactic reactions and compound 48/80-induced histamine release Induction of murine asthma model and calcium uptake into rat peritoneal mast cells (RPMC) [15, Mice were divided into 6 groups according to treatment: (1) 17]. The flavonoids such as baicalein and wogonin isolated saline as a vehicle control, (2) OVA-induced asthma mice, (3) from S. baicalensis have also various biological activities such 1.25 mg/kg/day dexamethasone as reference drug for posi- as anti-oxidative, anti-inflammatory and anti-allergic effects tive control, (4) 10 mg/kg/day baicalein, (5) 10 mg/kg/day [18-23]. However, despite studies on anti-allergic effects of S. wogonin, and (6) 200 mg/kg/day S. baicalensis ethanol extract baicalensis have progressed extensively, little is known about in OVA-induced asthma mice. Mice of groups 2, 3, 4, 5, and the detailed effects of baicalein, wogonin, and S. baicalensis 6 were immunized by intraperitoneal injection of 50 g OVA ethanol extract on ovalbumin (OVA)-induced Th2 cytokine- with 1 mg Imject Alum (Thermo Scientific, Rockford, IL, dependent murine asthma model and compound 48/80-in- USA) in a total volume of 200 l on days 1 and by intraperi- duced systemic anaphylaxis in vivo and compound 48/80-in- toneal injection of 50 g OVA without Alum on 14. On days duced mast cell activation in vitro. 27, 28, and 29 after the beginning of the sensitization period, In this study, we evaluated the levels of IgE, OVA-specific these mice were challenged for 30 minutes with an aerosol IgE and IgG1, IgG2a and balance of Th1/Th2 cytokines of of 5% (wt/vol) OVA in saline using ultrasonic nebulization OVA-induced murine asthma model in vivo to reveal the (NE-U12, Omron Crop., Tokyo, Japan). On days 15 to 26, the anti-asthmatic effect of S. baicalensis ethanol extract and its treatment groups were also orally treated once daily with ba- major flavonoids, baicalein, and wogonin. To further prove icalein, wogonin, S. baicalensis ethanol extract or dexametha- the effects of them on mast cell-mediated immune responses, sone. Saline group and OVA-induced asthma group were the inhibitory effect of them on compound 48/80-induced received only saline. Animals were sacrificed 24 hours after systemic anaphylaxis and mast cell activation was evaluated the last challenge on day 30 to investigate the inhibitory effect focusing on the histamine release from mast cells. of baicalein, wogonin, and S. baicalensis ethanol extract. Collection and analysis of bronchoalveolar lavage fluid Twenty-four hours after the final OVA challenge, bron- www.acbjournal.org https://doi.org/10.5115/acb.2017.50.2.124 Anat Cell Biol 2017;50:124-134 Thi Tho Bui, et al choalveolar lavage fluid (BALF) was collected by cannulat- density gradient, as described in detail elsewhere [25]. RPMC ing the upper part of the trachea and lavaging, as described preparation was approximately 95% pure as assessed by tolu- previously [24]. The total number of viable cells in BALF was idine blue staining and at least 98% of these cells were viable determined by trypan blue exclusion using a hemocytometer. as assessed by trypan blue exclusion. Differential cell counts were determined with cytospin (Cen- trifuge 5403, Eppendorf, Hamburg, Germany) preparation, Observation of mast cell degranulation followed by Diff Quik staining (Sysmex Co., Kobe, Japan). Purified RPMC (1×10 cells/ml) were resuspended in HEPES-Tyrode buffer. The RPMC were pretreated with vari- ous concentrations of baicalein, wogonin, or S. baicalensis Histopathologic analysis Histopathologic analysis of lung was performed as previ- ethanol extract for 10 minutes at 37°C and observed for 10 ously described [24]. Lung were fixed in 10% formalin and minutes after addition of compound 48/80 (5 g/ml) un- embedded in paraffin. Serial 5 m thickness sections were der phase contrast microscopy and photographed [25]. The stained with congo red for eosinophils and inflammatory mast cells were classified (×1,000) as follows: (1) extensively cells, periodic acid-Schiff (PAS) for goblet cells and mucus degranulated (>50% of the cytoplasmic granules exhibiting and Masson trichrome for collagen fiber deposits. fusion, staining alterations and extrusion from the cell), (2) slight to moderately degranulated (10%–50% of the granules exhibiting fusion or discharge), or (3) normal. Measurement of Th1, Th2 cytokines and OVA-specific IgE, IgG1, and IgG2a The levels of Th1 cytokines such as interferon (IFN- ) γ γ Histamine assay and Th2-related cytokines such as TNF- , IL-4, and IL-6 RPMC (2×10 cells/well) were pre-incubated with various levels in the BALF from each mouse using the appropriate concentrations of baicalein, wogonin, or S. baicalensis etha- enzyme-linked immunosorbent assay (ELISA; BioSource In- nol extract at 37°C for 10 minutes and then incubated with ternational, Camarillo, CA, USA) were measured as described compound 48/80 (5 g/ml) for 30 minutes. The cells were earlier [24]. Also OVA-specific IgE, IgG1, and IgG2a were separated from the released histamine by centrifugation at measured according to the manufacturer’s instructions. 150 ×g for 10 minutes at 4°C. Residual histamine in the cells was released by boiling cells. After centrifugation, histamine content was measured by using ELISA. Induction of systemic anaphylaxis Mice intraperitoneally received 8 mg/kg body weight (BW) of mast cell degranulator Compound 48/80 or saline as previ- Statistical analysis ously described [17]. Baicalein (10 mg/kg BW), wogonin (10 Results were expressed as mean±SD for the number of mg/kg BW), or S. baicalensis ethanol extract (100, 200, and experiments. Student’s t test and ANOVA with Dunnett’s test 400 mg/kg BW) were dissolved in saline and administered were used for statistical comparison among the groups. Re- orally at 24, 12, and 1 hour prior to injection of compound sults with P<0.05 were considered statistically significant. 48/80 (n=20/group). Mortality was monitored for 1 hour af- ter induction of anaphylactic shock. After the mortality test, Results blood was obtained from the heart of each mouse. After cen- trifugation of blood from mouse heart, the plasma was with- Effect of baicalein, wogonin, and S. baicalensis drawn and histamine content was measured by ELISA kit. ethanol extract on infiltration of eosinophils and other inflammatory cells in BALF As shown in Fig. 1, the numbers of eosinophils, lympho- Preparation of RPMC RPMC were isolated as previously described [25]. Briefly, cytes, macrophages and total cells in BALF of mice treated rats were anesthetized with ether and peritoneally injected 10 with OVA was significantly increased compared with those of 2+ ml of Ca -free HEPES-Tyrode buffer, following which the mice treated with saline alone. However, these increases in the abdomen was gently massaged for approximately 90 seconds. numbers of eosinophils, lymphocytes, macrophages, and total The peritoneal cavity was opened, and fluid was aspirated cells were significantly reduced by the administration of ba- using a Pasteur pipette. RPMC were purified using a Percoll icalein, wogonin, or S. baicalensis ethanol extract. Dexameth- https://doi.org/10.5115/acb.2017.50.2.124 www.acbjournal.org Anat Cell Biol 2017;50:124-134 Anti-asthmatic effects of baicalein, wogonin, and Scutellaria baicalensis AB C Fig. 1. Effect of baicalein, wogonin, and Scutellaria baicalensis ethanol extract on DE F infiltration of inflammatory cells (A– F) and differential cellular components and total cells (G) in bronchoalveolar lavage fluid (BALF) of mice induced by ovalbumin (OVA) sensitization and Dexamethasone OVA Control challenge. (A) Control. (B) Saline. (C) Baicalein Wogonin Scutellaria baicalensis ### Dexamethasone 1.5 mg/kg. (D) Bai- 15 calein 10 mg/kg. (E) Wogonin 10 mg/ kg. (F) S. baicalensis ethanol extract 200 mg/kg. Values are presented as the ### *** 10 mean±SD (n=6 per group). Data were analyzed using ANOVA followed by ### # Student’s t test. P<0.001, P<0.05, *** *** *** *** sig ni ficantly different from the value *** *** * * of saline group. ***P<0.001, *P<0.05, *** *** *** ### sig ni ficantly different from the value *** *** of OVA group. Results are presented as Eosinophil Neutrophil Lymphocyte Macrophage Total cell the mean±SD (n=6 per group). Control OVA Dexamethasone Baicalein Wogonin Scutellaria baicalensis Fig. 2. Effect of baicalein, wogonin, and Scutellaria baicalensis ethanol extract on ovalbumin (OVA)-induced histopathological changes. Mice were sensitized on days 1 and 14, and challenged on days 27, 28, and 29 by OVA. On days 15 to 26, the treatment groups were also orally treated once daily with saline, dexamethasone 1.5 mg/kg, baicalein 10 mg/kg, wogonin 10 mg/kg, and S. baicalensis ethanol extract 200 mg/kg. Lung tissues from each group were stained with congo red for eosinophils, periodic acid-Schiff (PAS) for goblet cells (white arrows) and mucus (white asterisk) and Masson trichrome for collagen fiber deposits (black arrows). Only a representative picture is shown for each group. Scale bars=100 m. www.acbjournal.org https://doi.org/10.5115/acb.2017.50.2.124 Cells in BALF (x10 /ml) Trichrome PAS Congo red Anat Cell Biol 2017;50:124-134 Thi Tho Bui, et al asone also significantly inhibited the levels of eosinophils and of bronchioles in OVA-challenged mice compared to control inflammatory cells in BALF. mice (Fig. 2). However, mice treated with baicalein, wogonin, or S. baicalensis ethanol extract showed significantly reduc- tions in the thickening of the airway epithelium, the infiltra- Effect of baicalein, wogonin, and S. baicalensis ethanol tion of eosinophils around bronchioles and blood vessels, the extract on histopatholocial changes Histopathological analyses revealed typical pathological amount of mucus in the airway lumen (Fig. 2). This increased features of asthma in OVA-sensitized and -challenged mice. peribronchial and perivascular lung inflammation was mark- There are increased in the thickness of airway epithelium, edly alleviated by the administration of baicalein, wogonin, the infiltration of eosinophils around bronchioles and blood or S. baicalensis ethanol extract. Dexamethasone also signifi- vessels, the accumulation of mucus and debris in the lumen cantly decreased peribronchial and perivascular lung inflam- A B 25 3 ### ### ** ** ** ** ** 10 ** ** 0 0 C D 150 200 ### *** ** ** *** *** ### *** 0 0 Fig. 3. Effect of baicalein, wogonin, and Scutellaria baicalensis ethanol extract on the serum levels of total IgE (A), ovalbumin (OVA)-specific IgE ### (B), OVA-specific IgG1 (C), and OVA-specific IgG2 (D). Data were analyzed using ANOVA followed by Student’s t test. P<0.001, significantly different from the value of saline group. P<0.001, P<0.01, P<0.05, significantly different from the value of OVA group. Results are presented *** ** * as the mean±SD (n=6 per group). https://doi.org/10.5115/acb.2017.50.2.124 www.acbjournal.org Control OVA Dexamethasone Baicalein Wogonin Scutellaria baicalensis Control Control OVA OVA Dexamethasone Dexamethasone Baicalein Baicalein Wogonin Wogonin Scutellaria baicalensis Scutellaria baicalensis Control OVA Dexamethasone Baicalein Wogonin Scutellaria baicalensis OVA-specific lgG1 (mU/ml) Total lgE (pg/ml) OVA-specific lgG2a (mU/ml) OVA-specific lgE (pg/ml) Anat Cell Biol 2017;50:124-134 Anti-asthmatic effects of baicalein, wogonin, and Scutellaria baicalensis mation (Fig. 2). increased compared to control mice. The numbers of goblet cells with stained with PAS in airway epithelium was mark- edly higher in OVA-challenged mice than in control mice Effect of baicalein, wogonin, and S. baicalensis (Fig. 2). However, mice treated with baicalein, wogonin, or S. ethanol extract on mucus accumulation and goblet cell baicalensis ethanol extract significantly inhibited the accumu- hyperplasia Mucus hypersecretion which contributes significantly to lation of mucus and debris in the lumen of bronchioles, and airflow restriction is accompanied by goblet cell hyperplasia hyperplasia of goblet cells in the epithelium of bronchioles (Fig. 2). In OVA-challenged mice, the accumulation of mu- (Fig. 2). Dexamethasone also significantly suppressed the ac- cus and debris in the lumen of bronchioles, and hyperplasia cumulation of mucus and debris in the lumen of bronchioles, of goblet cells in the epithelium of bronchioles significantly and hyperplasia of goblet cells in the epithelium of bronchi- A B C 60 250 60 ### ### ## 150 * ** * 30 30 ** ** ** ** *** 0 0 0 D E F 2.5 150 150 ** * * 2.0 100 100 1.5 1.0 50 50 0.5 0 0 0 Fig. 4. Effect of baicalein, wogonin, and Scutellaria baicalensis ethanol extract on the levels of Th1/Th2 cytokines in bronchoalveolar lavage fluid. (A) Tumor necrosis factor α (TNF-α). (B) Interleukin (IL)-1β. (C) IL-4. (D) IL-5. (E) IL-12. (F) Interferon γ (IFN-γ). Data were analyzed using ### ## # ANOVA followed by Student’s t test. P<0.001, P<0.01, P<0.05, significantly different from the value of saline group. P<0.001, P<0.01, *** ** *P<0.05, significantly different from the value of ovalbumin (OVA) group. Results are presented as the mean±SD (n=6 per group). www.acbjournal.org https://doi.org/10.5115/acb.2017.50.2.124 Control OVA Dexamethasone Baicalein Wogonin Scutellaria baicalensis Control OVA Dexamethasone Baicalein Control Wogonin OVA Scutellaria baicalensis Dexamethasone Baicalein Wogonin Scutellaria baicalensis Control OVA Dexamethasone Baicalein Control Wogonin OVA Scutellaria baicalensis Dexamethasone Baicalein Wogonin Scutellaria baicalensis Control OVA Dexamethasone Baicalein Wogonin Scutellaria baicalensis IL-5 (pg/ml) TNF- (pg/ml) IL-12 (pg/ml) IL-1 (pg/ml) INF- (pg/ml) IL-4 (pg/ml) Anat Cell Biol 2017;50:124-134 Thi Tho Bui, et al oles (Fig. 2). with control mice. The administration of baicalein, wogonin, or S. baicalensis ethanol extract reduced the levels of total IgE and both the OVA-specific IgE and IgG1 in serum. However, Effect of baicalein, wogonin, and S. baicalensis ethanol neither baicalein, wogonin nor S. baicalensis ethanol extract extract on collagen deposit around bronchiole Collagen deposit among inflammatory cells around inhibited the production of OVA-specific IgG2a (Fig. 3D). bronchiole was significantly increased in the lung of OVA- challenged mice compared to that of control mice (Fig. 2). Effect of baicalein, wogonin, and S. baicalensis ethanol The treatment of baicalein, wogonin, or S. baicalensis ethanol extract on the levels of Th1/Th2 cytokines in BALF extract reduced the collagen deposit around bronchiole by Next, to further delineat the anti-asthmatic effects of ba- OVA. Dexamethasone also inhibited the levels of peribron- icalein, wogonin S. baicalensis ethanol extract, we investigated chial fibrosis. the levels of Th1/Th2 cytokines including IFN- , TNF- , IL- γ α 4, IL-5, and IL-6 that known to regulate IgE-mediated allergy and asthma [26, 27] in the BALF using appropriate ELISA. Effect of baicalein, wogonin, and S. baicalensis ethanol The levels of Th1 cytokines such as IFN- and IL-12 in BALF extract on the serum levels of total IgE and γ were decreased and the levels of Th2 cytokines IL-1 , IL- OVA-specific IgE, IgG1, and IgG2a β To investigate the underlying immunoregulatory mecha- 4, IL-5, and TNF- in BALF were significantly increased in nism of baicalein, wogonin, and S. baicalensis ethanol extract OVA-challenged compared with control mice (Fig. 4). The de- on anti-asthma, we detected serum levels of total IgE and creased levels of IFN- and IL-12 were significantly increased OVA-specific IgE, IgG1 (Th2 related Ig), and IgG2a (Th1 re- and the increased levels of IL-1 , IL-4, IL-5, and TNF- were β α lated Ig). As shown in Fig. 3, the levels of total IgE (Fig. 3A), reduced by the administration of baicalein, wogonin or S. ba- OVA-specific IgE (Fig. 3B), and OVA-specific IgG1 (Fig. 3C) icalensis ethanol extract, which were similar to the increment were markedly increased in OVA-challenged mice compared of IFN- and IL-12 and the reduction of IL-1 , IL-4, IL-5, and γ β A B Control Compound 48/80 Baicalein Wogonin 100 200 400 Scutellaria baicalensis (mg/kg) ### ** ** *** *** 0 0 0 51015202530354045505560 Times after compound 48/80 injection (min) Scutellaria baicalensis (mg/kg) Fig. 5. Inhibitory effect of baicalein, wogonin, and Scutellaria baicalensis ethanol extract on compound 48/80-induced systemic anaphylaxis. Five groups of mice (n=7/group) were orally administered saline, baicalein (10 mg/kg body weight [BW ]), wogonin (10 mg/kg BW ) or S. baicalensis ethanol extract (100, 200, and 400 mg/kg BW), at 24, 12, and 1 hour prior to injection of compound 48/80. The compound 48/80 was intraperitoneally given to mice. (A) Survival rates (%) were monitored for 1 hour after induction of anaphylactic shock. (B) Histamine concentrations in serum were measured by enzyme-linked immunosorbent assay. Each data represents the mean±SD (n=7 per group). Data were analyzed using ANOVA followed by Student’s t test. P<0.01, P<0.001, significantly different from the value of saline treated group. Results ** *** ### are presented as the mean±SD. P<0.001, significantly different from the value of saline group. https://doi.org/10.5115/acb.2017.50.2.124 www.acbjournal.org Control Compound 48/80 Baicalein Wogonin Survival (%) Histamine (ng/ml) Anat Cell Biol 2017;50:124-134 Anti-asthmatic effects of baicalein, wogonin, and Scutellaria baicalensis TNF- in BALF by dexamethasone (Fig. 4). pound 48/80-induced plasma histamine release in mice was investigated. The content of plasma histamine in mice treated with compound 48/80 was 315.7±21.8 ng/ml. As shown in Effect of baicalein, wogonin, or S. baicalensis ethanol Fig. 5B, the inhibition effects on histamine release by oral extract on compound 48/80-induced systemic administration of S. baicalensis ethanol extract was significant anaphylaxis To investigate the effect of S. baicalensis ethanol extract at doses of 200 to 400 mg/kg BW. In addition, baicalein and in anaphylactic shock, we first used the in vivo model of wogonin significantly decreased the plasma histamine release systemic anaphylaxis using compound 48/80. As shown in induced by compound 48/80 (Fig. 5B). Fig. 5, the intraperitoneal injection of compound 48/80 (8 mg/kg BW) resulted in 93.33% death of mice. However, oral Effect of baicalein, wogonin, and S. baicalensis ethanol administration of S. baicalensis ethanol extract (200 to 400 extract on rat mast cell degranulation and histamine mg/kg BW) reduced compound 48/80-induced mortality in release from RPMC dose-dependent manner (Fig. 5A). Also, baicalein (10 mg/kg Mast cells in asthma have been known to exert their patho- BW) and wogonin (10 mg/kg BW) significantly suppressed physiological role through histamine release by degranulation the compound 48/80-induced mortality (Fig. 5A). The effect [28]. To investigate whether baicalein, wogonin, or S. ba- of baicalein, wogonin S. baicalensis ethanol extract on com- icalensis ethanol extract directly suppressed histamine release AB C Fig. 6. Effect of baicalein, wogonin, and Scutellaria baicalensis ethanol extract on rat mast cell degranulation and his- ta mine release from rat peritoneal mast DE F cells (RPMCs). RPMCs were prein- cubated with dexamethasone, baic alein, wogonin, or S. baicalensis ex tract at 37°C G H for 10 minutes before in cu ba tion with 80 100 ### compound 48/80. The RPMCs were ### micrographed within 10 minutes after the addition of saline or compound 48/80 by using inverted phase contrast microscopy. (A) Saline as a control. (B) Compound 48/80 5 μg/ml. (C) Dexamethasone 1.5 mg/ml. (D) Baicalein 10 mg/ml. (E) ** *** *** ** Wogonin 10 mg/ml. (F) S. baicalensis ethanol extract 200 mg/ml. Scale bars=10 20 μm (A–F). (G) Quantitation of mast cell deg ra nulation. (H) Histamine release (%). Data were analyzed using ANOVA 0 0 ### followed by Student’s t test. P<0.001, sig ni fi cantly different from the value of saline group. ***P<0.001, **P<0.01, *P<0.05, significantly different from the value of ovalbumin group. Results are pre- sented as the mean±SD (n=6 per group). www.acbjournal.org https://doi.org/10.5115/acb.2017.50.2.124 Control Compound 48/80 Dexamethasone Baicalein Wogonin Scutellaria baicalensis Control Compound 48/80 Dexamethasone Baicalein Wogonin Scutellaria baicalensis Mast cell degranulation (%) Histamine release (%) Anat Cell Biol 2017;50:124-134 Thi Tho Bui, et al from RPMC, we used compound 48/80 that has been known [22-24]. Eosinophils are markers of allergic airway inflam- as a stimulator the degranulation of RPMC through perturba- mation in asthma and play a critical role in the pathogenesis tion of the membrane [29]. of asthma [29, 32]. It has been known that there are a correla- Compound 48/80-treated RPMC showed cell swelling, cy- tion between pulmonary eosinophilia and asthma, as well as toplasmic vacuoles, irregular membrane, and extruded gran- a correlation with the level of eosinophils in the BALF [24, ules near the cell surface and in the surrounding media that 29, 32]. Similar to some studies [18, 23], baicalein, wogonin, is referred as mast cell degranulation (Fig. 6B). The normal and S. baicalensis ethanol extract significantly inhibited the RPMC were generally oval or round in shape and were full of airway inflammation around bronchi and blood vessels, the many fine granules surrounding a prominent nucleus in their hyperplasia of goblet cells, the deposit of collagen fibers, the cytoplasm (Fig. 6A). However, pretreatment with baicalein, infiltration of inflammatory cells especially eosinophils in the wogonin or S. baicalensis ethanol extract inhibited RPMC OVA-induced asthmatic lung tissues. Also, we observed ba- degranulation by compound 48/80 (Fig. 6D–F), were similar icalein, wogonin, and S. baicalensis ethanol extract resulted in to normal RPMC. Dexamethasone also inhibited the mast cell a significant decrease in the number of eosinophils and lym- degranulation (Fig. 6C). Baicalein, wogonin, and S. baicalensis phocytes in BALF, these cells could be the targets of baicalein, ethanol extract might protect the lipid membrane via the in- wogonin and S. baicalensis. hibition of the perturbation induced by compound 48/80 (Fig. In murine asthma model, OVA challenges induced a sig- 6G). Moreover, baicalein, wogonin, and S. baicalensis ethanol nificant increase in the total serum IgE and OVA-specific extract diminished compound 48/80-induced histamine re- IgG1 and BALF IgE [33, 34]. Our present study showed oral lease from RPMC at concentrations used in this study (Fig. administration of baicalein, wogonin, and S. baicalensis etha- 6H). These results suggested that baicalein, wogonin, and S. nol extract markedly reduced the OVA-induced total IgE and baicalensis ethanol extract may alleviate compound 48/80-in- OVA-specific IgE in serum, which was similar to the reduc- duced histamine release from mast cells by the suppression of tion in dexamethasone treated mice. These results suggest mast cell degranulation. that baicalein and wogonin have therapeutic potential on the allergic asthma that developed in an IgE-dependent manner. Airway inflammation in asthma is associated with T-cell Discussion immune response in which Th2 cell derived cytokines includ- The radix of S. baicalensis has been used as a remedy in the ing IL-1 , IL-4, IL-5, and TNF- are thought to contribute β α traditional medicine due to its powerful anti-inflammatory to eosinophil recruitment, mucus hypersecretion, and airway activity and low toxicity to humans. S. baicalensis contains hyperresponsiveness [3-5] by controlling the key process of various components, and more than 60 flavonoids have IgE production, the growth of mast cells and the differentia- been identified from different sources of S. baicalensis [30, tion and activation of mast cells and eosinophil [6-8]. Asthma 31]. Many reports show that baicalein and wogonin are the and inflammation are associated with enhanced production of principal active components of S. baicalensis and have potent IL-4 and decreased production of IFN- . Compared with Th2 anti-cancer and anti-inflammatory effects [18-23, 31]. Here, cytokines, Th1 cytokines such as IFN- and IL-12 are associ- compared with their anti-inflammatory effects, we dem- ated to antagonism of Th2 cell responses and IgE synthesis to onstrated the anti-allergic and anti-anaphylactic effects of restrain the progress of asthma. In physiological condition, these flavonoids using OVA-induced asthma and compound immune responses of Th1 and Th2 cytokines maintain dy- 48/80-induced systemic anaphylaxis mice models and studied namic balance. Whenever this balance is disturbed, diseases with mast cell-mediated responses in vitro. will occur [35]. Accordingly, examination of levels of Th1/ In this study, we demonstrated the inhibitory effect of ba- Th2 cytokines is important index in the evaluation of asthma. icalein, wogonin and S. baicalensis ethanol extract on airway To confirm effects of baicalein, wogonin, and S. baicalensis inflammation using a classical asthmatic murine model in- ethanol extract on Th1/Th2 cytokines further, we examined duced by OVA. OVA-induced allergic asthma is well known the production of Th2 cytokines such as TNF- , IL-1 , IL- α β as a disease that results from chronic airway inflammation 4, and IL-5, and Th1 cytokines such as IFN- and IL-12. We typically associated with the infiltration of eosinophils, showed here that administration of baicalein, wogonin, and lymphocytes, and macrophages into the bronchial lumen S. baicalensis ethanol extract markedly reduced the produc- https://doi.org/10.5115/acb.2017.50.2.124 www.acbjournal.org Anat Cell Biol 2017;50:124-134 Anti-asthmatic effects of baicalein, wogonin, and Scutellaria baicalensis tion of IL-4 and IL-5, and also counteracted the OVA-induced production. These data suggest that baicalein, wogonin and production of IL-1 and TNF- , pro-inflammatory cyto- S. baicalensis ethanol extract may have preventive and thera- β α kines, induced by OVA challenges. Consistent with reduction peutic activities for Th2 type or mast cell-mediated allergic of the levels of Th2 cytokines, the infiltration of eosinophils diseases. were significantly suppressed in the BALF and lung tissues after treatment of baicalein or wogonin. However, oral ad- Acknowledgements ministration of baicalein, wogonin, and S. baicalensis ethanol extract enhanced the production of Th1 cytokines including This study was supported by research grants (E0121304-05) IFN- and IL-12. From these results, the imbalanced Th1/ from the Korea Food Research Institute and by Basic Science Th2 cytokines are confirmed to be a key factor for asthma Research Program through the National Research Foundation that increases airway inflammation. These results suggest that of Korea (NRF) funded by the Ministry of Education, Science baicalein and wogonin could regulate the balance of Th1/ and Technology (2012R1A1A303857). Th2 cytokines by suppression of the development of airway inflammation via shifting from a Th2 to Th1 response in the References OVA-induced asthma. However, detailed molecular and cel- 1. Walford HH, Doherty TA. 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Published: Jun 27, 2017

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