Effects of butylphthalide on bronchial asthma in guinea pigs and involvement of endothelin

Zhi-wang WANG, Xiao-yan FU, Yuan REN, Hai-jing DUAN, Xue-feng LIU, Xiao-li CHENG

Effects of butylphthalide on bronchial asthma in guinea pigs and involvement of endothelin

Zhi-wang WANG1,2, Xiao-yan FU1, Yuan REN1,2, Hai-jing DUAN1, Xue-feng LIU1, Xiao-li CHENG1

1. Gansu University of Traditional Chinese Medicine, Lanzhou 730000, China; 2. Key Laboratory of Pharmacology and Toxicology for Traditional Chinese Medicine of Gansu Province, Lanzhou 730000, China

Objecti ve:To study the eff ects of butylphthalide on bronchial asthma in guinea pigs, and investi -gate the involvement of endothelin.Methods:In guinea pigs, bronchial asthma was induced by injecti on of ovalbumin (OVA) and provoked by inhalati on of OVA, and the eff ects of butylphthalide on asthma were evaluated through the changes it induced by OVA, pulmonary functi on, endothelin-1 (ET-1) contents and acti vity of endothelin converti ng enzyme-1 (ECE-1) in bronchoalveolar lavage fl uid (BALF), serum and lung ti ssue, and the gene expression of ET-1 in lung ti ssue.Results:Butylphthalide signifi cantly improved pulmonary functi on, lowered asthmati c behavior score, inhibited the acti vity of ECE-1, and reduced ET-1 gene expression level in lung ti ssue.Conclusion:Butylphthalide has an anti -asthma eff ect and the mechanisms involve inhibiti on of ECE-1 acti vity and lowering of ET-1geng expression.

butylphthalide; anti -asthma; guinea pig; endothelin-1 (ET-1); endothelin converti ng enzyme-1 (ECE-1)

Introduction

“Shennong’s Herbal Classic”, the classical works of Chinese Medicine, states that Angelica sinensis (Oliv.) Diels. has the function of relieving cough and heavy pant. Under the theory of traditional Chinese Medicine, it is widely used in the prevention and treatment of respiratory diseases in the form of compound, such as Suzi Jiangqi Decoction, Baihe Gujin Tang, Jinshui Liujun Jian and so on. Pharmacodynamic study shows that Angelica sinensis (Oliv.) Diels and its volatile oil have the exact treatment effect on bronchial asthma， and butylphthalide, one of phthalide compounds in Angelica, has anti-spasm effect in vitro on tracheal smooth muscle of guinea pig [1,2]. On the basis of the above research we speculate that butylphthalide should have therapeutic effects on asthma. In this experiment, we intended to study for the first time whether butylphthalide has anti-asthmatic action and eff ect on endothelium to the asthmatic model in guinea pigs duplicated by ovalbumin.

Materials and Methods

Animals and experimental grouping

A total of 54 guinea pigs (provided by Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, SCXK (Gan) 2010-0001) weighing (180±20)g were used in this study. The guinea pigs were accommodated in the experimental environment for 7 days in SPF grade laboratory in Gansu University of Traditional Chinese Medicine (SCXK (Gan) 2011-0001). The room temperature and humidity were maintained at 22°C-24°C and 55%-65% with 12h light and dark cycle. According to the gender and body weight, the guinea pigs were hierarchically and randomly divided into blank control group (BC), asthma model group (AM), dexamethasone group (DXM, 1 mg/kg), butylphthalide high dose group (BH, 120 mg/kg), butylphthalide middle dose group (BM, 60 mg/kg), butylphthalide low dose group (BL, 30 mg/kg) with 9 guinea pigs in each group.

Experiment methods

On the first and eighth day (a total of 2 times), the guinea pigs were hypodermically and intraperitoneally injected 1.0ml sensitizing solution (including OVA 100μg and aluminum hydroxide 10mg) totally each time to sensitize, and then inhaled

atomized 1% OVA solution daily for 25 min, at a speed of 1.0 ml/min, with every day from 15th day to 21st day to excite. The guinea pigs in the BC group were handled with normal saline [3]. Before the excitation 1h each time, the guinea pigs in the DXM group, BH group, BM group and BL group were given corresponding drugs through lavage once daily, and the guinea pigs in the BC group and AM group were treated with blank control solution (1% Twain-80).

Indexes and examination methods

On the 21st day, after inhalation of OVA, the guinea pigs were observed for 35 minutes to obtain a behavior score by counting tickling and nose grabbing and scoring asthma attack. Specifi c scoring criteria: The guinea pigs do not tickle or grab nose recorded as 0 points, the number of tickling or grabbing noses recorded is 1-3 as 1 points, the number of tickling or grabbing noses recorded is 4-6 as 2 points, and the number of tickling or grabbing noses recorded is above 7 as 3 points; the guinea pigs without asthma symptoms record 0 points, the guinea pigs have mild asthma symptoms record 3 points, the guinea pigs have shortness of breath, respiratory frequency increased or anxiety and other asthmatic symptoms record 6 points, the guinea pigs have severe respiratory problems such as respiration accompanied by rapid nod, abdominal breathing and other asthmatic symptoms record 9 points [4]. The guinea pigs were placed in the lung function instrument (EMKA, Beijing Guangyuanda science and Technology Development Co., Ltd.) to measure lung function after excitation four hours when experiments were carried out to the 21st day. At the end of animal experiment, aft er anesthetized by the intraperitoneal administration of 50 mg/kg pentobarbital solution the guinea pigs were collected blood from femoral vein, and after centrifuging (3 000 r/min×10 min) the serum were collected to mensurate ET-1. On the basis of consulting relative experimental methods the BALF was drawn and centrifuged (3 000 r/min×10 min) to obtain supernatant to determine ET-1 [2]. The lung without lavage was removed and cut into two parts in fixed position. The first part of lung tissue was observed the expression of ET-1 and ECE-1 under microscope aft er formalin fi xation, biopsy and immunohistochemical staining and then the average optical density (OD) was calculated through medical image analysis system. Th e second part of lung tissue was used to determine the expression of ET-1 mRNA. As a template for real-time fl uorescence quantitative RT-PCR analysis, the first-strand cDNA was synthesized after the total RNA was extracted from lung tissue and judged its concentration and purity. Established 10 μl reaction system, the expression of ET-1 mRNA was determined. Th e upstream primer of β-Actin is 5'-ACTCTCCACCTTCCTAGCAGA-3', and the downstream primer of it is 5'-CACCTTCACCGTTCTCAGTTT-3'. The upstream primer of ET-1 is 5'-GGAGTGCGTCTACTTCTGCC-3', and the downstream primer of it is 5'-TTCTTGTGGTTATCCCAGCC-3'. The fluorescence intensity of ET-1 and β-Actin was detected, and aft er calculating 2-ΔΔCtthe diff erence of ET-1 expression was compared between groups.

Statistical analysis

The data of quantity response were expressed as“mean ± SD”, and analyzed the statistical signifi cance with the one-way ANOVA or with Ridit if the data were the grade data. Th e data of qualitative response were analyzed with Chi square test and P<0.05 indicated that there was statistical signifi cance.

Results

Effect of butylphthalide on asthmatic behaviors in guinea pigs during the process of bronchial provocation test

Cough, phlegm, and asthma are clinical symptoms of bronchial asthma, and the guinea pigs appear tickling, grabbing nose and asthma attack during replicating the model of allergic asthma. On the 21st day it was found that the number of asthmatic guinea pigs and the complex score of AM group were markedly increased compared with BC group (P<0.01) during and after inhaled 1% OVA. Under interference of butylphthalide, the above-mentioned abnormal changes had been greatly alleviated. (P<0.05, 0.01, Tab.1).

Effect of butylphthalide on lung function in asthmatic guinea pigs

Aft er repeated stimulation by OVA, respiratory rate (F) and enhanced expiratory pause (Penh) obviously increased and tidal volume (TV) declined sharply compared with the BC group (P<0.01). Compared with the AM group, the anomalous changes of BH group and BM group in the lung function indexes above were obviously relieved under the action of butylphthalide (P<0.05, 0.01, Tab.2).

Tab. 1 Effect of butylphthalide on asthmatic behaviors in guinea pigs during the process of bronchial provocation test (mean±SD, n=9).

Tab. 2 Effect of butylphthalide on lung function in asthmatic guinea pigs (mean±SD, n=9).

Effect of butylphthalide on ET-1 in BALF and serum of asthmatic guinea pigs

The data in the Table 3 were displayed that the expression of ET-1 significantly increased after sensitization and excitation with OVA, and ET-1 in BALF and serum of three dose groups of butylphthalide emerged a clear downward trend compared with AM group (P<0.05, 0.01).

Effect of butylphthalide on ET-1 in lung tissue of asthmatic guinea pigs

Th e positive reactive product of ET-1 in lung tissue of asthmatic guinea pigs showed brownish blue in cytoplasm and distributed mainly in smooth muscle cells of bronchial tube wall, mucosal epithelial cells, alveolar epithelial cells and vascular endothelial cells. The expression of ET-1 in AM group was obvious (Fig.1A) but that in butylphthalide groups was lighter to some degree (Fig.1D, Fig.1E, Fig.1F). Semiquantitative analysis results were also shown that the average optical density of ET-1 in butylphthalide groups had significantly descendent tendency compared with AM group (P<0.05, 0.01, Tab.4).

Tab. 3 Effect of butylphthalide on ET-1 in BALF and serum of asthmatic guinea pigs (mean±SD, n=9).

Tab. 4 Effect of butylphthalide on ET-1 in lung tissue of asthmatic guinea pigs (mean±SD, n=9).

Effect of butylphthalide on ET-1 mRNA in lung tissue of asthmatic guinea pigs

Six samples in each group were randomly extracted and determined ET-1 mRNA in lung tissue of asthmatic guinea pigs. Th e expression of ET-1 mRNA in AM group rose obviously and appeared diff erence compared with BC group (P<0.01). Under the treatment of butylphthalide, the expression of ET-1 mRNA went down signifi cantly (P<0.01, Tab.5).

Effect of butylphthalide on ECE-1 in lung tissue of asthmatic guinea pigs

The expression of ECE-1 appeared pale brown in cytoplasm through regular method of sectioning and immunohistochemical staining. The expression of ECE-1 in BC group was slightly but that in AM group was deeper (Fig.2A, Fig.2B). The image analysis results showed that the average optical density of ECE-1 expression in BH group and BM group was lower than AM group (P<0.05, Tab.6).

Discussion

Fig. 1 Effect of butylphthalide on ET-1 in lung tissue of asthmatic guinea pigs (×40). A: Blank control group (BC group); B: Asthma model group (AM group); C: Dexamethasone group (DXM group); D: Butylphthalide high dose group (BH group); E: Butylphthalide middle dose group (BM group); F: Butylphthalide low dose group (BL group).

Fig. 2 Effect of butylphthalide on ECE-1 in lung tissue of asthmatic guinea pigs (×40). A: Blank control group (BC group); B: Asthma model group (AM group); C: Dexamethasone group (DXM group); D: Butylphthalide high dose group (BH group); E: Butylphthalide middle dose group (BM group); F: Butylphthalide low dose group (BL group).

Tab.5 Effect of butylphthalide on ET-1 mRNA in lung tissue of asthmatic guinea pigs (mean±SD, n=6).

Tab.6 Effect of butylphthalide on ECE-1 in lung tissue of asthmatic guinea pigs (mean±SD, n=9).

As the pollution of human survival environment and the increase in allergic population, bronchial asthma (abbreviated as asthma), the most common chronic airway inflammatory disease, has becomean important disease aff ected human health [5]. Th e respiratory tract of asthmatic patients has hyperresponsiveness to allergic substances and very low concentration of them can cause sneezing, itching nose，and even result in asthma attacks. Injecting ovalbumin (OVA) to sensitize, and inhaling OVA to stimulate, the asthmatic guinea pig models were established and the above symptoms were evaluated. The results showed that butylphthalide could obviously reduce the asthmatic score value of the asthmatic behavior in guinea pigs and improve asthma symptoms. The respiratory rate (F), tidal volume (TV) and enhanced expiratory pause (Penh) were mensurated based on the indexes of pulmonary function testing and the physiological characteristics of guinea pigs. Th e study showed that butylphthalide could promote pulmonary function through reducing F and Penh and increasing TV.

As an active polypeptide found by Yanagisawa in 1988, Endothelin (ET) is transmuted under endothelin converting enzyme (ECE) in the respiratory tract and other parts [6]. ET can cause airway smooth muscle contraction directly or through promoting the release of the media and acting nervous system on the respiratory tract [7]. Also, ET-1 participates in airway remodeling by stimulating smooth muscle cells and fi broblasts proliferation and thickening airway wall and basement membrane, improves sputum viscosity through promoting secretion of submucosal glands, and worsens airway inflammation by inducing the release of inflammatory mediators such as prostaglandin, histamine and platelet activating factor [8]. So, ET widely involves in asthmatic airway inflammation, airway high reactivity, mucus hypersecretion, and airway remodeling so on many aspects, and has a direct relationship with asthma [9]. In this experiment, ET-1 significantly increased in BALF and serum of asthmatic guinea pigs, and ET-1 mRNA expression in lung tissue also increased. Th is is consistent with some similar previous studies [10]. Under the action of butylphthalide, the above changes were obviously alleviated. ECE-1 significantly increased in lung tissue of asthmatic guinea pigs and this is not consistent with previous studies [11]. Th e possible reason is that the inhibitory eff ect of elevated ET-1 on ECE is not yet appeared in the early stage of asthma. Th ere was an obvious decline on the expression of ECE-1 under butylphthalide intervention.

Conclusion

Butylphthalide has obvious inhibitory effect on asthmatic pulmonary infl ammation and trachea high reactivity and one of mechanisms is to inhibit high expression of endothelin. These findings suggest that butylphthalide has potential use value to the asthmatic patients in clinical.

Acknowledgements

This work was supported by the National Natural Science Fund (81460668), the Natural Science Foundation of Gansu Province (1310RJZA086, 1606RJZA011) and the open fund project of Key Laboratory of Pharmacology and Toxicology for Traditional Chinese Medicine of Gansu Province (ZDSYS-KJ-2015-002).

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doi 10.13459/j.cnki.cjap.2016.06.006

Zhi-wang WANG, MD, Professor, Pharmacology Laboratory, Lanzhou, 730000, China. Tel: 86-931-8765395; E-mail: wzw0933@126.com. Yuan REN, Tel: 86-931-8762653; E-mail: leyuan988@163.com.

2016-07-24; accepted 2016-10-24