Effects of the Traditional Chinese Medicine Dilong on Airway Remodeling in Rats with OVA-induced-Asthma

1 Introduction

More recently, the mechanisms of airway remodeling are closely associated with incrassation of the airway wall, extracellular matrix apposition, hypertrophy and hyperplasia of airway smooth muscle cells (ASMCs), incrassation of basement membranes, angiogenesis, and gland enlargement. These conditions cause irreversible luminal narrowing, airway hyper responsiveness and recurrent asthma attacks [1,2].

Traditional Chinese medicine, which has a documented history dating back X-thousand years, has produced several therapies for asthma. This was based on the understanding that the lungs provide gas exchange and connects all vessels, and the heart governs blood flow through the vessels, and together control blood circulation. As asthma patterns involved the lungs, asthma attacks were reasoned to be based on impaired function of lung gas exchange and the circulation of blood through vessels [3]. Many medical experts in ancient Chinese medicine noticed the relation between asthmatic coughs and blood stasis, and it is now known that microcirculation-improving drugs can prevent fibroblasts from synthesizing collagen to cause connective tissue thinning, reduce puffing, and resolve proliferative lesions. Despite a lack of knowledge regarding the exact mechanisms of action, traditional treatments were effective at resolving asthmatic symptoms [4].

One such traditional treatment was based on the use of Dilong, a preparation made from the dried body of the earthworm Pheretima aspergillum, and functioned to reduce body temperature and tranquilizing the mind, in addition to its anti-asthmatic and diuretic effects. Dilong also has also been shown to exhibit many pharmacological effects, such as decreasing blood pressure, preventing thrombus formation, and prevent cancer [5]. Dilong has also been suggested to inhibit or partly reverse the proliferation of fibrous tissues, and its thrombolytic effects involve plasminogen, lumbrukinase and collagenase [6]. Certain preparations of Dilong have been approved for clinical use in China, and are marketed mainly as ‘Guang Dilong’, is produced in Guangdong and Guangxi provinces.

Dilong is an anti-inflammatory drug that inhibits the proliferation of eosinophils, and inhibits the proliferation of smooth muscle cells in asthma patients from the early stage by inhibiting histamine and thrombin release, and acts to dissolve collagen-based components in the extracellular matrix [7]. It contains hypoxanthine and succinic acid, and is effective in spasmolysis. It. Animal experiments show that Guang Dilong extracted with 90% ethanol can inhibit bronchoconstriction induced by histamine and pilocarpine to increase pulmonary ventilation 3 to 4 times. The microcirculation-improving actions are beneficial in relieving microcirculatory disturbances by increasing oxygen saturation and inhibiting pathological changes caused by chronic asthma.

The objective of this study was to examine the effects and possible mechanisms of Dilong, alone and in combination with dexamethasone (Dex), on airway remodeling in rats with chronic asthma.

2 Materials and methods

2.4 Blood and tissue samples

All subjects were euthanized by use of 10% chloral hydrate (0.35 ml/100 mg) after the last OVA inhalation. Blood was drawn from the inferior vena cava after laparotomy, then heart and lungs were excised. Alveoli in the left lung were washed with 2.5 ml saline. Broncho-alveolar lavage fluid (BALF) was stored in EP tubes (1.5 ml) that had been refrigerated with DEPC at -80°C. Portions of the right lung were stored in paraformaldehyde (4%), while other portions were dissected immediately to dissociate the bronchus smooth muscle (Fig. 1). These samples were homogenized, suspended in 1:10 saline in 1.5 ml EP tubes, and stored at a low temperature (-80°C).

Figure 1

Dissociated bronchus sample of a control group.

3 Results

Asthma was observed in all OVA-treated subjects, who showed dysphoria, abdominal respiration, shortness of breath, abdominal retraction, prostration and immobility, while the rats in control group didn’t show these symptoms. Subjects in the high- and medium-dose Dilong, and Dilong-Dex treatment groups, showed milder symptoms than those in low-dose Dilong treatment group.

3.2 Pathology

The airway wall, basement membrane and smooth muscle were thicker in group with asthma than controls. The surface epithelium of the airway shed, and eosinophils and lympholeukocytes infiltrated in the airway wall (Fig. 2).

Figure 2

Lung tissue pathology (H&E staining, magnification 10 × 10).

The ratio of inner to outer diameter of the airway wall was greater in rats with high- and medium-dose Dilong and Dex than in rats with asthma. The airway smooth muscle and airway wall basement membrane were thinner in rats with medium- and high-dose Dilong, Dex, and Dilong and Dex than in rats with asthma (Table 2).

Table 2

Ratio of inner to outer diameter, thickness of smooth muscle and basement membrane in lung tissue of rats with asthma and treated rats.

Group (n)	Inner to outer diameter ratio	Thickness of smooth muscle	Basement membrane thickness
Control (13)	0.77 (0.06)	3.70 (0.47)	2.52 (0.49)
Asthma (10)	0.60 (0.07)▵▵	11.8 (1.44)▵▵	3.93 (0.46)▵▵
Dilong
Low dose (10)	0.61 (0.06)	9.83 (3.0)9	3.81 (0.44)
Medium dose (10)	0.63 (0.10)	6.19 (1.45)**	3.36 (0.42)**
High dose (9)	0.68 (0.04)**	5.65 (1.21)**	3.32 (0.6)3**
Dex (9)	0.63 (0.08)	6.70 (1.30)**	3.37 (0.75)
Medium-dose Dilong & Dex (8)	0.69 (0.04)** ▴	5.11 (0.93)**▴▴	3.26 (0.60)**

Data are mean (SD). ▵ ▵ P < 0.01 compared with control; * P < 0.05;** P < 0.01, compared with asthma; ▴ P < 0.05;▴ ▴ P < 0.01 compared with Dex

3.4 c-Myc Expression

The mRNA expression of c-Myc was stronger in rats with asthma than in those with medium- and high-dose Dilong, Dex, and Dilong and Dex (P < 0.01). The expression was weakest in rats with Dilong and Dex and controls (Fig.3). In situ hybridization revealed the expression of c-Myc mostly in smooth muscle, basal membrane, endothelial cells, and infiltrative inflammatory cells (Fig. 4). The expression of c-Myc was lower in rats with medium- and high-dose Dilong, Dex, and Dilong and Dex than in rats with asthma (P < 0.01) (Table 4).

Figure 3

RT-PCR analysis of mRNA expression of c-Myc in airway smooth muscle of rats with asthma and treated rats. Lane 1, asthma; 2, low- dose Dilong; 3, medium-dose Dilong; 4, high-dose Dilong; 5, Dex; 6, medium-dose Dilong and Dex; 7, control.

Figure 4

Lung tissue pathology (in situ hybridization, magnification 10 × 40)

Table 4

Expression of c-Myc by in situ hybridization in rats with asthma and treated rats.

Group (n)	c-Myc expression
Control (13)	132.39 (13.43)
Asthma (10)	100.12 (5.90)▵▵
Dilong
Low dose (10)	103.73 (3.53)
Medium dose (10)	117.86 (7.43)**
High dose (9)	129.06 (4.78)**▴▴
Dex (9)	122.43 (3.89)**
Medium-dose Dilong & Dex (8)	128.08 (7.38)**

Data are mean (SD). ▵▵P < 0.01 compared with normal control; * P < 0.05;** P < 0.01, compared with asthma; ▴ P < 0.05; ▴▴ P < 0.01 compared with Dex.

4 Discussion

Dilong (earthworm) is an effective anti-asthma traditional Chinese drug. Its pharmacological actions have been widely investigated in recent years. Here we investigated whether Dilong has a role in airway remodeling in asthma. Dilong decreased the number of eosinophils in inferior vena cava blood and BALF fluid of rats with asthma. It inhibited the thickened airway wall, as well as airway wall basement membrane and airway smooth muscle in asthmatic rats. Dex could reinforce Dilong’s effects. Dilong decreased the levels of TGF-β1, MMP-9/TIMP-1, uPA, PAI-1 and c-Myc. The cellularity of eosinophils, thickness of the airway wall basement membrane and airway smooth muscle were positively associated with levels of TGF-β1, uPA, and c-Myc. Thus, Dilong may have a role in ameliorating airway remodeling in asthma [10].

Airway remodeling of asthma mainly includes loss of airway epithelial cells, extracellular matrix deposition, vascular proliferation, and increased tracheal smooth muscles and secretion. Eosinophils play a significant role during the generation and development of bronchial asthma characterized by allergies and airway inflammation triggered by environmental agents. A growing body of evidence suggests that eosinophils are also important in airway remodeling. Eosinophils can synthesize many tissue remodeling factors, such as TGF-a, TGF-β1, VEGF, and MMP-9. We found that Dilong could decrease the number of eosinophils in the inferior vena cava blood and BALF of rats with asthma. During asthma attacks, the expression of TGF-β1 is strong; the factor participates in abnormal reparation of epithelial tissues after damage, proliferation of myofibroblast and fibrous degeneration of fibrous tissues under the basal membrane. This results in chronic airway inflammation and remodeling. Hoshino et al. found the basement membrane of asthmatic patients thicker than that of healthy controls [11]. Electron microscopy of the basement membrane revealed thickened subepithelial lamina reticularis, which was associated with the number of fibroblasts in the submucosa in asthmatic patients but not controls. Moreover, with chronic inflammation, fibroblast proliferation (likely caused by increased TGF-β1 level) leads to collagen deposition, airway remodeling and aggravated asthma [12]. We found that Dilong could decrease the expression of TGF-βi in our rats with asthma.

The imbalance between MMP-9 and TIMP-1 contributes to airway remodeling in bronchial asthma, which can be shown by reorganization of interstitial substances, angiogenesis, and hyperplasia of bronchial smooth muscle. MMPs may be key factors in altering lung extracellular matrix [11]. By digesting components of the ECM, including the basement membrane, MMP-9 leads to airway remodeling [13]. However, increased TIMP-1 expression leads to airway remodeling due to the composition of extracellular matrix components [14]. So the imbalance between MMP-9 and TIMP-1 is a marker of airway remodeling. Here, we found that Dilong could alter the expression of MMP-9 and TIMP-1 in rats with asthma.

Lumbrukinase in Dilong promotes fibrinolysis and is clinically effective against thrombosis. Zhang et al. [15] reported that increased uPA and uPAR may act on matrix degradation in the early fibrotic liver. uPAR and tPA were associated with overall inhibition of matrix degradation in advanced cirrhotic liver. However, a similar relationship among the expression of PAI-1, uPA, uPAR and airway remodeling has not been reported in experimental rat models of asthma, as in liver cirrhosis. Here, we found that Dilong could decrease the expression of uPA and PAI-1 in rats with asthma [15].

Myc, the product of protein expression of protooncogene c-Myc, may induce the proliferation, growth and differentiation of smooth muscle cells and fibroblasts in airways, an important factor of airway remodeling [16]. C-Myc is the early gene necessary for mRNA transcription of some inflammatory factors (endothelial growth factor, TGF-β1, interleukin 4 and 5), and the common final passage during signal transduction. Recent studies confirmed that the induction of c-Myc expression can release many cytokines and mediators of inflammation, which stimulate the further release of c-Myc and perpetuates airway remodeling. The proliferation of smooth muscle cells and fibroblasts depends on the function of several growth factors, but some cytokines can also promote the expression of c-Myc [17]. We found that Dilong could decrease the expression of c-Myc in rats with asthma.

5 Conclusion

This study explored the inhibitive action of Dilong on airway remodeling in asthmatic rats and suggested mechanisms by which this may occur. We analyzed basement membrane thickness because airway remodeling from incrassation of the basement membrane is the feature of asthma that distinguishes it from other diseases. Dilong inhibited the thickened airway wall, as well as airway wall basement membrane and airway smooth muscle, in asthmatic rats and decreased the levels of TGF-β1, MMP-9/TIMP-1, uPA, PAI-1 and c-Myc, features of asthma. Thus, Dilong may have a role in ameliorating airway remodeling in asthma.