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Introduction

Asthma is a lung disease characterized by chronic inflammation of the airways (1). An oxidant/antioxidant imbalance in asthma is one of the important mechanisms underlying the inflammation process. Therefore, supplementation of antioxidants in order to maintain the oxidant/antioxidant balance can significantly reduce airway inflammation and improve ventilation and airway remodeling. In traditional Chinese medicine, there are a large number of natural antioxidant substances, including phenolic acids and saponins (2). Over the years, the use of traditional Chinese medicine in the treatment of asthma and other respiratory inflammatory diseases has exhibited unique effects when compared with conventional treatments, including the use of inhaled corticosteroids and oral leukotriene receptor antagonists (3). Chinese medicine not only produces an anti-inflammatory response, it also exhibits immunoregulatory effects and reduces the release of oxygen free radicals (4). Xiao-qing-long-tang is a well-known drug that has been shown to be effective for the treatment of coughs and asthma and has a far-reaching impact in the history of Chinese medicine development. Paeonia and Schisandra extracts are administered as adjuvant drugs in xiao-qing-long-tang; this combination has exhibited considerable success. However, the anti-inflammatory mechanisms of the two herbs in the treatment of asthma remain unclear. Previous studies have demonstrated that Paeonia contain saponins, while Schisandra contain other antioxidants, including schisandrin B. However, the effects of Paeonia and Schisandra in an in vivo model of asthma remain unclear (5–7). Thus, the present study aimed to investigate the underlying mechanisms of Paeonia and Schisandra in the treatment of asthma.

Materials and methods

Ethics statement

This study was approved by the Ethics Committee of the 324th Hospital of PLA (Chongqing, China). Animal care guidelines were strictly followed for all experimental procedures in the study.

Extract preparation

Schisandra and peony original drugs were extracted from Chinese medicine solution, in accordance with the ratio of 1:1 using ethanol extraction methods. The reflux time was 5 h and the extracts were placed in a sealed container at 4°C.

Preparation of asthmatic rats

Rats (n=200) were purchased from the Third Military Medical University Laboratory Animal Center (Chongqing, China) and were maintained under specific pathogen free conditions. The rats were randomly divided into treatment (group A), asthma (group B) and saline control groups (group C). For the rats in the asthma and treatment groups, 200 mg aluminum hydroxide, (Hebei Xinhua Pharmaceutical Company, Shijiazhuang, China) 1 mg ovalbumin (Hebei Xinhua Pharmaceutical Company) and 0.2 ml saline (0.9%; Hebei Xinhua Pharmaceutical Company) were administered subcutaneously. Once every two days, an additional subcutaneous injection of 0.2 ml inactivated Bordetella pertussis (Qilu Pharmaceutical Co., Ltd. Jinan, China) was administered. Following sensitization, 2% ovalbumin inhalation was applied for stimulation once a day for one week. In the control group, normal saline was used to replace the subcutaneous injection of antigen solution. In the asthma and treatment groups, the rats exhibited symptoms such as sneezing, scratching heads, shortness of breath, abdominal muscle contraction, agitation, hair removal and hair thinning. The treatment group received 2 ml herbal extracts via daily gavage for 10 days. Blood samples (2 ml) were centrifuged for analysis prior to treatment and at day 5 and 10 following treatment in the rats of the three groups. At day 10 following treatment, the rats were sacrificed by cervical dislocation and the trachea tissues were removed for hematoxylin and eosin staining and NF-κB activity analysis in the three groups.

In vitro hydroxyl radical, total antioxidant activity and total phenolic content determination in the herbal extracts

A total antioxidant activity assay was performed with the herbal extracts using a kit from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Determining the hydroxyl radical suppression of the herbal extracts was conducted according to the Fenton reaction principle. The procedure was performed using a hydroxyl radical assay kit, according to the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute). Determination of the total phenolic content in the herbal extracts was measured using Lowry reagents (Sigma-Aldrich, St. Louis, MO, USA).

Serum malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) measurement

These parameters were measured using ELISA kits, according to the manufacturer’s instructions (Biovalue Co., Ltd., Shanghai, China.

Immunohistochemistry

Paraffin-embedded sections, 4-μm thick, were obtained from the lung tissues. Following dewaxing, xylene and graded ethanol dehydration was performed. Hydrogen peroxide (3%) was used to inactivate endogenous peroxidase antigens. Following serum sealing for 30 min, primary antibodies against NF-κB p65 (Beyotime Institute of Biotechnology, Haimen, China) were added and incubated for 1 h. Staining was then performed using a 3,3′-diaminobenzidine staining kit (Biotek Co. Ltd. Beijing, China). Phosphate-buffered saline was used in the negative control group instead of primary antibody. A positive reaction was indicated by brown staining in the cytoplasm and nuclei. In three high magnification fields, the positive rate was calculated as the ratio of positive cells/total cells.

Statistical analysis

Statistical analysis was performed using the Statistical Package for Social Sciences computer software (version 17.0; SPSS, Inc., Chicago, IL, USA) for Windows. Analysis was conducted using the χ2 test and the analysis of variance method. P<0.05 was considered to indicate a statistically significant difference.

Results

Histology

As shown in Fig. 1A, the tracheal mucosa and columnar epithelium underwent exfoliation and evident eosinophil infiltration was observed prior to treatment. As shown in Fig. 1B, infiltration of a large number of eosinophils, neutrophils and lymphocytes occurred at day 10 following saline treatment. However, in the traditional Chinese medicine treatment group, the trachea cilia and muscle layer were normal, and marked eosinophil infiltration was not observed, as shown in Fig. 1C.

Figure 1 Immunohistochemistry images of the (A) control group at day 0 and the (B) control and (C) treatment (traditional Chinese medicine) groups at day 10 following treatment (magnification, ×100).

In vitro antioxidant activity

As shown in Table I, the total antioxidant activity, hydroxyl free radical level and total phenolic content in the mixed extract were higher than those in the single herb extracts.

Table I Antioxidant activity of the herbal extracts.

Table I

Antioxidant activity of the herbal extracts.

Parameter	Schisandra extract	Herbaceous peony extract	Mixed extract
Total antioxidant activity (mmol/g)	1.482±0.115a	0.556±0.031a	1.963±0.211
Hydroxyl free radical (U/ml)	316.24±1.46a	89.09±3.76a	368.55±1.27
Total phenol content (μg/ml)	1138.26±6.53a	1253.28±5.91a	2055.32±4.65

{ label (or @symbol) needed for fn[@id='tfn1-etm-08-05-1479'] } Total antioxidant activity was determined using the ABTS method.

a P<0.05 vs. the mixed extract.

Determination of erythrocyte antioxidant activity

As shown in Table II, serum and erythrocyte SOD activity and GSH-Px levels in group A were higher than those in group B, while the level of MDA in group A was lower than that in group B (P<0.05). Serum and erythrocyte SOD activity and GSH-Px levels in group B were lower than those in group C, and the MDA level in group B was higher than that in group C (P<0.05).

Table II Serum antioxidant activity.

Table II

Serum antioxidant activity.

Parameter	Group A	Group B	Group C
SOD activity (U/l)	0.5±0.012a	0.33±0.025a	0.62±0.025
GSH-Px (μmol/l)	3166±252a	1650±125a	3250±203
MDA (μmol/l)	125.93±13.05a	511.334±15.363a	324.162±16.472

a P<0.05 vs. group C.

{ label (or @symbol) needed for fn[@id='tfn4-etm-08-05-1479'] } SOD, superoxide dismutase; GSH-Px, glutathione peroxidase; MDA, malondialdehyde.

Determination of cytokine levels

IL-4, IL-6 and IL-13 levels in the serum (Table III), bronchoalveolar lavage fluid (Table IV) and lung tissue homogenates (Table V) were not significantly different between groups A and B (P>0.05). However, IFN-γ levels significantly increased in group A as compared with group B, and IL-22 levels decreased significantly (P<0.05). The levels of IL-4, IL-6, IL-13 and IL-22 in the lung tissue, bronchoalveolar lavage fluid and serum of group B were significantly higher than those in group C, while the IFN-γ level decreased significantly (P<0.05).

Table III Cytokine levels in the serum.

Table III

Cytokine levels in the serum.

Cytokine (pg/ml)	Group A	Group B	Group C
IL-4	115.06±30.53a	173.78±32.85a	123.67±12.55
IL-6	997.92±145.31a	2005.23±125.62a	759.54±102.47
IFN-γ	1434.07±115.63a	766.83±84.05a	1226.14±125.01
IL-13	766.54±35.24a	859.32±55.08a	583.28±39.12
IL-22	50.20±11.31a	123.47±20.25a	62.35±15.18

a P<0.05 vs. group C.

{ label (or @symbol) needed for fn[@id='tfn6-etm-08-05-1479'] } IL, interleukin; IFN, interferon.

Table IV Cytokine levels in the bronchoalveolar lavage fluid.

Table IV

Cytokine levels in the bronchoalveolar lavage fluid.

Cytokine (pg/ml)	Group A	Group B	Group C
IL-4	40.81±8.46a	39.75±8.35a	28.09±7.66
IL-6	83.52±12.65a	187.19±13.62a	51.09±10.21
IFN-γ	259.96±25.31a	148.69±19.34a	235.61±22.04
IL-13	46.64±11.22a	45.32±10.07a	28.96±9.62
IL-22	92.68±15.24a	125.96±20.34a	82.95±10.21

a P<0.05 vs. group C.

{ label (or @symbol) needed for fn[@id='tfn8-etm-08-05-1479'] } IL, interleukin; IFN, interferon.

Table V Cytokine levels in the lung homogenates.

Table V

Cytokine levels in the lung homogenates.

Cytokine (pg/ml)	Group A	Group B	Group C
IL-4	43.29±3.06a	47.35±5.33a	30.57±4.31
IL-6	89.62±8.69a	187.19±10.08a	59.61±9.64
IFN-γ	389.14±25.93a	202.08±29.18a	354.35±35.77
IL-13	47.26±8.41a	46.86±6.22a	25.75±5.93
IL-22	88.56±12.03a	125.37±15.84a	67.42±8.67

a P<0.05 vs. group C.

{ label (or @symbol) needed for fn[@id='tfn10-etm-08-05-1479'] } IL, interleukin; IFN, interferon.

Discussion

When asthma occurs, airway inflammation results in increased levels of proinflammatory cytokines, NADPH oxidase and reactive oxygen species or reactive nitrogen radicals. Under normal circumstances, antioxidants in the lung tissue can remove small amounts of reactive oxygen species or reactive nitrogen radicals. Only an excessive amount of reactive oxygen species or reactive nitrogen radicals causes damage to airway proteins, lipids and DNA, resulting in airway inflammation, airway hyperresponsiveness, airway microvascular hyperpermeability and airway mucus hypersecretion. However, continued inflammation increases the levels of apoptosis and necrosis, which is followed by lung tissue oxidative damage (8). Therefore, oxidative stress is an important mechanism in the pathogenesis of asthma. To reduce oxidative stress or increase the antioxidant function, decreasing airway eosinophil infiltration, mucus secretion, airway hyperresponsiveness and changes in airway remodeling is required. Antioxidant supplementation has become a new method for the treatment of asthma. Chinese traditional medicine, including phenols, polysaccharides, alkaloids and saponins, are recognized as strong antioxidants that exhibit a free radical scavenging effect (9,10). Water and ethanol extracts containing these Chinese medicinal ingredients have been shown to exhibit strong anti-inflammatory and anticytotoxic effects. In vitro experiments have demonstrated that these extracts can inhibit nitric oxide (NO) and tumor necrosis factor-α production (11). The antioxidant activity of Chinese medicinal raw herbs is low (12), however, a water or ethanol extract of these Chinese medicinal raw materials exhibits significantly increased antioxidant activity. Therefore, extracting the active ingredients of Chinese medicinal raw materials is becoming increasing popular in the development of Chinese medicine. In our preliminary study, an ethanol extract reflux time of 5 h was shown to produce the highest antioxidant activity in vitro.

The traditional Chinese medicinal formula, xiao-qing-long-tang, has a strong therapeutic effect on asthma and has been shown to regulate protein secretion in airway club cells (13). In addition, the formula has been shown to reduce the proportion of eosinophils in the serum and thus, the levels of IL-5 and histamine release. The medicine can stabilize mast cell membranes, inhibit mast cell degranulation and also inhibit endothelin-1 levels and NO synthesis (14). Schisandra species, that are in xiao-qing-long-tang, are a type of superior medicine that have been used in China for almost one thousand years. Schisandra has been shown to exhibit antioxidant, cough and asthma inhibiting and liver function protective effects (15). Modern pharmacological studies have shown that Schisandra exhibits biological and pharmacological effects predominantly due to lignans (16). These lignans include schisandrin, deoxyschisandrin, schisandrin B, schisandrin C, schisandrol B, schisantherrin A, schisantherrin B and gomisin A. Levels of lignans in Schisandra water extract can reach ~99% (17). Paeonia also has a very long history of use in China and can significantly reduce eosinophil chemokine expression and secretion. In addition, Paeonia can inhibit the migration of eosinophils in A549 culture medium and the activation of NF-κB. Paeonia species have a therapeutic effect on asthma (18). A study of 44 types of traditional Chinese medicine revealed that there were relatively high phenolic and flavonoid levels in Akebia, Aster and water or ethanol extract of Paeonia. Therefore, Paeonia is one of the three traditional Chinese medicines with the highest anti-inflammatory and antioxidant activities (19). In the present study, following the joint use of Paeonia and Schisandra, the total antioxidant activity was 1.963±0.211 mmol/g, hydroxyl radical inhibition was 368.55±1.27 U/ml and the total phenolic content was 2055.32±4.65 μg/ml, which was significantly improved as compared with the single herb extracts. Levels of oxidative stress are closely associated with airway inflammation (20).

The level of oxidative stress in vivo is reflected through the oxide levels and antioxidant capacity in the body. During an asthma attack, the vitality of inflammatory cells increases, the function of airway epithelial cilia is impaired, mucus secretion increases and a large number of free radicals are produced. Active substances, including SOD, GSH-Px and vitamin C and E, are involved in the pathophysiological process of asthma (21). SOD exists in the cytoplasm and lysosome and can transform O2 into H2O; GSH-Px is also involved in this process. Free radicals induce body injury mainly through lipid peroxidation, following the reaction of unsaturated fatty acids, ethylene, pentane and MDA (22). Thus, following lipid injury of the cells, the end products increase. Therefore, asthma treatment should focus on increasing SOD and GSH-Px enzyme activity and reducing the increase in oxidation products. During an asthma attack, MDA levels increase, while GSH-Px levels decrease (23).

In the present study, serum and erythrocyte SOD activity and GSH-Px levels were lower in group B than in group C, while MDA levels in group B were higher than in group C (P<0.05). These results further illustrate the effect of oxidative stress on asthma. Paeonia and Schisandra extracts were used in the treatment of asthmatic rats, and the levels of serum and erythrocyte SOD activity and GSH-Px in the treatment group were found to be higher than those in group B (asthma group). In addition, the MDA level was lower than that in group B (P<0.05). Therefore, Paeonia and Schisandra extracts can significantly increase the antioxidant levels and reduce oxidative damage.

Cytokines, inflammatory cells and inflammatory mediators interact with each other, resulting in the infiltration of a large number of airway eosinophils and CD4+ T cells, as well as mucus hypersecretion, airway hyperresponsiveness, airway remodeling and increased IgE production. An imbalance in the cytokine levels produced by Th1, Th2 and Th17 cells leads to asthma. Therefore, regulating cytokine levels is the focus for asthma treatment (24). T lymphocytes play a major regulatory role in asthmatic airway inflammation, with Thl cell subsets secreting IFN-γ, which mediates immune responses and delayed-type hypersensitivity. Th2 cell subsets primarily produce IL-4, IL-5 and IL-13, which may induce the production and aggregation of eosinophils, as well as stimulate B cells to produce IgE and mediate humoral immune responses and engineering-type hypersensitivity. IL-22 is a proinflammatory cytokine that exhibits anti-inflammatory activity. IL-22 belongs to the IL-10 cytokine family and has two functional receptors, IL-22R1 and IL-22R2 (25). IL-22 may be activated by a variety of signaling pathways, the most important being the signal transducer and activator of transcription 3 pathway (26). In an antigen-sensitized rat model, IL-22 levels significantly increased (27). Neutralizing IL-22 antibodies can increase the production of Th2 cytokines, causing eosinophil infiltration and airway hyperresponsiveness (28). By contrast, IL-22 is a protective agent for airway inflammation, and recombinant IL-22 can inhibit eosinophilic airway inflammation and the production of Th2 cytokines (29,30). IL-22 also inhibits IFN-γ-inducible expression of proinflammatory cytokines and human airway epithelial cell adhesion (31). Therefore, the function of IL-22 is closely associated with the external environment and changes with the body disease state.

In conclusion, this study investigated the antioxidant and anti-inflammatory effects of Schisandra and Paeonia extract and showed that these effects could be beneficial in alleviating the asthmatic condition. Therefore, Schisandra and Paeonia extract could be used in the future treatment of asthma.

Acknowledgements

The study was supported by grants from the 12th Five-Year Plan of Chengdu (no. C1203) and the Chongqing Gongguan Projects (no. cstc2012gg-yyjs0627).

References