Asthma is a common chronic airway inflammatory disorder which already affects an estimated 300 million individuals1 and the prevalence of asthma is increasing.2,3 The major characteristics of asthma are airway inflammation, airway hyperresponsiveness (AHR), and airway remodeling; with airway inflammation considered to be the basic pathological change and one of the key pathological mechanisms of repeated attacks of asthma. Corticosteroids and β2-adrenoceptor agonist treatments are significantly effective in relieving asthma symptoms.4,5 However, there are still a number of people with asthma symptoms which are difficult to control and continue to experience persistent symptoms and asthma exacerbations.6 Bu-Shen-Yi-Qi-Tang (BSYQT), which is prescribed on the basis of clinical experience, is commonly used in clinics of traditional Chinese medicine (TCM) for asthma treatment. The components of BSYQT include Radix Astragali (RA), Herba Epimedii (HE), and Radix Rehmanniae (RR). Our previous studies revealed that BSYQT could increase the asthma control score, decrease daily asthma symptoms, and improve lung function. However, the variable effects of BSYQT from a granules decoction (GD) and herbs decoction (HD) on the airway inflammation of asthmatic mice have not been studied and compared. This study aimed to compare the effects of GD and HD of BSYQT on airway inflammation in asthmatic mice.
Pathogen-free, six-week-old female BALB/c mice (14–6 g) were purchased from Shanghai SLAC Laboratory Animal Co. Ltd. and experiments were performed following the guidelines approved by the Committee on the Ethics of Animal Experiments of Fudan University.
Ovalbumin (OVA, grade II and V), aluminum hydroxide (Al(OH)3) and methacholine (Mch) were purchased from Sigma-Aldrich. Herbs of RA, HE and RR were purchased from Shanghai Kangqiao Traditional Chinese Medicine Co. Ltd. Granules of RA, HE and RR were purchased from Jiangyin Tianjiang Pharmaceutical Co. Ltd. Dexamethasone (DEX) was purchased from Chenxin Pharmaceutical Co. Ltd. Mouse eotaxin ELISA kit (EK-033–40) was purchased from Phoenix, IL-17A (432507) and IL-4 (431107) were from BioLegend, and IL-5 (MBS355244) and IFN-γ (MBS262163) were purchased from Mybiosourse. TriZol Reagent was purchased from Invitrogen. SYBR Green kit was purchased from TaKaRa. RevertAidTM First Strand cDNA Synthesis kit was purchased from Fermentas.
Asthmatic model establishment and treatment
Sixty female BALB/c mice were randomly divided into 5 groups (12 mice/group). Except for the normal control (NC) group, mice were sensitized by multiple intraperitoneal (i.p.) injections of OVA at a dose of 20 μg OVA adsorbed to 2 mg Al(OH)3/0.5 ml saline solution on days 0, 7, 14 and 21. Starting on day 25, mice were challenged by inhalation of 3% (w/v) OVA nebulized solution, with an ultrasonic nebulizer, to challenge their airways (402AI, Yuyue medical equipment Co. Ltd., Jiangsu, China) for 30 minutes three times/week for 8 consecutive weeks.7 Mice in the NC group were sensitized with saline solution and exposed to a saline aerosol.
From day 24, mice in the NC and asthma model (A) groups were intragastrically administrated 0.3 ml of saline solution, mice of the other three groups were intragastrically administrated 0.3 ml of GD, HD (12 g/kg body weight) and DEX (1 mg/kg body weight) one hour prior to the OVA challenge per day for 8 consecutive weeks (Figure 1).
Measurement of AHR
AHR was evaluated by measuring the changes of airway resistance (RL) and lung dynamic compliance (Cdyn) by whole-body and invasive plethysmography (Buxco Electronics Inc., NY, USA). Briefly, mice were anesthetized with pentobarbital sodium (50 mg/kg) by i.p. injection 24 hours after the last OVA challenge. A 2 mm incision was made in the trachea after the tracheostomy and the tracheal tube was inserted with a suture around the trachea to prevent tracheal tube removal and air leak. Mice were put into the body plethysmograph chamber and the inserted tracheal tube was connected to the ventilator. PBS and progressive doses of Mch (3.125, 6.25 and 12.5 mg/ml) were challenged from the ventilator through an ultrasonic nebulizer after a stable baseline airway pressure was reached.
Immediately after the AHR measurement, blood collection was conducted from the orbital venous plexus; blood was stored at 4°C for 2 hours and then centrifuged at 5000 × g at 4°C for 15 minutes. The serum was collected, repackaged and stored at -80°C for ELISA assays.
Pulmonary histological analysis
The right lobe of the lung was removed, fixed in 4% paraformaldehyde for 24 hours and embedded in paraffin for histopathology analysis. Lung tissues were cut into 2 μm thick sections, stained with hematoxylin-eosin (HE), and examined for airway inflammation changes by light microscopy.
Inflammatory mediators assay in serum
The amounts of IL-4, IL-5, IL-17A, INF-γ, and eotaxin in serum were measured by ELISA-kit according to the manufacturer's instructions.
Quantitative real-time RT-PCR (qPCR) analysis
For analysis of IL-4, IL-5, INF-γ, and eotaxin mRNA expression, total RNA extraction of the left lung was performed with TriZol Reagent following the manufacturer's protocol. cDNA was synthesized from the total RNA using the RevertAidTM First Strand cDNA Synthesis kit following the instructions provided by the manufacturer. The mRNA expression was determined by using qPCR through SYBR Green kit. Primers used are presented in Table 1. The fold change in expression of each gene was calculated using the ΔΔCt method, with the housekeeping gene GAPDH mRNA as an internal control.
Data are expressed as the mean±standard deviation (SD). Statistical analyses were performed by one-way analysis of variance (ANOVA) for multiple comparisons followed by the LSD test for comparisons between groups. P <0.05 was considered statistically significant.
Effect of GD and HD of BSYQT on AHR to Mch in asthmatic mice
The effect of GD and HD of BSYQT on AHR to Mch was evaluated by measuring the changes of RL and Cdyn. As shown in Figure 2A, our results demonstrated that OVA challenge significantly increased RL to increasing doses of Mch compared with the NC group (P <0.05). Both GD and HD treatment decreased RL to Mch compared with the A group. However, HD treatment significantly decreased RL at the three Mch doses (P <0.05), and GD treatment only significantly decreased RL at Mch doses of 6.25 and 12.5 mg/ml (P <0.05). Although both GD and HD treatment decreased RL at Mch doses of 3.125 and 6.25 mg/ml compared with the A group, there was a significant difference in their efficiency in RL reduction (P <0.05). As the positive control drug, DEX treatment significantly decreased RL to increasing doses of Mch compared with asthmatic mice (P <0.05). There was not a marked difference in RL reduction between mice treated with HD and DEX (P >0.05).
As shown in Figure 2B, OVA challenge significantly decreased Cdyn to increasing doses of Mch compared with the NC group (P <0.05). Both GD and HD treatment could increase Cdyn to Mch compared with the A group (P <0.05). Although both GD and HD treatment increased Cdyn at Mch doses of 6.25 and 12.5 mg/ml compared with the A group, the efficiency in Cdyn promotion of HD was markedly more significant than that of GD (P <0.05). Mice in the DEX group manifested a greater increase of Cdyn than that of the asthmatic mice (P <0.05). The effect of HD on Cdyn improvement was the same as that of DEX (P <0.05).
Effects of GD and HD of BSYQT on pulmonary pathology of asthmatic mice
As shown in Figure 3, OVA sensitization and challenge resulted in large number of inflammatory cells infiltrating, secreted mucus, and epithelial damage in the lung compared with mice in the NC group. GD, HD, and DEX treatment markedly attenuated lung inflammation (P <0.05). It is worth noting that HD treatment demonstrated better alleviation of inflammation than that of GD and DEX treatment (P <0.05).
Effects of GD and HD of BSYQT on inflammatory cytokine levels in serum of asthmatic mice
We also examined the level of IL-4, IL-5, IL-17A, INF-γ, and eotaxin in serum of mice in each group. There was a significant increase in the serum level of IL-4, IL-5, IL-17A, and eotaxin compared with the NC group (P <0.05). A significant reduction in IL-4 and IL-17A levels in serum was observed in the GD, HD and DEX treatment groups compared with the A group (P <0.05). The effect of HD in lowering IL-4 and IL-17A levels was more prominent than that of GD (P <0.05). HD treatment significantly reduced serum levels of IL-5 and eotaxin compared with the A group, however, mice in the GD treatment group did not demonstrate this effect. DEX treatment remarkably reduced IL-5 levels compared with the asthmatic mice (P <0.05). HD treatment was more effective in lowering IL-5 expression than was DEX (P <0.05). OVA challenge resulted in a remarkable decrease in IFN-γ in serum compared with mice in the NC group (P <0.05). GD, HD and DEX treatment markedly elevated IFN-γ levels compared with mice in the A group (P <0.05), however, there was no significant difference in the IFN-γ level among the GD, HD, and DEX treatment groups (P <0.05).
Effects of GD and HD of BSYQT on mRNA expression of inflammatory cytokines in lung tissue of asthmatic mice
The effects of GD and HD of BSYQT on expression of IL-4, IL-5, INF-γ, and eotaxin mRNA were further compared. As shown in Figure 5, OVA sensitization and challenge increased IL-4, IL-5, and eotaxin mRNA expression levels and decreased IFN-γ mRNA expression level significantly in the lungs compared with the NC group (P <0.05). GD and HD treatment significantly reduced IL-4 and eotaxin mRNA expression compared with the A group (P <0.05). HD and DEX treatment significantly reduced IL-5 mRNA expression compared with the A group (P <0.05). There was a significant difference between the GD and HD treatment groups in reducing IL-5 and eotaxin mRNA expression levels (P <0.05). Compared with the A group, an obvious increase in mRNA expression of IFN-γ was observed in the GD, HD, and DEX treatment groups (P <0.05). However, the effect of HD treatment on increase of IFN-γ mRNA expression was greater than that of GD and DEX treatment (P <0.05).
In this study, the effect of GD and HD of BSYQT on airway inflammation in asthmatic mice was investigated and compared. AHR to Mch, lung histopathology analysis, inflammatory mediators in serum (IL-4, IL-5, IL-17A, IFN-γ, and eotaxin) and lung (IL-4, IL-5, IFN-γ, and eotaxin) were selected for investigation and comparison.
Excessive airway constriction following provocative stimuli is a prominent feature of clinical asthma.8 In our study, OVA challenge resulted in markedly increased RL and decreased Cdyn, and both GD and HD treatment reduced RL and increased Cdyn, indicating the relief of AHR by GD and HD treatment. At doses of 3.125 and 6.25 mg/ml, the effect of HD on RL was more obvious than that of GD, and at doses of 6.25 and 12.5 mg/ml, the effect of HD on Cdyn was greater than that of GD, indicating a greater therapeutic effect of HD than GD on AHR. DEX is the most frequently used agent in the clinic for asthma treatment. There were no marked differences in RL reduction and Cdyn improvement between mice in the HD and DEX groups, indicating the obvious reduction effect of AHR in asthma. There are always inflammatory cells infiltrating into the airways and the lungs during chronic asthma.9 We compared the effect of GD and HD on changes in pulmonary pathology and our results proved that both GD and HD treatment attenuated lung inflammation. There was a remarkable difference in attenuating lung inflammation between the GD and HD treatment groups, and mice in the HD treatment group manifested less inflammatory damage, which was more obvious than with DEX treatment.
Asthma is traditionally viewed as an eosinophilic airway inflammatory disorder. The focus on eosinophilia in part reflects a view of asthma as being associated with an immune response biased towards a Th2 response, which is characterized by the production and secretion of Th2 cytokines such as interleukin IL-4, IL-5, and IL-13.6,10 Dysregulation of Th1/Th2 cytokines production is considered to be one of the mechanisms of asthma.11 In this study, IL-4 and IL-5 were selected for comparison of the effect of GD and HD of BSYQT on airway inflammation in asthma. Our results demonstrate that both GD and HD treatment are effective in reducing IL-4 and IL-5 levels, and HD treatment is more effective than GD treatment. IL-4 is involved in differentiation and stimulation of Th2 cells, synthesis of IgE, and activation of macrophages.12 IL-5 is reported to induce genes involved in the growth, survival, and activation of eosinophils13–16 and sputum IL-5 protein levels are reported to correlate with frequent asthma exacerbations.17 The reduction in IL-4 and IL-5 during GD and HD treatment might account for its effect in attenuating inflammation in asthma. The mass of evidence supports a role for Th1 cells in asthma and IFN-γ is the signature cytokine of Th1 cells. In a chronic OVA exposure model, mice deficient in IFN-γ and IFN-γ receptor 1 (IFN-γR1) failed to develop increased airway resistance.18 Our results reveal that the Th1 cytokine IFN-γ is decreased during asthma and both GD and HD treatment could elevate IFN-γ mRNA expression and serum level, and HD treatment has a better effect in increasing IFN-γ mRNA expression than GD and DEX. Increased expression of IL-17 has been found in asthma19 and was most closely associated with asthma severity.20,21 An increase in IL-17A in serum was observed in both GD and HD treatment groups, and the increase in the HD treatment group was more remarkable than that in the GD group, indicating a stronger role of HD in lowering IL-17A. The levels of the chemokine eotaxin are markedly increased in sputum and plasma of asthmatic patients and correlated with the percentage of eosinophils in induced sputum.22 It is noted that HD treatment was more effective in down-regulation of IL-5 and eotaxin expression as well as up-regulation of IFN-γ mRNA levels than was DEX. The variety of chemical components in HD may account for its superiority to DEX. Our study proved that both GD and HD treatment could reduce eotaxin mRNA expression and serum level. However, inconsistent results were found with the effects of GD and HD on eotaxin between mRNA and serum levels. At the level of mRNA expression, the effect of GD treatment was more obvious than that of HD, on the contrary the effect of HD treatment was more marked than that of GD at the serum level. This indicates there is some posttranslational modification process in the effect of GD and HD on eotaxin expression.
In summary, the present study indicates that both GD and HD treatment could reduce airway inflammation in asthmatic mice, and HD treatment has greater inhibitory effects on inflammation, which may due to differences in the active ingredients between GD and HD. Further work will be done to ascertain the differences in the active ingredients between GD and HD.
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