Altered Frequency of NK Cells and Treg Cells by Astragalus Polysaccharide Combined with Budesonide in Asthma Model Mice

Objective. We investigated the ecacy of astragalus polysaccharide (APS) combined with budesonide and the eect on expressions of peripheral NK cells and Treg cells and the molecular mechanism in mice with bronchial asthma. Methods. In this study, we established a mouse model of asthma. Four groups of BaLB/C mice were developed; control group had no asthma induction, and the other three groups of mice were sensitized by OVA (Ovalbumin), OVA + budesonide, and OVA + APS + budesonide. Flow cytometry was used to determine the proportion of NK cells and Treg cells. Levels of cytokines IL-4 and IL-10 were detected using RT-PCR and ELISA. Results. Asthma mice treated with APS + budesonide showed alleviated airway resistance compared to model mice ( < 0.05). e percentage of dendritic cells (DCs) was reduced ( < 0.05), while anti-in¡ammatory NK cells and Treg cells signi¢cantly increased a£er APS + budesonide treatment ( < 0.05). Further, APS + budesonide treatment resulted in improvements in IL-4 and IL-10 mRNA and protein levels ( < 0.05). Conclusion. APS combined with budesonide medication may regulate expressions of DCs cells and related cytokines, reliving clinical symptom of bronchial asthma.


Introduction
Bronchial asthma is a common chronic airway in ammatory disease, and its pathogenesis includes a variety of cells and components. Based on epidemiological survey data, elderly people have a higher incidence of asthma [1]. At present, the main treatment is drug therapy, which can be classi ed as control patient's condition and relief symptoms. Long-term oral or intravenous use of glucocorticoid can cause immunosuppression and Cushing's syndrome and other side e ects. Inhalation of common doses of glucocorticoid generally does not cause adverse reactions [2]. But a er glucocorticoids inhalation, approximately 80-90% of the drug is deposited in the pharynx and swallowed into the gastrointestinal tract, eventually depositing glucocorticoid will cause pharyngeal or systemic adverse reactions.
Over the last decades, traditional Chinese medicine extracted from the root of Astragalus membranaceus was documented to have biological activities. Astragalus polysaccharide (APS), the extraction, is a polysaccharide consisting of glucose and arabinose sugar, possessing very low toxicity, with a wide range of immune enhancement and antiviral e ect, which can relieve the plant hemagglutination (PHA) toxicity and resist the inhibitory e ects of immunosuppressive drugs such as prednisolone and cyclophosphamide [3]. e therapeutic implication of APS has been con rmed in patients with lung cancer [4]. Budesonide, which is a commonly used inhaled hormone, has fewer systemic adverse reactions. However, there are a few clinical studies on the combined therapy for adult asthma.
Given the NK cells and regulatory T cells contribute to asthma pathogenesis, it is believed that combination of APS and budesonide may in uence immune cell counts. In this study, the e ects of APS combined with budesonide on pulmonary function and immune response in mice with bronchial asthma were observed, aiming to provide theoretical basis for clinical treatment.  [5]. Brie y, 25 μg OVA (SIGMA-ALDRICH, O1641) dissolved in 200 μL PBS were injected intraperitoneally into the mice except those in control group. Mice in the control group were injected with same volume of PBS. On day 14, mice in the non-control group were kept in a closed container and inhaled 6% OVA solution by ultrasonic atomization to induce asthma 30 min/day for 7 days while mice in the control group inhaled PBS. On day 15, control and untreated asthma group was administered with atomized PBS; budesonide group, 3 μg/day budesonide; APS + budesonide group, 100 mg/kg body weight/day APS injected intraperitoneally and 3 μg/day budesonide inhaled. On day 29, mice were sacri ced to evaluate airway response and other biomarkers.

Bone-Marrow-Derived DCs (BMDCs) Generation.
e tibial and femoral bone marrow was isolated from the mice and the BMDCs were prepared as previous reported [6]. Isolated BMDCs were cultured in RPMI-1640 containing 5% fetal bovine serum and stimulated with 1000 U/mL IL-4 and 500 U/mL granulocyte-macrophage colony-stimulating factor (GM-CSF). On day 7, non-adherent cells were harvested.

Flow Cytometric Analysis of NK, DCs and Treg Cells.
A FC500 (Beckman Coulter, USA) was used to analyze the proportions of NK, DCs and Treg cells. e data was analyzed with CXP analysis so ware 2.3. NK cells were stained with FITC-CD16 and PE-CD56 (eBioscience, SanDiego, CA).

Determination of Airway Hyper-Responsiveness.
Airway hyper-responsiveness was measured using the FinePointe™ RC animal airway resistance and pulmonary compliance system (BUXCO, USA). On day 28, 60 mg/kg pentobarbital sodium was injected intraperitoneally, then a tracheal incision was performed. Respiratory frequency was set at 160 times/min. Mice were inhaled with 10 μL acetylcholine, at ascending concentrations of 0 mg/mL, 1 mg/mL, 2 mg/mL, 4 mg/mL, 8 mg/mL. e maximum airway resistance within 3 minutes was recorded.

Statistical Analyses.
All data were analyzed using SPSS13.0 so ware. e data are expressed as mean ± standard deviation. e inter-group comparisons of means were performed using univariate analysis of variance and pairwise comparison between groups, with < 0.05, < 0.01 or < 0.001 as signi cantly di erent.

Body Weight and Airway Resistance in Mice with Asthma
In uences by APS and Budesonide. Compared to control group, mice in model group, and drug treated group had a loss and slight uctuation in body weight (Figure 1(a)). Airway hyper-responsiveness was assessed in 4 groups. In untreated group, mice exposed to OVA showed signi cant airway resistance at acetylcholine concentrations of 1, 2, 4, and 8 mg/mL ( < 0.01; Figure 1(b)). To verify airway resistance, the airway resistance a er acetylcholine challenge between untreated model mice and various treatment groups was compared. e mice of three various treated groups exhibited signi cantly less airway resistance at an acetylcholine concentration of 1, 2, 4, and 8 mg/mL ( < 0.05; Figure 1(b)). e mice in APS + budesonide showed signi cantly lower airway resistance than that in budesonide group ( < 0.05; Figure 1(b)).

Percentage of DCs, NK Cells, and Treg Cells In uences by APS and Budesonide.
e frequency of NK cells and Treg cells in BALF has been analyzed. Compared with healthy controls, animals with experimental asthma showed attenuated DCs and attenuated cell numbers of NK cells and Treg cells ( < 0.05; Figure 2). However, the percentages of NK cells and Treg cells, were markedly increased in mice with drug treatment whether combined with APS ( < 0.05; Figure 2). e frequency of DCs in untreated asthma group was elevated when compared to control group ( < 0.05; Figure 2). By comparison, frequency of DCs in two treated groups was signi cantly alleviated ( < 0.05; Figure 2). In addition, the frequency of DCs in APS + budesonide group was signi cantly lower than that in budesonide group ( < 0.05 ; Figure 2).

mRNA Levels of IL4 and IL10 in Mice In uences by APS and
Budesonide. As NK cells in the lungs have been considered to be regulators of 1 or 2 cytokine production, we analyzed the mRNA levels of in ammatory mediators: IL4 and IL10 in lung tissues of mice. 2 cytokines IL4 and anti-in ammatory cytokines IL10 in mice model of asthma were signi cantly increased, compared to control group ( < 0.05; Figure 3). Administration of budesonide signi cantly reduced the mRNA levels of IL4 in mice with asthma ( < 0.05; Figure 3). Levels of IL4 and IL10 in APS + budesonide group were signi cantly decreased compared to budesonide group ( < 0.05; Figure 3).

Cytokine Levels in APS and Budesonide Treated
Mice. Levels of the important 2 cytokine IL-4 and in ammatory cytokine IL-10 in BALF and the spleen were measured by ELISA. As shown in Figure 4, levels of these cytokines in BALF signi cantly declined in APS + budesonide-treated mice, when compared with budesonide treated mice ( < 0.05; Figure 4). Levels of IL-4 and IL-10 in spleen were consistent with that in BALF.

Discussion
e etiology of asthma is complex, and airway hyperresponsiveness is the characteristic pathological manifestation of asthma. Leukotriene, a metabolite of arachidonic acid, is widely present in the airway of patients with bronchial asthma and mediates and induces in ammatory reactions. Previous clinical practice has shown that the clinical e ect of montelukast alone as a leukotriene regulator in the treatment of bronchial asthma needs to be further improved. Polysaccharide served as anti-tumor drug has been well studied in various types of tumor cells [7,8]. In the present study, we established a murine asthmatic model and demonstrated that asthma model mice, challenged with APS and budesonide, exhibited improved airway hyper-responsiveness.
Current studies have con rmed that the pathogenesis of bronchial asthma is closely related to cellular immune dysfunction. e cell types involved in the pathological process of asthma include 2 cells, 17 cells, NK cells, antigen presenting cells, B cells, etc. [9][10][11][12][13]. As an important natural immune cell, NK cells are the third type of lymphocytes along with T cells and B cells. Recent studies have shown that compared with healthy controls, NK cells isolated from the peripheral blood of asthmatic patients have a reduced killing ability against tumor cell lines, although they secrete more cytotoxic regulators [14]. e airway in ammatory immune response of bronchial asthma is associated with decreased cytotoxicity of NK cells and increased leukocyte count. Some studies have con rmed that NK cell subsets are signi cantly reduced in patients with allergic asthma, suggesting that the occurrence of asthma is related to the inhibition of NK function [15]. Moreover, the NK cells content in the remission phase of asthma was higher than that in the acute attack phase [16,17]. NK cells is a necessary factor for naive T cells di erentiating to 2 cells.
Treg plays an important role in balancing the in ammatory response and regulating the immune response. Studies have found that in allergic patients, Treg content is reduced and function is impaired. In addition, Treg cells can negatively feedback regulate T cells through a variety of inhibitory mechanisms, such as down-regulating the expression of T cytokines and inhibiting the activation and di erentiation of T cells [18]. DCs can be directly activated and indirectly activated by Advances in Polymer Technology 4 Impaired capacity of DCs to activate T cells results in incomplete maturation of speci c inhibitory cytokines such as IL-10, which will suppress T-cell immune reactivity [20][21][22]. In this study, we observed the mRNA levels of IL-4 and IL-10 were alleviated by APS and budesonide treatment. Furthermore, both local (BALF) and systemic (spleen) levels of IL-4 and IL-10 in mice treated with APS and budesonide were signi cantly lower than in untreated model mice.
erefore, it is possible that APS and budesonide suppressed the production of IL-4 and IL-10 in gene and protein levels.
epithelium when inhaling harmless allergens, which are sucient to initiate 2 immunity. Our results shed light upon how the polysaccharide in uence the asthma model mice. Using the OVA-induced asthma model, our data showed that combination of APS and budesonide modulated the proportion of DCs, NK cells, and Treg cells. Notably, APS and budesonide had signi cant decrease of DCs compared to budesonide alone, suggesting the reliving immune response in treated-asthma mice.
It is documented that patients with asthma o en had increased frequencies of IL-4-producing NK cells [19].

Conflicts of Interest
e authors declare that there are no con icts of interest regarding the publication of this paper.
Taken together, these ndings suggest that budesonide with or without APS treatment might induce protective immunity against asthma by the regulation of DCs, NK cells and Treg cells through IL-4 and IL-10 production. Apart from that, combination of APS and budesonide shows bene ts in improving airway pressure maybe due to the modulation of DCs, IL-4 and IL-10, which provide a new strategy in the therapy of asthma.
Data Availability e data used to support the ndings of this study are available from the corresponding author upon request. 3: APS and budesonide suppressed mRNA levels of in ammatory mediators. Relative expression of IL4 and IL10 mRNA levels were measured by RT-PCR and is represented above. Every independent experiment was triplicated. Mean ± SD is presented for each group. * < 0.01, compared with control group; # < 0.05, compared with untreated asthma group; △ < 0.05, compared with budesonide group. 4: Cytokine levels of IL-4 and IL-10 in BALF and spleen were analyzed by ELISA. Every independent experiment was repeated twice. Mean ± SD is presented for each group. * * < 0.01, compared with control group; # < 0.05, ## < 0.01, compared with untreated asthma group; △ < 0.05, compared with budesonide group.