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Abstract

Allergic rhinitis (AR) is an inflammatory disease with a hypersensitivity response to environmental stimulus. The aim of this study was to evaluate the effect of Yiqi Wenyang Fang (YWF) on AR and investigate the underlying mechanism. A total of 48 female Sprague-Dawley rats were randomly divided into six groups (normal control, model control, YWF at low dose, YWF at median dose, YWF at high dose, and loratadine). Rats were injected with antigen for sensitization. Then, rats in the YWF groups were treated with different dose of YWF for 28 days. Loratadine was used as a positive control. Number of sneezes, degree of runny nose, nasal rubbing movements, and tissue damage were scored. The protein and mRNA expression of Foxp3 were determined by western blot and real time-PCR analysis, respectively. Flow cytometry was used to detect the number of CD4⁺CD25⁺Foxp3⁺ Treg cells. The content of interleukin (IL)-10, transforming growth factor β1 (TGF-β1), IL-13, and IL-4 in the serum were detected by enzyme-linked immunosorbent assay (ELISA). Scores of symptoms were significantly reduced and nasal mucosa damage was alleviated after YWF administration. YWF increased the expression of Foxp3, IL-10, TGF-β1, and number of CD4⁺CD25⁺Foxp3⁺ Treg cells which were reduced by antigen injection. The expression levels of IL-13 and IL-4 were increased after antigen administration while decreased after YWF treatment. YWF may ameliorate AR through inhibiting inflammatory response and promoting Foxp3 expression.

Keywords

allergic rhinitis cytokines Foxp3 inflammatory response Yiqi Wenyang Fang

Introduction

Allergic rhinitis (AR) is a chronic inflammatory airway disease, which affects the health of 10–20% of the population.¹ AR is clinically considered a symptomatic disorder of the nose induced by an immunoglobulin E (IgE)-mediated hypersensitivity reaction against allergens and involving mucosal inflammation driven by type 2 helper T (Th2) cells.² The four cardinal symptoms of AR are nasal itching, sneezing, watery rhinorrhea, and nasal congestion.³ The incidence of AR is increasing and AR has been shown to affect quality of life and productivity at school or work and cause severe financial losses.⁴ Besides, AR has been reported to underlie many complications and be a major risk factor for poor asthma control.⁵ Thus, there has been a growing interest in the treatment of AR.

The pathophysiology of AR is complex and AR should be considered a constellation of the underlying mechanisms and not a simple acute response to allergen exposure.⁶ Moreover, patients’ main symptom and compliance, the severity and duration of AR, safety of medication, and cost-effectiveness should be taken into consideration when treatment options for AR are chosen. It has been reported that an anti-Ig-E antibody, Omalizumab, can provide effective control of seasonal and perennial symptoms in patients through lowering free IgE levels.⁷ Recently, Passalacqua et al. had demonstrated that immunotherapy could be an effective option of treatment for respiratory allergy, and novel methods for various forms of allergen immunotherapy were currently under evaluation.⁸ Besides, antihistamines, intranasal corticosteroids, intranasal heparin, and anti-leukotrienes treatments have been shown to be effective for AR.^9,10 However, studies have reviewed that antihistamines can cause some sedation that worsens work performance while intramuscular corticosteroid injection is related with systemic side effects and muscular necrosis, both of which can act as rescue medication.¹ Therefore, more medications and therapies with safety and efficacy are needed to be developed for the treatment of AR.

Recently, many investigations on immunologic changes especially after sublingual immunotherapy (SLIT) have been carried out because of low incidence of adverse effects and convenience of self-administration of SLIT.¹¹ Several lines of evidence have shown that regulation of antigen-specific responses (an increase in the IgG4/IgE ratio), inhibition of activation/recruitment of inflammatory cells, shift of Th2 to Th1 responses, and activation of regulatory T (Treg) cells are the main mechanisms of SLIT.¹² Treg cells, which are known to play a critical role in immune tolerance, are reported to be related to the mechanism of SLIT.¹³ Additionally, with CD25 and Forkhead box p3 (Foxp3) as specific molecular markers for detecting and manipulating naturally arising Treg cells, there is now evidence that the population of CD4⁺CD25⁺Foxp3⁺Treg cell is actively engaged in the negative control of immune responses and can be exploited for the induction of immunological tolerance to non-self-antigens and negative control of aberrant immune responses such as allergy and immunopathology.¹⁴ The finding of Frew et al. had demonstrated that high-dose allergen extracts for SLIT could induce Treg cells, which could inhibit allergic inflammatory reactions via producing interleukin (IL)-10 and transforming growth factor-β (TGF-β) and inhibiting Th2 cells.¹³ Correspondingly, the concentration of Th2 cytokines such as IL-4 and IL-13 that were secreted by Th2 cells were decreased.¹⁵ On the other hand, several studies and clinical trials revealed that traditional Chinese medicine (TCM) was effective for the treatment of AR.^16,17 From the point of view of TCM, the lung and spleen, which are highly associated with qi deficiency, are the two important locations of diseases and studies have shown that symptoms of yang deficiency among AR patients included manifestations of qi deficiency with prominent fear of cold and with cold extremities.¹⁸ Yiqi Wenyang Fang (YWF) was designed to treat the cold deficiency of the lung and spleen of AR patients and was demonstrated to have marked therapeutic effects.¹⁹ Moreover, Astragalus membranaceus, which is used as a composition of YWF, had been demonstrated to significantly increase the CD4⁺CD25⁺Foxp3⁺ Treg cell population and promote Foxp3⁺ mRNA expression in a rat model of asthma.²⁰ However, the underlying mechanisms of the effect of YWF on AR are still not fully understood and whether YWF can promote Fox3 expression and induce immunological tolerance need further investigations.

In this study, AR model rats were induced by antigen injection. Then we evaluated the symptoms of AR after YWF administration. Further, the expression of cytokines in serum was observed and CD4⁺CD25⁺Foxp3⁺Treg levels in the CD4⁺ T cells were also detected. In addition, we detected the expression of Fox3 to explore the underlying action mechanism of YWF associated with immune tolerance in the rat model of AR.

Materials and methods

Rats

A total of 48 female Sprague-Dawley rats, which were provided by Laboratory Animal Center of Zhejiang University (Certificate SCXK [Zhe]: 2008-0033) and bred in a specific pathogen-free animal facility, were randomly assigned to six groups (n = 8 per group). Animals were housed two per cage in polycarbonate boxes, covered with filter lids, and had free access to tap water and food (Xietong Bioengineering Co., Ltd., PR China; product batch number: 20131210). Rats were housed in a room at a temperature of 22–25°C and relative humidity of 40–55%. Room lights were set on a 12 h light/dark cycle beginning at 06:00. All the experiments were carried out according to the ethical guidelines on animal care and approved by Experimental Animal Committee of Zhejiang University.

Ovalbumin sensitization and challenge protocols

Rats were sensitized by intraperitoneal injection with antigen ovalbumin (0.3 mg) mixed with aluminum hydroxide adjuvant (30 mg) in 1 mL saline, which was performed once every 2 days, lasting for 14 days. Sham immunization for the normal control rats (n = 8, randomly selected) was done with saline instead of ovalbumin in the same manner. Subsequent intranasal challenge was carried out on day 15. Briefly, 50 μL of an ovalbumin solution (0.5%, prepared in pyrogen-free saline) was instilled into each nasal airway passage of rats for 7 consecutive days. Sham-challenged (normal control) rats were instilled with pyrogen-free saline.

Medication

The main ingredients of YWF included huangqi (Astragalus membranaceus Bunge), dangshen (Codonopsis pilosula), ganjiang (rhizoma zingiberis), guizhi (Ramulus Cinnamomi), mahuang (ephedra sinica Stapf), and wuwenzi (Schisandra chinensis). These ingredients were proportionally mixed¹⁹ followed by water extraction, ethanol precipitation, and vacuum concentration, and then were sterilized and preserved at 4°C. The final concentration was 1.6 g/mL. Loratadine, a second-generation non-sedating antihistamine,²¹ was purchased from Shanghai Schering-Plough Pharmaceutical Co., Ltd.

Treatment procedures

In this study, the dosage in a rat was reasonably calculated by converting the human dosage with the conversion rate at 1:6.3 (human:rat).^22,23 The human body weight was taken as 60 kg.²³ Eventually, the other 40 rats which had intranasal challenge with ovalbumin were then randomly divided into five groups: the model control; YWF at low dose (clinical dose); YWF at median dose (five-fold of clinical dose); YWF at high dose (10-fold of clinical dose); and loratadine (as positive control drug) with eight rats in each group. YWF was given by gavage once a day lasting for 28 days. Loratadine was given by gavage for 28 days with 6.3-fold of the clinical dose. The rats in the model control and normal control groups were given the same amount of sterile normal saline by gavage. Along with medicine treatment, intranasal challenge was continually performed once a week (on days 7, 14, and 28) after medicine treatment.

Evaluation of nasal symptoms

Nasal symptoms of rats were observed for 30 min after nasal challenge on days 1 and 7 after sensitization and days 7, 14, and 28 after being given drugs. The frequency of sneezes and nasal rubbing movements, and the degree of runny nose were evaluated in accordance with the score system used by Narita et al.²⁴ Sneezes were scored on a scale of 1 (1–3 sneezes) to 3 (⩾11 sneezes). In addition, the score of runny nose degree was scored as: 1 (flowing to anterior nostril); 2 (flowing through anterior nostril); and 3 (very severe rhinorrhea). Itchiness of the nose was also scored as 1 (scratching few times) or 2 (scratching continually).

Histopathological examination

Rats were sacrificed by dislocation of the neck 24 h after the last intranasal provocation. Nasal mucosa was taken, routinely processed with 10% formaldehyde solution, and embedded in paraffin for histopathological examination. Tissues were then stained with hematoxylin and eosin (H&E). The lesion extent of tissue was scored on a scale of 0 to 4 (4, extremely severe; 0, none) for epidermal cell degeneration, sub-epithelial hyperemia, inflammatory cells infiltration, and inflammatory exudate as shown in Table 1. Individual lesion scores were summed from each samples to create an overall histopathology score for each group. Remaining tissues were taken and frozen in liquid nitrogen and stored at −70°C for subsequent analysis.

Table 1.

The scoring criteria of the lesion scores.

Lesion extent				Score
Epidermal cell degeneration and necrosis	Sub-epithelial hyperemia	Inflammatory cells infiltration	Inflammatory exudate	Score
None	None	None	None	0
Minimal	Minimal	Minimal	Minimal	0.5
Mild	Mild	Mild	Mild	1
Moderate	Moderate	Moderate	Moderate	2
Severe	Severe	Severe	Severe	3
Extremely severe	Extremely severe	Extremely severe	Extremely severe	4

Enzyme-linked immunosorbent assay (ELISA)

The amounts of serum cytokines including IL-10, TGF-β1, IL-4, and IL-13 were determined by commercially available double-antibody sandwich ELISA kits (eBioscience, USA), according to the manufacturer’s instructions.

Flow cytometry

CD4⁺CD25⁺Foxp3⁺ Treg levels were detected by flow cytometric analysis. Briefly, blood from the femoral artery was labeled with anti-CD4-fluorescein isothiocyanate (FITC) and anti-CD25-allophycocyanin (APC) monoclonal antibodies (eBioscience). For intracellular staining of Foxp3, cells were fixed and permeabilized using a Fixation/Permeabilization kit in accordance with the pro-tocol (eBiosciences), and then labeled with anti-Foxp3- phycoerythrin (PE) monoclonal antibodies (eBioscience). Non-specific fluorescence was determined by using an isotype-matched IgG as control. Flow cytometry was performed on a Cytomics FC500 flow cytometer (Beckman Coulter, Inc., USA). The frequency of CD4⁺CD25⁺ cell subset was calculated based on the percentage of positive cells in the total lymphocyte gate. For CD4⁺CD25⁺Foxp3⁺ cells, values were subsequently readjusted to represent percent of these cells within the CD4⁺ lymphocyte population. As the overall numbers of CD4⁺ cells remained constant,²⁵ the frequency of the CD4⁺CD25⁺Foxp3⁺ population was calculated as a percentage of positive cells in all CD4⁺ T lymphocytes.

Western blot analysis

Splenocyte tissue was cut into 3 × 3 mm pieces and lysed by phenylmethylsulfonyl fluoride (PMSF) lysis buffer. The quantity of protein was detected by Bradford method (Bio-Rad). Subsequently, 40 μg protein was loaded per line and were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and then were transferred to nitrocellulose membranes (Millipore). The membranes were blocked by 5% non-fat milk (Bio-Rad) in TBST (Tris-buffered saline containing 0.05% Tween-20) for 1.5–2 h and then washed with 3 × 10 min using TBST. Subsequently, membranes were incubated with rabbit anti-mouse Foxp3 (1 μg/mL; E. Schmitt, Institute of Immunology, Mainz, Germany) at 4°C overnight, followed by TBST washing. HRP-labeled anti-rabbit IgG (1:1000, DAKO, Hamburg, Germany) was then used for 1 h at room temperature and washed three times in TBST for 10 min each time. The proteins were visualized by enhanced chemiluminescence (ECL) (G:BOX chemiXR5, SYNGENE, UK) and Gel-pro32 software was used for gray analysis. Also, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control.

Real time-PCR

Splenocyte tissue was homogenized in 1 mL of precool TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Then RNA was extracted according to the description in the TRIzol protocol. The RNA concentration was quantified with a Spectrophotometer (Eppendorf, Germany). Subsequently, RNA was transcribed to cDNA using reverse transcriptase (Takara Shuzo Co., Tokyo, Japan). RT-PCR was then performed with transcribed cDNA as template and real-time PCR Master Mix (SYBR Green) using DA7600 Sequence Detection System (Zhong-shan Da-An, PR China). Primers for GAPDH amplification were (103 bp) 5’-GGCCTTCCGTGTTCCTACC -3’ (sense) and 5’-CGCCTGCTTCACCACCTTC -3’ (anti-sense) with product of 103 bp. Primers for Foxp3 were 5-GGCTCTACTCTGCACCTTCC-3 (sense) and 5-GCAGTGGGTAGGATCCTTGT-3 (anti-sense) with product of 86 bp. Amplification was then performed in a thermocycler (Eppendorf, Germany) as follows: 2 min at 94°C, followed by 36 cycles of 30 s each at 94°C, 30 s at 60°C, and 1 min at 72°C. After cycling, there was a DNA extension period of 10 min at 72°C. All PCR reactions were performed in triplicate. The results were analyzed by ABI Step one plus real-time PCR system (USA). The relative expression of Foxp3 was calculated according to 2^-CT with GAPDH as an internal control.²⁶

Statistical analysis

Data were expressed as mean ± standard deviation (SD). Least significant difference (LSD) method was used for multiple pair-wise comparisons of data which followed normal distribution. Rank sum test was performed for data that did not follow normal distribution. *P <0.05 was considered significant and **P <0.01 was considered extremely significant. All statistical analysis was performed on SPSS19.0 software.

Results

Effect of YWF on nasal symptoms in rats

The number of sneezes, degree of runny nose, and nasal rubbing movements were recorded in 30 min on days 1 and 7 after sensitization, and on days 7, 14, and 28 after medicine treatment; all these nasal symptoms were presented as scores (Figure 1). YWF inhibited nasal symptoms in a dose- and time-dependent manner. On the 28th day, the results showed that the decrease of the score in the YWF low, median, and high-dose groups was significant compared with the model control. The therapy effects in the YWF median and high-dose groups were superior to those in the loratadine group (P <0.01). However, there was no significant difference between the high-dose group and the median-dose group as well as the low-dose group and the loratadine group (P >0.05). Loratadine induced the inhibition of antigen-induced nasal symptoms at clinic dose (Figure 1).

Figure 1.

Scores of nasal symptoms were evaluated in normal control group (n = 8, □), model group (n = 8, ○), low dose of Yiqi Wenyang Fang (YWF) group (n = 8, ◊), median dose of YWF group (n = 8, ▽), high dose of YWF group (n = 8, △), and loratadine group (n = 8, ◁).

The score of the normal control group was persistently at a low level. The score of the other experimental groups peaked at 7 days after nasal sensitization, and gradually decreased after drug treatment. The scores in the high-dose and median-dose groups at day 28 were close to those in the normal control group (P >0.05). However, the scores in the low-dose group and the loratadine group at day 28 still had significant differences with the normal control group (P <0.01).

Effect of YWF on histology changes

The lesions were evaluated by epithelial disruption and infiltration of inflammatory cells (mainly eosinophils) in the nasal mucosa (Figure 2) and quantified by scores (Figure 3). No pathological abnormalities were observed in the nasal mucosa of normal control group (Figure 2). Ovalbumin-sensitized and challenged rats showed marked infiltration of eosinophils and gross alteration of epithelial cells in the inner wall of the nasal cavity. In particular, the tissue in the model control group showed marked epidermal cell degeneration and necrosis, significant eosinophils infiltration, sub-epithelial hyperemia, and inflammatory exudate (mainly granulocytes) in the nasal cavity (Figure 2). YWF at all doses significantly protected nasal mucosa against damage (less degenerative epithelium) compared with the model control (P <0.05). YWF treatment inhibited the inflammation by minimizing the inflammatory cell infiltration. Moreover, YWF administration resulted in less sub-epithelial hyperemia and inflammatory exudate (Figure 2). The total histological score of lesion extent was significantly reduced by YWF treatment (P <0.05) (Figure 3). However, the therapy effects of the YWF high-dose group and YWF median-dose group had no significant difference with those in the loratadine group (P >0.05). The YWF high-dose group had significant improvement in histological modification compared with other treatment groups (P <0.05) but still had significant difference with the normal controls (P <0.05).

Figure 2.

Histological findings of nasal mucosa in each group (hematoxylin and eosin (H&E) stain, 200×, scale bar = 50 μm). YWF treatment reduced the eosinophils infiltration. Moreover, YWF administration resulted in less degenerative epithelium, sub-epithelial hyperemia, and inflammatory exudate. The arrows represent eosinophil infiltration. The asterisks indicate inflammatory exudate.

Figure 3.

Scores of nasal tissue damage. *P <0.05; **P <0.01.

Expression of IL-10, TGF-β1, IL-4, and IL-13

ELISA was performed to detect the content of IL-10, TGF-β1, IL-4, and IL-13 in serum in all groups. The results showed that the expression of IL-10 and TGF-β1 was significantly reduced after antigen sensitization and YWF increased the content of IL-10 and TGF-β1 in a dose-dependent manner (P <0.01; Figure 4a and b). The content of IL-4 and IL-3 were significantly higher in the model group compared with the normal control group (P <0.05). YWF at high and median doses significantly decreased the content of IL-4 and IL-13 (P <0.05) compared with the model control, while there was no significant difference of IL-4 and IL-13 expression in the low dose group compared with the model group (P >0.05, Figure 4c and d).

Figure 4.

Content of interleukin (IL)-10, transforming growth factor (TGF)-β1, IL-4, and IL-13 detected in each group. **P <0.01.

Expression of Foxp3

Protein expression of Foxp3 was detected by western blot and the mRNA expression was detected by RT-PCR analysis. It was found that the expression level of Foxp3 was lower in the model group compared to the normal control group and YWF increased the protein expression of Foxp3 (Figure 5a). Similarly, YWF increased the mRNA expression of Foxp3 (Figure 5b).

Figure 5.

Protein (a) and mRNA (b) expression of Foxp3 in in the normal control group, model group, high dose of YWF group, median dose of YWF group, low dose of YWF group, and loratadine group. **P <0.01.

Functional CD4⁺CD25⁺Treg cells recruitment

The level of CD4⁺CD25⁺Foxp3⁺ iTreg cells in the model group was significantly decreased compared with normal controls (P <0.01; Figure 6). YWF at high and median doses increased the level of CD4⁺CD25⁺Foxp3⁺ Treg cells compared with the model group (P <0.01; Figure 6). The level of CD4⁺CD25⁺Foxp3⁺ iTreg cells by loratadine treatment was also higher than model group (P <0.05; Figure 6).

Figure 6.

Recruitment of CD4⁺CD25⁺Foxp3⁺ Treg regulatory cells in rats by YWF treatment. A. Twenty-eight days after the YWF, blood in the femoral artery was prepared and analyzed by flow cytometry in normal control group (a), model group (b), high dose of YWF group (c), median dose of YWF group (d), low dose of YWF group (e), and loratadine group (f). B. Statistical analysis of percentages of CD4⁺CD25⁺Foxp3⁺ T regulatory cells in six groups. *P <0.05; **P <0.01.

Discussion

Effective therapy of YWF on AR rats was investigated in this study. Number of sneezes, degree of runny nose, and nasal rubbing movements were alleviated by YWF. CD4⁺CD25⁺Treg cells were recruited and expression of Fox3 was significantly increased after YWF treatment.

The symptoms of AR consist of sneezing, rhinorrhea, and nasal itching along with release of granule constituents from eosinophils, thereby inducing damage of nasal mucosal tissue.³ In the present study, the scores of symptoms were significantly increased after antigen treatment compared with normal controls. YWF and loratadine treatment reduced the scores of symptoms obviously, especially for YWF at high and median doses. Histology changes demonstrated the damage alleviation of nasal epithelial cell and decrease of granulocyte filtration by YWF and loratadine treatment. Thus, YWF may be an effective therapy for AR in a rat model.

In the present study, the expression of IL-10 was decreased in the model group compared with normal controls while YWF increased the expression of IL-10. Similar to IL-10, the expression of TGF-β1 was reduced by antigen sensitization and increased by YWF treatment. The expression of IL-4 and IL-13 were both increased after antigen induction. YWF at high and median doses significantly decreased the content of IL-4 and IL-13 compared with the model controls. Studies reported that TGF-β could convert naïve CD4⁺CD25⁻ T cells into CD4⁺CD25⁺ anergic/suppressor T cells in the periphery, involving the induction of Foxp3 expression.^27,28 The work of Zheng et al. demonstrated that the suppressive effects of these secondary CD4⁺CD25⁺ cells depended on TGF-β and IL-10.²⁹ Evidence showed that the number of allergen-specific IL-10-secreting type I Treg cells was decreased in AR patients.³⁰ IL-4 and IL-13 production was related to lgE.³¹ It has been reported that IL-13 was suppressed while IL-10 and TGF-β were increased in AR patients in response to the major house-dust mite and birch pollen allergens.³² SLIT increased the expression of IL-10 in the later phase of therapy.³³ The expression of IL-13 was decreased in AR patients who received topical fluticasone propionate compared with those who received placebo.³⁴ Thus, we could conclude that YWF may act as immunotherapy for AR through an increase of IL-10 and TGF-β1 and suppression of IL-4 and IL-13.

In Lee’s study, the mRNA expression of Foxp3 by RT-PCR detection was significantly decreased in AR patients.³⁵ In our study, we could observe that the protein and mRNA expression levels of Foxp3 were both promoted after YWF administration. Foxp3 is a transcription factor that is genetically defective in inflammatory syndrome in humans and mice.³⁶ Besides, Scadding et al. had reported that Foxp3+ cells were increased in the sublingual epithelium of AR patients following grass pollen SLIT.³⁷ The function of CD4⁺CD25⁺ Treg cells that play important roles in the allergic inflammatory response is regulated by the transcription factor Foxp3.³⁸ The number of Fox3⁺CD25⁺CD4⁺ Treg cells was increased in the nasal mucosa of patients with AR after grass pollen immunotherapy.³⁹ In this study, level of Fox3⁺CD25⁺CD4⁺ Treg cells was decreased after antigen induction and increased after YWF treatment. The number of Fox3⁺CD25⁺CD3⁺ Treg cells was reported to be similar in AR patients compared with healthy participants.³⁰ Collectively, we suggest that Foxp3 may regulate the number of CD4⁺CD25⁺ Treg cells in the process of YWF treatment for AR, which may be a potential underlying mechanism of YWF on AR while further studies and experimental verifications are needed.

In summary, YWF ameliorates AR in allergen-induced rats by increasing IL-10 and TGF-β1, and decreasing IL-4 and IL-13, and recruitment of Fox3⁺CD25⁺CD4⁺ Treg cells, thereby inhibiting the immune response to allergens. The express-ion of Foxp3 was also involved in the process of YWF treatment. Further investigations are needed to confirm the results and clarify more precise mechanisms.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

This work was supported by grants from Jiangsu Provincial Administration of Traditional Chinese Medicine Program (LZ130055) and by grants from Jiangsu Province Hospital of Traditional Chinese Medicine Program (K2013Y29).

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Yiqi Wenyang Fang ameliorates allergic rhinitis through inhibiting inflammatory response and promoting the expression of Foxp3

Abstract

Keywords

Introduction

Materials and methods

Rats

Ovalbumin sensitization and challenge protocols

Medication

Treatment procedures

Evaluation of nasal symptoms

Histopathological examination

Enzyme-linked immunosorbent assay (ELISA)

Flow cytometry

Western blot analysis

Real time-PCR

Statistical analysis

Results

Effect of YWF on nasal symptoms in rats

Effect of YWF on histology changes

Expression of IL-10, TGF-β1, IL-4, and IL-13

Expression of Foxp3

Functional CD4+CD25+Treg cells recruitment

Discussion

Footnotes

Declaration of conflicting interests

Funding

References

Functional CD4⁺CD25⁺Treg cells recruitment