Mahuang Fuzi Xixin Decoction Attenuates Th1 and Th2 Responses in the Treatment of Ovalbumin-Induced Allergic Inflammation in a Rat Model of Allergic Rhinitis

Mengyue Ren, Qingfa Tang, Feilong Chen, Xuefeng Xing, Yao Huang, Xiaomei Tan, Mengyue Ren, Qingfa Tang, Feilong Chen, Xuefeng Xing, Yao Huang, Xiaomei Tan

Abstract

Allergic rhinitis (AR) is one of the most common allergic diseases, which adversely affect patients' quality of life. Mahuang Fuzi Xixin decoction (MFXD) has been widely used to treat AR in clinics in Asian countries. This study investigated the effect and possible therapeutic mechanisms of MFXD in the treatment of AR. A Wistar rat model of ovalbumin- (OVA-) induced AR was established and then treated with three doses of MFXD; AR symptoms, serum total immunoglobulin E, histamine, histopathological features, and release and expression of factors related to type 1 helper T (Th1) and type 2 helper T (Th2) responses were analyzed. Our study demonstrated that MFXD has a good therapeutic effect on OVA-induced allergic inflammation in an AR rat model as manifested in reduced frequencies of sneezing and nasal scratching and in reduced serum levels of total IgE and HIS. In addition, MFXD regulates imbalance in Th1/Th2 cells caused by AR by simultaneously attenuating Th1 and Th2 responses, such as by reducing the serum levels of IFN-γ and IL-4 and mRNA expression levels of IFN-γ, IL-4, GATA-3, and STAT-6. This study provided valuable information on the immunoregulatory effect of MFXD for the treatment of AR in future clinical studies.

Figures

Figure 1
Figure 1
Schematic diagram of preparation of AR model rats and MFXD administration. Rats were intraperitoneally sensitized with OVA and Al(OH)3 dissolved in physiological saline once every other day for 2 weeks and then challenged through nasal instillation with 5% OVA into each nasal cavity once daily from day 15 to day 21. Rats in MFXD, loratadine, and model groups were orally administered with three doses of MFXD, loratadine solution, and water once daily from day 22 to day 31, respectively, and were nasally instilled with OVA solution once every other day to maintain the nasal stimulation. Rats in the control group were sensitized with Al(OH)3, challenged with saline, and orally administered with water. All the rats were sacrificed on day 31.
Figure 2
Figure 2
Total ion chromatograms of MFXD. 10 groups of MFXD consisting of different batches of Mahuang, Fuzi, and Xixin were diluted to 0.06 g/mL and precipitated with equal methanol. The supernatants were obtained by centrifugation (10000 rpm) for 10 min and analyzed by UPLC-MS/MS using a mobile phase consisting of acetonitrile (a) and 0.1% formic acid aqueous solution (b) with a gradient program. The injection volume was 1 μL, the flow rate is 0.4 mL/min, and the column temperature was 25°C. (a) The total ion chromatograms of 10 groups of MFXD. (b) The MFXD fingerprint.
Figure 3
Figure 3
Effect of MFXD on OVA-induced allergic rhinitis in rat. (a) The frequencies of sneezing and nasal scratching of rats were counted for 30 min immediately after the last challenge by nasal instillation with OVA solution on day 31. Blood samples were collected and the serums were obtained by centrifugation. The total IgE (b) and HIS (c) levels in serum were detected by ELISA. The nasal mucosa samples were collected and stained with (d) hematoxylin and eosin (HE) and (e) toluidine blue (TB). Epithelial layer (El), ciliated cell layer (Ccl), leucocytes (represented by black arrows), and mast cells (represented by red arrows) were marked on the images. Data are expressed as mean ± SD; N = 8 rats; ∗p < 0.05, ∗∗p < 0.01 versus model.
Figure 4
Figure 4
Effect of MFXD on serum IFN-γ and IL-4 in OVA-induced rat of allergic rhinitis. Blood samples were collected after the last nasal challenge and the serums were obtained by centrifugation. The IFN-γ (a) and IL-4 (b) levels in serum were detected by ELISA. (c) The IFN-γ/IL-4 values in all groups were calculated. Data are expressed as mean ± SD; N = 8 rats; ∗p < 0.05, ∗∗p < 0.01 versus model.
Figure 5
Figure 5
Effect of MFXD on the percentages of CD3+CD4+IFN-γ+ Th1 and CD3+CD4+IL-4+ Th2 cells in peripheral blood mononuclear cells (PBMCs) of AR rats. PBMCs of rats were separated from anticoagulant blood sample, stained with fluorescently labeled anti-rat antibodies, and analyzed by flow cytometry. (a) Representative flow cytometry dot plots for each groups, and the plots in the upper right quadrant indicate the percentage of CD3+CD4+IFN-γ+ Th1 and CD3+CD4+IL-4+ Th2 cells among PBMCs. Percentages of (b) CD3+CD4+IFN-γ+ Th1 cells and (c) CD3+CD4+IL-4+ Th2 cells in each groups. (d) Ratios of CD3+CD4+IFN-γ+ Th1 and CD3+CD4+IL-4+ Th2 cells were calculated. Data are expressed as mean ± SD; N = 6 rats; ∗∗p < 0.01 versus model.
Figure 6
Figure 6
Effect of MFXD on the mRNA expression of IFN-γ, IL-4, T-bet, GATA-3, STAT-1, and STAT-6 in the nasal mucosa. Nasal mucosa samples from rats in different groups were obtained after the last nasal challenge, and total RNA was isolated and analyzed with qRT-PCR. (a) The mRNA expression of IFN-γ, T-bet, and STAT-1 in the nasal mucosa. (b) The mRNA expression of IL-4, GATA-3, and STAT-6 in the nasal mucosa. (c) The ratios of IFN-γ/IL-4, T-bet/GATA-3, and STAT-1/STAT-6 are shown. Data are expressed as mean ± SD; N = 6 rats; ∗p < 0.05, ∗∗p < 0.01 versus model.

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