Effects of t Ma-Xin-DiTan decoction on ovalbumin-induced allergic asthma in mice

Purpose: To investigate the effect of the Ma-Xin-Di-Tan (MXDT) decoction on ovalbumin-induced allergic asthma (AA) in mice. Methods: Asthma was induced in mice by ovalbumin (OVA) injection, and different doses of MXDT (150, 300, and 600 mg/kg/day) were administered orally for 28 days. Pathological changes in lung tissues were examined, while levels of cytokines, including interleukin (IL)-4, IL-6, IL-17, interferon (IFN)-γ, and transforming growth factor (TGF)-β, were determined using enzyme-linked immunosorbent assays (ELISAs) of the bronchoalveolar lavage fluid. Toll-like receptor (TLR)-4, GATA-binding protein (GATA)-3, Ox40 ligand (OX40L), indoleamine 2,3-dioxygenase (IDO), forkhead box P3 (Foxp3), and T box expressed in T cells (T-bet) levels were determined in lung tissues by western blot analysis. Results: MXDT inhibited the inflammatory reaction of lung tissues in OVA-challenged mice. After treatment with MXDT, levels of IL-4, IL-6, IL-17, and TGF-β were downregulated, whereas IFN-γ levels were upregulated. In addition, MXDT decreased TLR-4, GATA-3, and OX40L levels in lung tissues but increased the expression of Foxp3, T-bet, and IDO. Conclusion: MXDT has antiallergic effects on OVA-induced AA in mice; the possible molecular mechanisms might involve the inhibition of inflammatory reactions and modulation of Th1/Th2 cytokine balance.


INTRODUCTION
Allergic asthma (AA), characterized by serious airway inflammation, airway hyperreactivity, shortness of breath, and excessive mucous production in pulmonary airways, is a common and intractable allergic disorder that affects the life quality for millions of individuals [1][2][3].It is estimated that AA affects more than 300 million patients worldwide, with an increasing prevalence in recent years [4,5].Currently available drugs, such as glucocorticoids, can only temporary control or relieve asthma symptoms, and long-term usage of such drugs can result in serious adverse reactions [2,5].Thus, more effective therapies for AA are urgently required.
It has been reported that traditional herbal medicines are a potential source of effective agents for the treatment of allergic diseases, including AA, allergic rhinitis, and eczema [2,6,7].The Ma-Xin-Di-Tan (MXDT) decoction is derived from the clinical experiences of Professor Jianer Yu in the treatment of AA (Shanghai Municipal Hospital of Traditional Chinese Medicine, Shanghai, China).MXDT is mainly composed of the whole Ephedra sinica Stapf herb, the seeds of Amygdalus Communis Vas, rainworm, and the seeds of Prunus persica (L.) Batsch.In the present study, the antiallergic effects of the Ma-Xin-Di-Tan decoction on ovalbumin (OVA)induced AA was evaluated in mice, and its molecular mechanisms were also explored.

Animal studies
A total of 60 mice (18 -22 g) were obtained from the Shanghai Laboratory Animal Research Center (Shanghai, China).All animals received humane care according to the Declaration of Helsinki, which was promulgated in 1964 and amended in 1996 [8].All experimental protocols were approved by the Animal Care and Use Committee of Shanghai Municipal Hospital of Traditional Chinese Medicine, Shanghai, China (approval no.a201611-27#).Mice were randomly divided into six groups (n = 10): normal group, control group (AA mice), positive drug treatment group (Dex), and three MXDT treatment groups (150, 300, and 600 mg/kg/day).The experimental protocol was carried out according to previous reports with some modifications [2,9].
The mice in the control, Dex, and MXDT groups were immunized intraperitoneally with a mixture of 50 mg OVA and 2 mg aluminum hydroxide on days 1 and 14.Subsequently, on days 21 through 28, the mice were challenged with a 5 % OVA solution for 40 min using an ultrasonic nebulizer (OMRON Co, Tokyo, Japan).The Dex (1.5 mg/kg/day) and MXDT (150, 300, and 600 mg/kg/day) groups were administered agents orally on days 28 through 56.Twenty-four hours after the final drug treatment, the mice were sacrificed by cervical dislocation, and the bronchoalveolar lavage fluid (BALF) and lung tissues were collected and stored at -70 °C for further analysis.

Histochemical analysis
Lungs tissues were fixed in 10 % paraformaldehyde for 24 h, then embedded in paraffin and subsequently sectioned (5-μm-thick slices).The lung tissue sections were subsequently stained with hematoxylin and eosin for routine histochemical analysis.Finally, pathological changes in the lung tissues were examined using an optical microscope (Olympus 2H12003, Tokyo, Japan).

ELISA
The levels of IL-4, IL-6, IL-17, IFN-γ, and TGF-β were quantified in the supernatant of the BALF with commercial ELISA kits following the manufacturer's instructions.

Western blot analyses
Lung tissues were homogenized in lysis buffer.Total protein was then extracted from the lung tissues.Subsequently, 35 μg total lung tissue protein were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis and transferred to a PVDF membrane, which was then probed with primary antibodies against TRL-4, GATA-3, OX40L, IDO, Foxp3, and T-bet.The PVDF membrane was incubated with horseradish peroxidase-conjugated secondary antibodies.The target protein bands were then visualized by chemiluminescence, and GAPDH was used as an internal reference.

Statistical analysis
Data are presented as the mean ± standard deviation (SD).Statistical comparisons were made by one-way analysis of variance using SPSS software (version 18.0, USA), followed by Dunnett's multiple comparison test.A P value less than 0.05 was considered statistically significant.

MXDT treatments alleviate inflammatory reactions in lung tissues of OVA-challenged mice
Results of the pathological examinations of the lung tissues are shown in Figure 1.In normal mice, bronchial wall tissues were normal and the structural integrity was intact; no obvious pathological changes were observed (Figure 1A).
In contrast, in the control mice, the bronchial walls appeared thick and damaged, and the airway lumen was obviously narrower; inflammatory cell infiltration could be observed in the airway walls and other lung tissues (Figure 1B).Interestingly, for Dex-and MXDT-treated mice, the inflammatory and pathological changes mentioned above had been significantly alleviated; no obvious bronchial wall thickening was observed, but moderate inflammatory cell infiltration could be seen in the lung tissues (Figure 1C-F).

MXDT treatments regulate cytokine levels in the BALF of OVA-challenged mice
Results of the ELISAs are shown in Figure 2. The cytokine levels of IL-4 (P < 0.01), IL-6 (P < 0.01), IL-17 (P < 0.01), and TGF-β (P < 0.01) were significantly higher in the BALF of control mice than in that of normal mice; however, the cytokine levels of IFN-γ (P < 0.01) were significant decreased in the BALF of control mice  Protein expression was determined using western blot assays.All drugs were administered orally for 28 days.Data are expressed as the mean ± SD (n = 10); **p < 0.01, compared to controls compared to in that of normal mice.Interestingly, after treatment with MXDT (150, 300, and 600 mg/kg/day) for 28 days, the increased levels of IL-4 (p < 0.01), IL-6 (p < 0.01), IL-17 (p < 0.01), and TGF-β (p < 0.01) were downregulated, whereas the decreased levels of IFN-γ (p < 0.01) were upregulated, compared to control mice.

DISCUSSION
To the best of our knowledge, the present report is the first animal research study to describe the anti-allergic effects of the MXDT decoction on ovalbumin-induced AA in mice.Animal models are a very important tool to explore human disease pathology and identify more useful and effective drugs to treat human diseases.
The mouse model of OVA-challenged AA is a broadly used tool for the investigation of asthma and for the screening of candidate antiasthma drugs [10,11].Thus, the present study successfully induced AA in mice after treatment with OVA.Inflammatory reactions play important roles in the development of AA, and various studies have demonstrated that the alleviation of inflammatory reactions is a useful way to treat asthma [12,13].The present results demonstrate that MXDT inhibits inflammatory reactions in lung tissues of mice with OVA-induced AA.
Th1/Th2 balance is recognized as an important mediator of asthma; modulation of Th1/Th2 balance is thus considered beneficial for the treatment of asthma [2,14,15].The present study revealed that after treatment with MXDT, increased levels of Th2 cytokines (IL-4 and IL-6) were downregulated, whereas decreased levels of the Th1 cytokine IFN-γ was upregulated, suggesting that MXDT can modulate Th1/Th2 balance.Clinical research has reported high serum/BALF levels of IL-7 and TGF-β in asthma patients [16,17].IL-17 is an early proinflammatory cytokine that can activate TGF-β; these two cytokines can then cause proinflammatory cytokine release, airway inflammation, goblet cell proliferation, and airway remodeling [16,17].The present results show that MXDT is able to decrease the levels of IL-7 and TGF-β in the BALF of mice with OVAinduced AA.TLR4, an important pattern recognition receptor, plays a crucial role in the reactions and secretion of inflammatory cytokines.The ratio of GATA-3, OX40L, and Tbet levels was reported to be important for Th1/Th2 balance.GATA-3 and OX40L may activate the release of Th2 cytokines, whereas Tbet commonly activates the release of Th1 cytokines [18][19][20].The relative increase in GATA-3 levels may also moderate Th1/Th2 balance [19,20].In addition, IDO and Foxp3 are reportedly able to alleviate the allergic symptoms of AA patients [21,22].The results of the present study suggest that MXDT can moderate Th1/Th2 balance by regulating TRL-4, GATA-3, OX40L, IDO, Foxp3, and T-bet levels.

CONCLUSION
The present results suggest that MXDT has potential anti-allergic effects on OVA-induced AA in mice; possible molecular mechanisms may involve the inhibition of inflammatory reactions and the modulation of Th1/Th2 balance.The present work implies that MXDT is a promising candidate for the treatment of asthma.

Figure 2 :
Figure 2: Cytokines levels in BALF.All drugs were administered orally for 28 days.Data are expressed as the mean ± SD (n = 10); **p < 0.01 compared to controls

Figure 3 :
Figure 3: TLR-4 expression in the lung tissues of OVA-challenged mice.The protein expression levels were determined using western blot assays.All drugs were administered orally for 28 days.Data are expressed as the mean ± SD (n = 10); **p < 0.01 compared to controls

Figure 4 :
Figure 4: GATA-3, OX40L, IDO, Foxp3, and T-bet expression levels in the lung tissues of OVA-challenged mice.Protein expression was determined using western blot assays.All drugs were administered orally for 28 days.Data are expressed as the mean ± SD (n = 10); **p < 0.01, compared to controls