International Immunopharmacology 24 (2015) 88–94

Contents lists available at ScienceDirect

International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp

Paeoniflorin attenuates allergic inflammation in asthmatic mice Jing Sun a,1, Jinfeng Wu b,1, Changqing Xu c, Qingli Luo a, Bei Li a, Jingcheng Dong a,⁎ a b c

Department of Integrative Medicine, Huashan Hospital, Fudan University, 12 Middle Urumqi Road, Shanghai 200040, China Department of Dermatology, Huashan Hospital, Fudan University, 12 Middle Urumqi Road, Shanghai 200040, China Department of Respiration, Affiliated Hospital of Hangzhou Normal University, 126 Wenzhou Road, Hangzhou 310015, China

a r t i c l e

i n f o

Article history: Received 29 April 2014 Received in revised form 27 October 2014 Accepted 14 November 2014 Available online 26 November 2014 Keywords: Paeoniflorin Asthma IL-5 IL-13 IL-17 MAPK

a b s t r a c t Paeoniflorin (PF), one of the major active ingredients of Chinese peony, has demonstrated anti-inflammatory and immunoregulatory effects. However, it has remained unclear whether PF treatment can inhibit allergic inflammation in asthma. In this study, we evaluated the effects of PF on pulmonary function and airway inflammation in asthmatic mice. The allergic asthma models were established in BALB/c mice. The mice were sensitized and challenged with ovalbumin. Airway hyperresponsiveness was detected by direct airway resistance analysis. Lung tissues were examined for inflammatory cell infiltration. IL-5, IL-13, IL-17, and eotaxin in bronchoalveolar lavage fluid (BALF) and their mRNA expression in lung tissue were examined by ELISA and realtime PCR, respectively. The total IgE level in serum was measured by ELISA. The protein expression of p-ERK and p-JNK was detected by western blot. Our data showed that PF oral administration significantly reduced airway hyperresponsiveness to aerosolized methacholine and decreased IL-5, IL-13, IL-17 and eotaxin levels in the BALF, and decreased IgE level in the serum. Histological studies showed that PF administration markedly decreased inflammatory infiltration. Similarly, treatment with PF significantly inhibited IL-5, IL-13, IL-17 and eotaxin mRNA expression in lung tissues. The protein expression levels of p-ERK and p-JNK were substantially decreased after oral administration of PF. In summary, PF displayed anti-inflammatory effects in the OVA-induced asthmatic model by decreasing the expression of IL-5, IL-13, IL-17 and eotaxin. These effects were mediated at least partially by inhibiting the activation of MAPK pathway. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Bronchial asthma is one of the most common chronic inflammation diseases, characterized by airway hyperresponsiveness (AHR), chronic eosinophilic inflammation, episodic airflow obstruction that is at least partially reversible and airway remodeling that includes subepithelial fibrosis, increased airway smooth muscle, increased vascularization of the airway wall, and goblet cell and submucosal gland hyperplasia [1–3]. Many inflammatory cells and mediators are well-known as critical participants in allergic diseases [4]. In asthma, increased numbers of eosinophils are observed in the circulation and sputum [5]. More and more evidence suggests that eosinophils participate in a wide variety of functions in lung allergic inflammation, including airway epithelial cell damage and loss, airway dysfunction of cholinergic nerve receptors, airway hyperresponsiveness, mucus hypersecretion, and airway remodeling, characterized by fibrosis and collagen deposition [6–8]. In addition, eosinophils express surface membrane receptors with high ⁎ Corresponding author. Tel.: +86 2152888301; fax: +86 2152888265. E-mail address: [email protected] (J. Dong). 1 These two authors contributed equally to this work and should be considered as cofirst authors.

http://dx.doi.org/10.1016/j.intimp.2014.11.016 1567-5769/© 2014 Elsevier B.V. All rights reserved.

affinity and specificity for IgE. The interaction of antigen-bound IgE in surface membrane receptors releases histamine, prostaglandins, leukotrienes and cytokines. These cytokines activate chemotaxis and phagocytosis of neutrophils and macrophages. Finally, cytokine-induced reactions cause tissue inflammation [9]. The number of eosinophils in the lung is associated with disease severity and has been used to guide therapy in severe asthma. Eosinophils are considered as a potential target for the treatment of asthma [6]. A biased immune response towards Type 2 T helper (Th2) cells is observed in asthma, which is characterized by their production and secretion of cytokines including IL-4, IL-5, IL-9, and IL-13 [10]. These cytokines play an important role in driving eosinophilic inflammation and tissue damage, leading to AHR and the release of additional mediators [11]. Chinese herbal medicines have been used for treating allergic diseases for thousands of years. The effectiveness of herbal medicine has received increasing attention [12,13]. Paeoniflorin (PF) is one of the main active ingredients of Chinese peony which is also known as Paeonia lactiflora Pall. PF has demonstrated anti-inflammatory and immunoregulatory effects [14]. A recent study showed that PF could improve IgE-induced anaphylaxis and scratching behaviors [9]. Little is known about the effectiveness of PF treatment in allergic asthma. The aim of this study was to obtain in vivo evidences to show that PF can

J. Sun et al. / International Immunopharmacology 24 (2015) 88–94

improve pulmonary function and reduce inflammation in allergic asthma mice. 2. Materials and methods 2.1. Reagents and animals PF (purity N 99.8%, Fig. 1) was purchased from Mansite Biotechnology Co. (Chengdu, China); molecular formula: C23H28O11; molecular weight: 480.45. Four-to-six-weeks-old female BALB/c mice weighing 18–22 g were obtained from Department of Laboratory Animal Science, Fudan University. The animals were housed under specific pathogen-free conditions in a temperature and humidity controlled environment and given ad libitum access to water and food. Mice were housed for 7 days for acclimation before experiments.

89

sodium pentobarbitone (wt/vol) at a dose of 50 mg/kg by intraperitoneal injection. AHR was evaluated by a Buxco's modular and invasive system (Buxco Electronics Inc., NY). Changes in airway resistance (RL) and lung dynamic compliance (Cdyn) were measured directly as described by Amdur and Mead [16]. Briefly, each anesthetized mouse was tracheostomized and intubated with an appropriate cannula, and then laid supine inside the body plethysmograph chamber connected to the ventilator. After a stable baseline airway pressure (b5% variation over 2.5 min) is reached, saline and increasing concentrations of methacholine (3.125, 6.12, and 12.5 mg/mL) in succession were administered via a jet nebulizer into the head chamber. Minimum values for RL were determined and expressed as percent change from the baseline value [17]. 2.5. Histopathological evaluation

The BALB/c mice were sensitized and challenged with OVA (grade V, Sigma, Taufkirchen, Germany) according to the protocol described previously [15]. Briefly, on day 0, mice were intraperitoneally injected with 20 μg of OVA precipitated with 2 mg of aluminum hydroxide gel in 0.2 mL saline. This was repeated on day 7, day 14 and day 21 for sensitization. On day 25, each animal was placed into an individual chamber and inhaled 3% OVA to challenge them for 6 consecutive days, every day for 30 min to replicate allergic asthma (Fig. 2).

The right upper lobe of the lung was removed and fixed in 10% neutral-buffered formalin, embedded in paraffin, cut into 4 μm sections and stained with hematoxylin and eosin (HE), another was stored at − 80 °C. The histological slides with H&E stain were read by a light microscope at high power (200×). Histopathological assessment (light microscopy) was performed blind on randomized sections. The severity of inflammatory cell infiltration in the lung was evaluated by a 5 point scoring system: 0, no cells; 1, a few cells; 2, a ring of cells 1 cell layer deep; 3, a ring of cells 2–4 cells deep; and 4, a ring of cells N 4 cells deep [18].

2.3. Animal treatment

2.6. Measurement of total IgE in serum

The whole protocol was approved by the Institutional Animal Care and Use Committee of Fudan University. Mice were randomly assigned to 6 experimental groups (n = 10 per group). Control Group (CON): Did not receive any treatment. Model Group (MOD): Mice were sensitized and challenged with OVA as described above. Low-dose PF Group (LDP), Medium-dose PF Group (MDP) and High-dose PF Group (HDP): Mice in these groups were sensitized and challenged with OVA as described in the model group. Simultaneously from days 24 to 30, they were treated with 10, 25, and 50 mg/kg PF solutions in 0.3 mL saline by gavages 1 h before OVA challenge, respectively.

After the methacholine challenge and assessment of AHR, the serum was separated by centrifuging at 1200 ×g for 15 min at 4 °C. Aliquot serum was stored at −80 °C. The concentrations of total IgE in serum were determined by using an ELISA kit (R&D, Minneapolis, MN), according to the manufacturer's instructions.

2.2. Allergic asthma model

2.4. Measurement of AHR Before the methacholine challenge and measurement of airway hyperresponsiveness (AHR), the mice were anesthetized with 1%

2.7. Preparation and analysis of bronchoalveolar lavage fluids (BALF) Mouse was anesthetized and a tracheal cannula was inserted via a midcervical incision, the right lung was ligated and the airway of each mouse was lavaged 3 times with 1 mL of PBS. The collected lavage fluid was centrifuged at 1000 ×g at 4 °C for 10 min. The supernatants were harvested and stored at − 80 °C for measurements of cytokine production. The levels of interleukin (IL)-5, IL-13, IL-17, and eotaxin were analyzed in BALF by using the ELISA kits (R&D, Minneapolis, MN), following the manufacturer's instructions. 2.8. RNA extraction and quantitative real-time PCR

Fig. 1. Chemical structure of paeoniflorin (PF).

Total RNA was extracted from the lung tissues with Trizol (TaKaRa Biotechnology Co., Ltd.), and the quality of RNA was subsequently evaluated by measuring the ratio of the absorbance at 260/280 nm. For reverse transcription, the First Strand cDNA Synthesis Kit was used. For PCR amplification, the following mouse-specific sense and antisense primers were used: IL-5, 5′ AAG GCT GAG GTT ACA GA 3′ (forward) and 5′ ATG AGG GGG AGG GAG TAT 3′ (reverse); IL-13, 5′ CCA CAC AGG GCA ACT GAG 3′ (forward) and 5′ G GCA TAG GCA GCA AAC CAT 3′ (reverse); IL-17, 5′ ATT CAG AGG CAG ATT CAG 3′ (forward) and 5′ AAA AAC AAA CAC GAA GCA G 3′ (reverse); eotaxin, 5′ CAC CCT GAA AGC CAT AGT 3′ (forward) and 5′ GT CAA GAG AGG AGG TTG TT 3′ (reverse); and GAPDH, 5′ TGG TGA AGG TCG GTG TG 3′ (forward) and 5′ GG TCA ATG AAG GGG TCG TT 3′ (reverse). The amplification was carried out in the ABI-7500 instrument (Applied Biosystems, USA) under the following conditions: initial denaturation at 95 °C for 10 min, 40 cycles of amplification at 95 °C for 15 s, annealing at 60 °C for 25 s, and then extension at 72 °C for 30 s.

90

J. Sun et al. / International Immunopharmacology 24 (2015) 88–94

Fig. 2. Protocol and measurements for the mice model of allergic asthma: mice were sensitized and challenged by OVA as described in the Materials and methods section.

2.9. Western blot analysis Proteins were extracted from the right lower lobe of the lung using the cell lysis buffer (150 mmol/L NaCL, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mmol/L Tris–Cl pH 8.0, 2 ug/mL aprotinin, 2 ug/mL leupeptin, 40 mg/mL of phenylmethylsulfonyl fluoride, 2 mmol/L DTT) and centrifuged at 12,000 rpm for 15 min to remove nuclei and cell debris. Supernatants were then quickly frozen at − 80 °C until use. The protein concentrations were determined by the Bradford assay (Biorad, Hercules, CA) and 30 μg of cellular proteins was electro-blotted onto a PVDF membrane following separation on a 10% SDS–polyacrylamide gel electrophoresis. The immunoblot was blocked for 1 h with 5% milk at room temperature followed by an overnight incubation at 4 °C with a 1:1000 dilution of primary antibody against p-ERK, p-JNK or GAPDH. Blots were washed twice with Tween 20/Tris-buffered saline (TTBS) before addition of a 1:1000 dilution of HRP-conjugated secondary antibody for 1 h at room temperature. Blots were again washed with TTBS before development by enhanced chemiluminescence using Supersignal West Femto Chemiluminescent Substrate (Thermo, Rockford, IL). Band intensities were quantified using UN-SCAN-IT gel analysis software (version 6). The optical density for target protein was shown as a proportion of GAPDH optical density. The western blot data were replicated three times.

Histological pictures of the lung tissue showed that there were more severe perivascular eosinophilia, peribronchiolar eosinophilia, epithelial damage and oedema in the model group, as compared with the control group. In contrast, PF treatment remarkably attenuated infiltration of inflammatory cells in airways at the doses of 50 mg/kg (Fig. 4). 3.3. Effect of PF on the serum IgE level of asthmatic mice IgE levels in serum were quantitated by using an ELISA's kit. As shown in Fig. 5, OVA-exposed mice presented significantly elevated levels of IgE, as compared with normal mice. PF 50 mg/kg treatment markedly attenuated the increases of IgE level in serum. The IgE levels did not significantly decrease after PF intervention at the doses of 10 mg/kg and 25 mg/kg. 3.4. PF reduced cytokine levels in BALF Airway inflammation in asthma is characterized by an imbalance of Th1/Th2 cytokines. Thus, to determine whether PF could modulate this

2.10. Statistical analysis Data were from three independent experiments and expressed as mean ± SEM. Statistical analyses were performed by the Student's t-test for paired data and the one-way Analysis of Variance (ANOVA) for differences between treatment groups. All analyses were undertaken using statistic software SPSS 16.0. A P value less than 0.05 was considered to be statistically significant. 3. Results 3.1. PF inhibits AHR in allergic asthma mice To evaluate the effect of PF on OVA-induced AHR, the airway responsiveness to aerosolized PBS or methacholine was assessed within 24 h after the final challenge. Only mild changes in airway resistance (RL) were observed in normal mice. However, there was a substantial enhancement of airway responsiveness in OVA-exposed model mice, with an obvious increase in RL, as compared with control mice (P b 0.05) (Fig. 3A). Oral administration of 50 mg/kg PF exhibited dramatically reduced bronchial responsiveness to inhaled methacholine, as compared with the model group. Whereas there were no significant differences in methacholine responsiveness between the PF 10 mg/kg treatment group, 25 mg/kg treatment group and the model group (Fig. 3B). 3.2. Effect of PF on OVA-induced lung tissue inflammation To demonstrate the inhibitory effects of PF on inflammation in the lung tissue, pulmonary pathology was observed by H&E staining.

Fig. 3. Airway responsiveness to aerosolized methacholine was evaluated by a Buxco's modular and invasive system, as described in the Materials and methods section. The mice were laid in a supine position inside the body plethysmograph chamber and were nebulized with PBS followed by increasing doses (3.125 to 12.5 mg/mL) of methacholine. The data were expressed as the mean ± S.E.M (A) Group CON versus Group MOD (P b 0.05); (B) Group MOD versus Group HDP (P b 0.05). The number of mice in each group was 10.

J. Sun et al. / International Immunopharmacology 24 (2015) 88–94

91

Fig. 4. Effects of PF on airway inflammation in the lung tissue. (A) Lung tissue slices were fixed, embedded, sectioned at 3–4 μm and stained with H&E, and observed under a microscope (200×). Representative pictures are shown for each group. (B) Graphs represent the inflammation score. The results are expressed as the mean ± S.E.M. #P b 0.05 versus CON group; *P b 0.05 versus MOD group.

imbalance, levels of IL-5 and IL-13 were measured by ELISA. BALF IL-5 and IL-13 levels, which were barely detectable in control mice, were significantly increased by OVA-sensitization and challenges. In contrast, treatment with PF significantly attenuated the up-regulation of IL-5 and IL-13 levels in BALF (Fig. 6). As IL-17 and eotaxin also play important roles in allergic asthma, we measured IL-17 and eotaxin levels in BALF. As compared with the control group, the expression levels of IL-17 and eotaxin were remarkably increased in the allergic asthma mice. In contrast, the oral administration of PF decreased IL-17 and eotaxin in BALF (Fig. 6). 3.5. Effects of PF on expression of cytokines mRNA in the lung tissue To verify the ELISA data, we further investigated IL-5, IL-13, IL-17 and eotaxin gene activation in the lung tissue of mice. As shown in

Fig. 7, OVA-exposed mice presented significantly elevated mRNA expression of IL-5, IL-13, IL-17 and eotaxin, as compared with the control group. In contrast, treatment with PF inhibited IL-5, IL-13, IL-17 and eotaxin mRNA expression. 3.6. PF inhibited p-ERK and p-JNK activation in the lung tissue It has been confirmed that mitogen-activated protein kinases (MAPKs) play significant roles in the growth and activation of inflammatory cells in the lung tissue. We also investigated whether PF treatment could interfere with the MAPK signaling pathways. The protein expression of p-ERK and p-JNK, which are key family members of MAPK, was detected by western blot. Western blot data showed that the protein expression of p-ERK and p-JNK was substantially increased after OVA exposure, as compared with normal mice. Whereas PF administration remarkably decreased the expression of p-ERK and p-JNK proteins, as compared with the model group (Fig. 8). 4. Discussion

Fig. 5. Effects of PF on IgE secretion in serum. The levels of IgE were measured by ELISA. The data are expressed as the mean ± S.E.M. #P b 0.05 versus CON group; *P b 0.05 versus MOD group.

OVA exposure remains a common method used to model asthma in mice [19]. In this study, OVA-exposed mice exhibited substantially enhanced bronchial responsiveness to inhaled methacholine, as compared with the control group. In addition, pictures of pathological lung tissue demonstrated significant infiltration of inflammatory cells into the peribronchiolar and perivascular connective tissues in the model group of mice. There were significant differences in inflammatory scores between normal mice and OVA-exposed mice. These findings confirmed that the mice model of allergic asthma was successful. The data of pulmonary function indicated that PF treatment at the dose of 50 mg/kg significantly reduced bronchial responsiveness to inhaled methacholine, as compared with OVA-exposed mice. It revealed that

92

J. Sun et al. / International Immunopharmacology 24 (2015) 88–94

Fig. 6. Effect of PF on cytokines and chemotactic factor secretion in BALF. The levels of the cytokines and chemotactic factor: IL-5, IL-13, IL-17 and eotaxin were measured by ELISA, respectively. The data are expressed as the mean ± S.E.M. #P b 0.05 versus CON group; *P b 0.05 versus MOD group.

PF treatment could protect pulmonary function in mice with allergic asthma. In this study, several mice had mild diarrhea after PF treatment, no significant side effects were observed. Clinical trials

demonstrated that the adverse events of total glucosides of peony were mainly gastrointestinal tract disturbances, mostly mild diarrhea [20].

Fig. 7. Effects of PF on mRNA expression of IL-5, IL-13, IL-17 and eotaxin in the lung tissue. The expression of IL-5, IL-13, IL-17 and eotaxin mRNA was measured by quantitative real-time PCR. The relative expression levels were calculated using 2−ΔΔCT methods. * indicates P b 0.05, as compared with MOD group.

J. Sun et al. / International Immunopharmacology 24 (2015) 88–94

Fig. 8. Effect of PF on protein expression of p-ERK and p-JNK in the lung tissue. The expression of p-ERK and p-JNK was measured by western blot. Total protein was isolated from the lung tissue of mice. Band intensities were quantified using UN-SCAN-IT gel analysis software (version 6). The optical density for the target protein was shown as a proportion of GAPDH optical density. # indicates P b 0.05, as compared with CON group, * indicates P b 0.05, as compared with MOD group.

Inflammation has been regarded as the main characteristics of asthma which leads to AHR and airway obstruction, mucus hyperproduction and airway wall remodeling [21]. Eosinophils have been viewed as key inflammatory cells in allergic asthma [22,23]. According to histological pictures of the lung tissue and inflammation scores, inflammatory cell infiltration, especially eosinophilia infiltration, was improved after PF administration at a dose of 50 mg/kg. Chemotactic factor, chemokine receptors and cytokines play important roles in the course that eosinophilia infiltrate inflammatory tissue from blood circulation [24]. Eotaxin is a member of the chemotactic factor family. Endothelial cells, smooth muscle cells, epithelial cells, alveolar macrophages and eosinophils can produce eotaxin. In asthma, eotaxin production is increased in the bronchial airway lumen and mucosa. Eotaxin actively participates in asthma pathogenesis by activating eosinophil recruitment [25,26]. In this study, the levels of eotaxin were remarkably increased after OVA stimulation, whereas PF treatment inhibited the production of eotaxin. In addition, IgE also participates in the activation of eosinophils. Eosinophils express surface membrane receptors with high affinity and specificity for IgE. The interaction of antigen-bound IgE in surface membrane receptors releases histamine, prostaglandins, leukotrienes and cytokines. These cytokines activate chemotaxis and phagocytosis of neutrophils and macrophages. Finally cytokine-induced reactions cause tissue inflammation [9]. The ELISA data indicated that IgE levels were significantly increased in OVA-exposed group mice, as compared with the control group. However, the IgE level in the PF 50 mg/kg treatment group was significantly decreased. Central to the pathogenesis of asthma, Th2 and Th17 lymphocytes of the adaptive immune system control many aspects of the disease by producing cytokines such as IL-5, IL-13, and IL-17 [21]. IL-5 has been proposed to be a key factor in regulating the growth, differentiation, recruitment, activation, and survival of eosinophils. Previous study has confirmed that IL-5 made the eosinophils accumulated in airways.

93

Anti-IL-5 treatment effectively reduced the need for oral corticosteroids while significantly reducing blood eosinophils and clinical symptoms [27]. In addition, anti-IL-5 also reduced the deposition of extracellular matrix proteins in the bronchial subepithelial basement membrane of mild atopic asthmatics [28]. At present, IL-5 has been proposed as a potential molecular target in the treatment of these diseases. Decades of research in animal models have provided abundant evidence to show that IL-13 is a key Th2 cytokine that directs many of the important features of airway inflammation and remodeling in patients with allergic asthma [29]. IL-13 participates in IgE synthesis, AHR, mucus hypersecretion, subepithelial fibrosis, eosinophilia, and the activation of certain chemokine molecules. IL-13 and its receptors have been proposed as attractive therapeutic targets for the treatment of allergic diseases. In this study, IL-5 and IL-13 were significantly increased in OVA-induced mice, as compared with control group mice. In contrast, IL-5 and IL-13 levels in PF treatment groups were significantly decreased, as compared with model group. Similar trend was observed in IL-5 mRNA and IL-13 mRNA [30]. These data revealed that PF attenuated airway inflammation via inhibiting the levels of Th2 cytokines. IL-17 is a key proinflammatory cytokine, which is produced by CD4+ T lymphocytes and eosinophils. In the lung tissue, IL-17 has been shown to contribute to the neutrophilic inflammation and airway remodeling of chronic respiratory conditions but the situation is increasingly complex [31]. According to the ELISA data, there was a significant difference in IL-17 between OVA-induced mice and normal mice. After PF treatment, IL-17 in BALF was decreased, as compared with OVA-induced group. The data of realtime PCR is similar to that of ELISA. It suggested that PF treatment markedly inhibited IL-17 expression in allergic asthma mice. The MAPK family includes three distinct stress-activated protein kinase pathways: p38, c-Jun N-terminal kinase (JNK), and extracellular regulating kinase (ERK) [32]. The ERK pathway is predominantly activated by mitogenic and proliferative stimuli, whereas the JNK and p38 MAPK pathways respond to environmental stresses. Of these kinases, much interest has been generated in terms of the potential involvement in chronic airway inflammatory diseases, such as asthma and COPD [33]. MAPKs have been shown to be important in the differentiation, activation, proliferation, degranulation and migration of various immune cells, and airway smooth muscle and epithelial cells [34]. We also investigated whether PF treatment could interfere with the MAPK signaling pathways. The western blot data indicated that the expression of p-ERK, p-JNK and p-p38 (Fig. 1S) was remarkably increased in the lung tissue of OVA-induced mice, as compared with that of model control group. In contrast, the expression of p-ERK and p-JNK proteins was significantly decreased after treatment with PF at medium and high doses. Only PF treatment at high dose altered the expression of p-p38, as compared with model group (Fig. 1S). No significant changes were observed in the expression of total ERK, JNK, and p38 (Figs. 1S, 2S) between different groups. This hinted that PF suppressed activation of MAPK pathways, leading to relieve airway inflammation in allergic asthma. In this study, the low dose of PF treatment could not protect pulmonary function and inhibit the airway inflammation in asthmatic mice. In pharmacokinetic studies, it was found that PF has a very poor absorption rate and thus a very low bioavailability (3%–4%) after oral administration. Poor permeation, p-gp-mediated efflux, and hydrolysis via a glycosidase contributed to the poor bioavailability of PF [35]. In this study, the ineffectiveness of PF treatment at low dose maybe was related to poor bioavailability of PF. In summary, the present study demonstrates that PF treatment can effectively improve pulmonary function and attenuate airway inflammation, especially eosinophilia infiltration in allergic asthma. Importantly, the levels of IgE and eotaxin expression are significantly decreased by PF administration. In addition, PF can inhibit the release of Th2 and Th17 cytokines and inactivate MAPK pathways. These

94

J. Sun et al. / International Immunopharmacology 24 (2015) 88–94

findings support PF as a potential agent for allergic asthma prevention and treatment. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.intimp.2014.11.016.

[14]

[15]

Acknowledgments

[16]

This project was funded by grants from the National Natural Science Foundation of China (81102541, 81173390), the Initial Scientific Foundation of Huashan Hospital, Fudan University (2013QD08), Natural Science Foundation of Zhejiang Province (Y2111111) and Zhejiang Province Traditional Chinese Medicine Health Science and Technology project (2012ZA105).

[17]

[18]

[19]

[20]

References [21] [1] Pascual RM, Peters SP. Airway remodeling contributes to the progressive loss of lung function in asthma: an overview. J Allergy Clin Immunol 2005;116:477–86 [quiz 87]. [2] Bergeron C, Al-Ramli W, Hamid Q. Remodeling in asthma. Proc Am Thorac Soc 2009; 6:301–5. [3] Shore SA. Modeling airway remodeling: the winner by a nose? Am J Respir Crit Care Med 2003;168:910–1. [4] Kikkawa Y, Sugiyama K, Obara K, Hirata H, Fukushima Y, Toda M, et al. Interferonalpha inhibits airway eosinophilia and hyperresponsiveness in an animal asthma model [corrected]. Asia Pac Allergy 2012;2:256–63. [5] Barnes PJ. Pathophysiology of allergic inflammation. Immunol Rev 2011;242:31–50. [6] Possa SS, Leick EA, Prado CM, Martins MA, Tiberio IF. Eosinophilic inflammation in allergic asthma. Front Pharmacol 2013;4:46. [7] Kanda A, Driss V, Hornez N, Abdallah M, Roumier T, Abboud G, et al. Eosinophilderived IFN-gamma induces airway hyperresponsiveness and lung inflammation in the absence of lymphocytes. J Allergy Clin Immunol 2009;124:573–82 [82 e1-9]. [8] Walsh GM. Targeting eosinophils in asthma: current and future state of cytokineand chemokine-directed monoclonal therapy. Expert Rev Clin Immunol 2010;6: 701–4. [9] Lee B, Shin YW, Bae EA, Han SJ, Kim JS, Kang SS, et al. Antiallergic effect of the root of Paeonia lactiflora and its constituents paeoniflorin and paeonol. Arch Pharm Res 2008;31:445–50. [10] Kung TT, Luo B, Crawley Y, Garlisi CG, Devito K, Minnicozzi M, et al. Effect of antimIL-9 antibody on the development of pulmonary inflammation and airway hyperresponsiveness in allergic mice. Am J Respir Cell Mol Biol 2001;25:600–5. [11] Chen X, Wen T, Wei J, Wu Z, Wang P, Hong Z, et al. Treatment of allergic inflammation and hyperresponsiveness by a simple compound, Bavachinin, isolated from Chinese herbs. Cell Mol Immunol 2013;10:497–505. [12] Bielory L. Complementary and alternative interventions in asthma, allergy, and immunology. Ann Allergy Asthma Immunol 2004;93:S45–54. [13] Yang SH, Hong CY, Yu CL. Decreased serum IgE level, decreased IFN-gamma and IL-5 but increased IL-10 production, and suppressed cyclooxygenase 2 mRNA expression

[22] [23] [24] [25] [26] [27] [28]

[29] [30] [31] [32] [33] [34] [35]

in patients with perennial allergic rhinitis after treatment with a new mixed formula of Chinese herbs. Int Immunopharmacol 2001;1:1173–82. Li X, Shen J, Zhong Z, Peng J, Wen H, Li J, et al. Paeoniflorin ameliorates schistosomiasis liver fibrosis through regulating IL-13 and its signalling molecules in mice. Parasitology 2010;137:1213–25. Kumar RK, Foster PS. Modeling allergic asthma in mice: pitfalls and opportunities. Am J Respir Cell Mol Biol 2002;27:267–72. Pichavant M, Goya S, Hamelmann E, Gelfand EW, Umetsu DT. Animal models of airway sensitization. In: Coligan John E, et al, editors. Current protocols in immunology; 2007 [Chapter 15:Unit 15 8]. Albertine KH, Wang L, Watanabe S, Marathe GK, Zimmerman GA, McIntyre TM. Temporal correlation of measurements of airway hyperresponsiveness in ovalbumin-sensitized mice. Am J Physiol Lung Cell Mol Physiol 2002;283:L219–33. Underwood S, Foster M, Raeburn D, Bottoms S, Karlsson JA. Time-course of antigeninduced airway inflammation in the guinea-pig and its relationship to airway hyperresponsiveness. Eur Respir J 1995;8:2104–13. Hirota JA, Hackett TL, Inman MD, Knight DA. Modeling asthma in mice: what have we learned about the airway epithelium? Am J Respir Cell Mol Biol 2011;44: 431–8. He DY, Dai SM. Anti-inflammatory and immunomodulatory effects of Paeonia lactiflora Pall., a traditional Chinese herbal medicine. Front Pharmacol 2011;2:10. Schuijs MJ, Willart MA, Hammad H, Lambrecht BN. Cytokine targets in airway inflammation. Curr Opin Pharmacol 2013;13:351–61. Jacobsen EA, Helmers RA, Lee JJ, Lee NA. The expanding role(s) of eosinophils in health and disease. Blood 2012;120:3882–90. Fulkerson PC, Rothenberg ME. Targeting eosinophils in allergy, inflammation and beyond. Nat Rev Drug Discov 2013;12:117–29. Palmqvist C, Wardlaw AJ, Bradding P. Chemokines and their receptors as potential targets for the treatment of asthma. Br J Pharmacol 2007;151:725–36. Conroy DM, Williams TJ. Eotaxin and the attraction of eosinophils to the asthmatic lung. Respir Res 2001;2:150–6. Hemelaers L, Louis R. Eotaxin: an important chemokine in asthma. Rev Med Liege 2006;61:223–6. Corren J. Inhibition of interleukin-5 for the treatment of eosinophilic diseases. Discov Med 2012;13:305–12. Flood-Page P, Menzies-Gow A, Phipps S, Ying S, Wangoo A, Ludwig MS, et al. Anti-IL-5 treatment reduces deposition of ECM proteins in the bronchial subepithelial basement membrane of mild atopic asthmatics. J Clin Invest 2003;112:1029–36. Ingram JL, Kraft M. IL-13 in asthma and allergic disease: asthma phenotypes and targeted therapies. J Allergy Clin Immunol 2012;130:829–42 [quiz 43–4]. Townley RG, Sapkota M, Sapkota K. IL-13 and its genetic variants: effect on current asthma treatments. Discov Med 2011;12:513–23. Tan HL, Rosenthal M. IL-17 in lung disease: friend or foe? Thorax 2013;68:788–90. Johnson GL, Lapadat R. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science 2002;298:1911–2. Chung KF. p38 mitogen-activated protein kinase pathways in asthma and COPD. Chest 2011;139:1470–9. Duan W, Wong WS. Targeting mitogen-activated protein kinases for asthma. Curr Drug Targets 2006;7:691–8. Liu ZQ, Jiang ZH, Liu L, Hu M. Mechanisms responsible for poor oral bioavailability of paeoniflorin: role of intestinal disposition and interactions with sinomenine. Pharm Res 2006;23:2768–80.