Oridonin (purity, >98.0%; Fig. 1) was purchased from Zelang Medical Technology Co. Ltd. (Nanjing, China). Oridonin was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA). In the cell assay-based studies, the final DMSO concentration was <0.01%.

A total of 32 male BALB/c mice (age, 4–6 weeks) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). The mice were maintained under specific pathogen-free conditions for at least 7 days prior to the ex vivo and in vivo studies. The mice had access to food and water ad libitum, and were maintained at 20–26°C under a 12-h light/dark cycle. The mice were divided into 4 groups of 8, as follows: i) phosphate-buffered saline (PBS; Invitrogen; Thermo Fisher Scientific, Inc.) group; ii) OVA group; iii) Oridonin-L; and iv) Oridonin-H. All animal studies were performed according to the Guide for the Care and Use of Laboratory Animals (26).

The BALB/c mice were sacrificed with carbon dioxide and their spleens were harvested. The spleens were sliced into small pieces and pulverized using a syringe. The cell suspension in RPMI 1640 medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was filtered through a 70-µm cell strainer (BD Biosciences, Franklin Lakes, NJ, USA). Red blood cells were lysed using 10 mM EDTA (Invitrogen; Thermo Fisher Scientific, Inc.), as previously described (27). The cells were then washed with PBS and re-suspended in PBS. NycoPrep™ 1.077A (Axis-Shield PoC, Oslo, Norway) was used to isolate the lymphocytes from the spleen cells as previously described (25). Finally, the lymphocytes were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum. The primary mouse spleen cells were stimulated with 5 µg/ml concanavalin A (Sigma-Aldrich) prior to Oridonin treatment.

Supernatant and bronchoalveolar lavage (BAL) fluid from the different groups was harvested by flushing the lung with 0.5 ml ice-cold PBS, following sacrifice with carbon dioxide, as described below and stored at −80°C. Cytokine and chemokine levels in the cell supernatant or BAL fluid were measured by ELISA assay. The ELISA kits were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

On day 0 and 14, mice (8 mice/group) were injected with 20 µg OVA (Sigma-Aldrich). On day 28, 29 and 30, the mice were administrated with Oridonin (10 or 20 mg/kg) or vehicle (0.5% sodium salt of carboxy methyl cellulose), 1 h after administration the mice were challenged with OVA (1%). The administration doses were established according to a previous study (24).

AHR was evaluated in the Oridonin-treated or vehicle group mice by whole-body plethysmography 24 h after the final OVA challenge. The mice were placed in separate chambers and exposed to 20, 40, 60 and 80 mg/ml aerosolized methacholine solution (Sigma-Aldrich). The bronchoconstriction was then measured for 5 min. The highest Penh value obtained in each group was expressed as a basal Penh value compared with the PBS challenge, which served as the control.

Twenty-four-hours subsequent to the final aerosol OVA challenge, the BALB/c mice were sacrificed with carbon dioxide and the trachea of the mice were incised. Ice-cold PBS was injected into the lungs of the mice through the trachea and the BAL fluid was subsequently harvested. Each mouse was injected with PBS three times. Cytospin slides (Thermo Fisher Scientific, Inc.) were stained in a modified Wright stain (Sigma-Aldrich); the number of inflammatory corpuscles and total cells were counted using a hemocytometer (Countess II FL Automated Cell Counter; Thermo Fisher Scientific, Inc.).

All the mice were sacrificed with carbon dioxide and the lungs were harvested. The lungs were infused, via the trachea, with formalin (Sigma-Aldrich) overnight. The tissues were then paraffinized (Sigma-Aldrich) and sliced into 7-µm sections. Hematoxylin and eosin (H&E; Beyotime Institute of Biotechnology) staining was performed to evaluate eosinophil infiltration and periodic acid-Schiff staining was performed to measure mucus production. Quantitative analysis of eosinophil infiltration and mucus production was performed as previously described (28). The scoring system for cell infiltration was: 0, no cells; 1, few cells; 2, a ring of cells that were one cell layer deep; 3, a ring of cells 2–4 cell layers deep; 4, a ring of cells >4 cells deep. Goblet cell hyperplasia in the airway epithelium was quantified based on a five-point system: 0, no goblet cells; 1, <25% of the epithelium; 2, 25–50% of the epithelium; 3, 50–75% of the epithelium; 4, >75% of the epithelium.

Data were presented as means ± standard deviation for the in vitro experiments and means ± standard error of the mean for in vivo study. Statistical analysis was performed with the Student's t-test using SPSS 14.0 (SPSS, Inc., Chicago, IL, USA) to determine the differences between groups. P<0.05 was considered to indicate a statistically significant difference.

A previous study indicated that Oridonin regulated the T_h1/T_h2 balance in rats (25). The present study aimed to investigate whether Oridonin modulates the T_h1/T_h2 cytokine balance in mice. Our preliminary experiment indicated that Oridonin dose-dependently decreased the viability of the primary mice spleen cells, and that the half maximal inhibitory concentration is ~37 mM (data not shown). In the present study primary mice spleen cells were cultured, stimulated with concanavalin A and treated with Oridonin (12.5 or 25 mM). The supernatant was then harvested for ELISA. The results indicated that Oridonin treatment significantly decreased the T_h1 cytokine level [interferon (IFN)-γ and IL-2; P<0.001] in a dose-dependent manner, and significantly increased the T_h2 cytokine level (transforming growth factor; TGF-β and IL-10; P<0.001), also in a dose-dependent manner, in the mice spleen cell culture supernatant (Fig. 2). Thus, the results indicate that Oridonin regulates the T_h1/T_h2 cytokine balance in mice.

Previous studies have indicated that asthma is associated with the T_h1/T_h2 balance (29), and that Oridonin may modulate the rat T_h1/T_h2 balance in vitro (25). The present study demonstrated that Oridonin modulated the mice T_h1/T_h2 balance in vitro, therefore, the effect of Oridonin was investigated in a mouse model of asthma. Asthma is defined as excessive narrowing of the airways when challenged with contractile agonists; OVA is a contractile agonist. To investigate the anti-asthmatic effect of Oridonin on asthma in mice, a mouse model of asthma was established with methacholine to induce the AHR in OVA-challenged mice, and AHR was measured. Mice that had been sensitized with OVA continuously for three days were stimulated with methacholine to induce AHR. The control group was stimulated with PBS. The results indicate that Oridonin significantly inhibited methacholine-induced AHR when compared with the vehicle group (P<0.05; Fig. 3).

Eosinophil recruitment is an important characteristic in asthma (30). When challenged with OVA, numerous leukocytes were recruited in the lung (31), therefore, the effect of Oridonin on eosinophil recruitment was investigated in BAL fluid of OVA-challenged mice. The OVA-challenged mice lungs were flushed with cold PBS 48 h after the last challenge and cells in the BAL fluid were counted. The results indicated that Oridonin significantly decrease the number of eosinophils, neutrophils and total cells in BAL fluid when compared with the control group (P<0.05). The effects of Oridonin on the number of macrophages and lymphocytes were marginal (Fig. 4).

Humoral immune response usually causes the allergic reaction, when exposed to an allergen the immune cells secrete a series of cytokines and these cytokines, such as IL-4 and IL-13 contribute to the allergic condition (32). Kim et al (33) indicated that the imbalance of IL-5, eotaxin and IFN-γ was associated with asthma. Therefore, the present study analyzed the effect of Oridonin on cytokine levels in the BAL fluid of mice. The lungs of OVA-challenged mice were lavaged with cold PBS 48 h after the final challenge and the BAL fluid was collected. The ELISA assay was performed to evaluate the level of the associated cytokines in the mice BAL fluid according to the manufacturer's instructions. The findings demonstrated that the level of IL-4, IL-13, IL-5 and eotaxin increased significantly in the BAL fluid of the OVA-challenged mice (P<0.05). The results indicated that Oridonin treatment significantly inhibited the IL-4, IL-13, IL-5 and eotaxin levels in BAL fluid, but exerted a marginal effect on IFN-γ (Fig. 5). These results indicate that Oridonin modulated the level of cytokines produced by immune cells in asthmatic mice.

Significant migration of inflammatory cells (eosinophils) to peribronchiolar tissues was observed in the mice that had been challenged with OVA (31). Therefore, the effect of Oridonin on the eosinophil infiltration and mucus production was investigated in the lungs of asthmatic mice. Twenty-four hours after the last OVA challenge, mice were sacrificed and their lungs were harvested. Lung tissues were stored in formalin and histologically examined by a pathologist (Fig. 6A). The histopathological examination results indicated that following challenge with OVA, the eosinophil infiltration and mucus production were significantly increased compared to the PBS group (P<0.05). However, following administration of oridonin the inflammation score decreased from 3.8 to 2.1 (P<0.05) and the mucus score decreased from 3.7 to 2.3 (P<0.05). These results indicate that Oridonin significantly inhibited eosinophil infiltration (Fig. 6B) and mucus production (Fig. 6C) in the asthmatic lung when compared with that of the control group.