Phillyrin attenuates airway inflammation and Th2 cell activities in a mouse asthma model

ABSTRACT

Phillyrin is isolated from the fruit of Forsythia suspensa, and exhibits multiple pharmacological effects, including anti-tumor, anti-inflammation, and anti-oxidation activities. Here, we investigated whether phillyrin could alleviate airway hyperresponsiveness (AHR) and eosinophil infiltration in the lungs of asthmatic mice, and mitigate inflammatory responses in tracheal epithelial BEAS-2B cells. IL-4/TNF-α-stimulated BEAS-2B cells were treated with various phillyrin doses. Female BALB/c mice were sensitised and challenged with ovalbumin (OVA), and then treated with intraperitoneal injection different phillyrin doses. In IL-4/TNF-α-stimulated BEAS-2B cells, phillyrin effectively reduced proinflammatory cytokines, chemokines, and eotaxin (CCL11) levels. In the lungs of asthmatic mice, phillyrin treatment relieved AHR, airway inflammation, eosinophil infiltration, and goblet cell hyperplasia. Phillyrin also reduced serum OVA-IgE, and Th2-associated cytokine levels in splenocyte culture medium, and in bronchoalveolar lavage fluid of asthmatic mice. Our results indicate that phillyrin attenuated airway inflammation and eosinophil infiltration in asthmatic mice by suppressing Th2 cytokine production.

KEYWORDS:

• Airway hyperresponsiveness
• asthma
• cytokine
• eosinophil
• airway inflammation
• phillyrin

1. Introduction

Asthma is a chronic bronchial inflammation, characterised by recurrent attacks that cause dyspnoea and wheezing. Statistics from the World Health Organization show an increasing annual prevalence of asthma, mainly due to serious air pollution and climate warming (Stern et al., 2020). Notably, the most common type of asthma is allergic asthma (Maciag & Phipatanakul, 2020), with clinical features including chronic airway inflammation, airway remodelling, airway hyperreactivity (AHR), and elevated serum IgE concentrations. Allergic asthma is characterised by excessive activation of Th2 cells to release more asthma-related Th2 cytokines, including IL-4, IL-5, and IL-13 (Celakovska et al., 2017; Lambrecht et al., 2019). IL-4 can induce B-cell activation, and a switch from the expression of IgM class antibodies to IgE antibodies. IL-5 is a cytokine that stimulates bone marrow cells to form differentiated and activated eosinophils (Lambrecht et al., 2019). Excessive IL-13 secretion by Th2 cells in the airway will induce AHR and mucus hyperplasia, and enhance airway remodelling (Hammad & Lambrecht, 2021). Therefore, inhibiting Th2 cell overactivation in the respiratory system will effectively reduce the pathological symptoms of asthma.

Two main types of medicine are used to treat or prevent asthma: long-term control medicines and attack relief medicines (Maciag & Phipatanakul, 2020). During asthma attacks, bronchodilators are often used to relieve excessive airway smooth muscle contraction, to reduce shortness of breath and dyspnoea (Brusselle & Koppelman, 2022). Anti-inflammatory drugs are commonly taken to prevent and control asthma attacks, including inhaled steroids, oral corticosteroids, cromolyn, and nedocromil, which can control airway inflammation, and reduce airway swelling and mucus hypersecretion (Brusselle & Koppelman, 2022). To avoid severe side effects, only low doses of oral or inhaled steroids are used to prevent asthma attacks. However, steroids are immunosuppressants, which can suppress the immune function of Th1 and Th2 cells, leading to the increasing incidence of microbial infections (Guerau-de-Arellano & Britt, 2022). Therefore, some people are hesitant to use steroid agents to treat allergic and asthma diseases.

Traditional Chinese medicine uses herbal formulas containing ephedra to treat, improve or relieve asthma symptoms, including Ding Chuan Tang, Ma Xing Shi Gan Tang, and Xiao-Qing-Long Tang (Zhang et al., 2021). The main active ingredient of ephedra, ephedrine, can slow airway constriction, and improve airway obstruction and dyspnoea in asthmatic patients (Zheng et al., 2023). However, ephedrine can be refined to produce the drug amphetamine, which poses dangers to physical and mental health (González-Juárez et al., 2020). Thus, there is interest in identifying other herbal medicines or plant pure compounds to replace ephedra for improving asthma symptoms.

Forsythia suspensa (Thunb.) Vahl is a traditional herbal medicine commonly used in China, Japan, and Korea (Wang et al., 2018). Phillyrin is isolated from the fruit of F. suspensa (Zhou et al., 2022). In recent years, studies have demonstrated that phillyrin exerts a variety of pharmacological activities, including anti-inflammation, anti-oxidation, anti-obesity, and anti-cancer effects (Zhou et al., 2022).

F. suspensa fruit extracts can ameliorate allergic reaction in an allergic dermatitis mouse model (Sung et al., 2016). Phillyrin has been reported to reduce LPS-induced lung inflammation and lung injury in mice (Zhong et al., 2013), but the effects of phillyrin on airway inflammation and AHR in asthmatic mice remain unclear. Hence, we would investigated whether phillyrin could relieve inflammatory responses in airways, and regulate Th2-associated cytokine expression, in an OVA-induced asthmatic mouse model.

2. Materials and methods

2.1. Animals

Female BALB/c mice (age, 6–8 weeks; weight, 20–22 g) were obtained from the National Laboratory Animal Center (Taiwan). Mice were housed in a standard animal room (12:12 light–dark cycle) with food and water ad libitum. The animal experiment protocols were approved by the animal care and protection committee of Chang Gung University of Science and Technology (IACUC approval number: 2020-001).

2.2. Sensitisation and phillyrin treatment

Phillyrin (≥98% purity) was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA), and was dissolved in DMSO solution. Mice were sensitised by administering a sensitising solution, containing 50 μg OVA and 0.8 mg AlOH₃ adjuvant, via intraperitoneal injection on days 1, 2, 3, and 14. These mice were then subjected to a respiratory allergen challenge using inhaled 2% atomised OVA, on days 14, 17, 20, 23, and 27. Mice were administered phillyrin, prednisolone, or DMSO by intraperitoneal injection at 1 h before OVA challenge or methacholine treatment. On day 29, mice were anaesthetised and sacrificed to investigate airway inflammation, immunomodulatory symptoms, and lung pathology. All mice were randomly divided into five groups as follows: normal controls (N group); OVA sensitisation/challenge (OVA group); OVA sensitisation mice treated with 5 and 10 mg/kg phillyrin by intraperitoneal injection (Ph10 and Ph30 groups, respectively); and OVA-sensitised mice treated with 5 mg/kg prednisolone by intraperitoneal injection (P group).

2.3. AHR measurement

On day 28, all mice inhaled aerosolised methacholine (0–40 mg/ml) for 3 min (Wu et al., 2022). Mice were placed in single chamber that was connected to a whole-body plethysmograph (Buxco Electronics, Troy, NY, USA) for measurement of the enhanced pause (Penh) to evaluate AHR, as previously described (Wu et al., 2022).

2.4. Serum collection and splenocyte cultures

Mice were anesthetised with isoflurane, and serum was collected and stored at −80°C. Serum OVA-specific antibodies were detected by ELISA, as previously described (Liou et al., 2020). Mice were euthanised by inhalation of excessive CO₂, and then the spleens were removed and single splenocytes were isolated, as previously described (Huang et al., 2021). Next, the splenocytes were treated with 100 μg/ml OVA for 5 continuous days, followed by detection of cytokine expressions.

2.5. Bronchoalveolar lavage fluid (BALF) collection

The mice were euthanised and BALF was collected, as previously described (Huang et al., 2022). Briefly, an indwelling needle was inserted into trachea, and the lungs were lavaged three times with 1 ml of normal saline. In the collected BALF, we measured cytokine and chemokine concentrations by ELISA. Additionally, BALF was analysed by cell staining using Giemsa stain (Sigma) to measure different immune cell counts.

2.6. Histologic analysis

The lungs were removed from each mouse, fixed in 4% formaldehyde, and embedded in paraffin. Lung tissue was cut into 6-μm-thick sections, and stained with haematoxylin/eosin (H&E) to observe eosinophil infiltration. Additionally, periodic acid-Schiff (PAS) staining was performed to observe goblet cell hyperplasia of the trachea. Briefly, the slides were deparaffinised and stained with periodic acid solution. Next, the slides were treated with Schiff’s reagent and haematoxylin solution. Finally, the goblet cells of the trachea were examined using a light microscope (Olympus, Tokyo, Japan).

2.7. Cell viability assay

The BEAS-2B human bronchial epithelial cell line was purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were maintained in DMEM/F-12 containing 10% FBS. Cell viability was evaluated using CCK-8 assay reagent (Enzo Life Sciences, NY, USA). BEAS-2B cells were treated with 0–200 μM phillyrin for 24 h. Next, the culture plates were treated with CCK-8 reagent for 4 h at 37°C. Finally, the absorbance at 450 nm was measured using a multimode reader (Thermo Fisher Scientific, Grand Island, NY, USA).

2.8. Phillyrin treatment of BEAS-2B cells

BEAS-2B cells were treated with phillyrin (0–30 μM) for 1 h. Next, the cells were stimulated with 10 ng/ml IL-4 /TNF-α for 24 h. Finally, we used specific ELISA kits to detect the chemokine and cytokine levels in the culture supernatant.

2.9. Elisa

Cytokine and chemokine levels in BALF samples and cell culture supernatants were measured using specific ELISA kits (R&D Systems, Minneapolis, MN, USA) (Wang et al., 2022). Serum antibodies were assessed using specific antibody ELISA kits (BD Biosciences). The levels of chemokines, cytokines, and OVA antibody were detected using a microplate reader (Thermo Fisher Scientific).

2.10. Statistical analysis

Each experiment was independently performed at least three times. Statistical significance was measured using a parametric Student’s t test, and data are presented as mean ± SEM. A P value of <0.05 was considered statistically significant.

3. Results

3.1. Phillyrin suppressed the expression of inflammatory mediators in BEAS-2B cells

We used CCK8 reagent to assess the cytotoxicity of phillyrin in BEAS-2B cells. Phillyrin did not exhibit significant cytotoxic effects at a concentration of ≤50 μM, and subsequent cell experiments used phillyrin at concentrations of 0–30 μM concentrations (Figure 1(A)). Next, BEAS-2B cells were treated with phillyrin, and then stimulated with TNF-α/IL-4. Compared to untreated TNF-α/IL-4-stimulated BEAS-2B cells, phillyrin treatment yielded significant decreases of IL-6, IL-8, MCP-1, CCL5, and CCL11 levels, in a concentration-dependent manner (Figure 1(B–F)).

Figure 1. Effects of phillyrin (Ph) on cytokine and chemokine production in BEAS-2B cells. (A) Cell viability of Ph-treated BEAS-2B cells. (B–F) ELISA results show the levels of IL-6 (B), IL-8 (C), MCP-1 (D), CCL5 (E), and CCL11 (F) in BEAS-2B cells treated with TNF-α/IL-4. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, compared to BEAS-2B cells stimulated with TNF-α/IL-4.

3.2. Phillyrin improved AHR and reduced inflammatory cells in BALF

In our AHR experiment, we demonstrated that OVA sensitisation dose-dependently increased Penh values compared to mice in the Normal group. Compared to mice in the OVA group, phillyrin-treated OVA-sensitised mice (Ph10 and Ph30 groups) exhibited lower Penh values when exposed to 40 mg/ml methacholine. Prednisolone-treated asthmatic mice also exhibited lower Penh values compared to OVA group mice (Figure 2(A)). Next, we found that phillyrin-treated OVA-sensitised mice exhibited lower levels of eosinophils and total cells, compared to mice in the OVA group. On the other hand, phillyrin treatment did not significantly inhibit neutrophils, lymphocytes, or macrophages compared to OVA group mice. Additionally, prednisolone-treated asthmatic mice also exhibited significantly suppressed eosinophils and total cells compared to OVA group mice (Figure 2(B–F)).

Figure 2. Effects of phillyrin (Ph) on airway hyperresponsiveness (AHR) and cell counts in bronchoalveolar lavage fluid (BALF) of asthmatic mice. (A) Mice inhaled increasing doses of methacholine, and AHR was assessed (shown as Penh values). (B–F) The inflammatory cells in BALF were counted, including total cells (B), eosinophils (C), neutrophils (D), lymphocytes (E), and macrophages (F). Three independent experiments were analysed. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01 compared to the OVA control group.

3.3. Phillyrin modulated cytokine and chemokine expressions in BALF

Compared with OVA-sensitised mice, phillyrin-treated mice (Ph30 group) exhibited significantly suppressed IL-4, IL-5, IL-6, IL-13, TNF-α, and CCL11 levels (Figure 3(A–F)). On the other hand, phillyrin treatment (Ph30 group) increased IFN-γ expression in asthmatic mice (Figure 3(G)).

Figure 3. Effects of phillyrin (Ph) on the levels of cytokines and chemokines in bronchoalveolar lavage fluid (BALF). The concentrations of IL-4 (A), IL-5 (B), IL-13 (C), IL-6 (D), TNF-α (E), CCL11 (F), and IFN-γ (G) were measured in BALF. Three independent experiments were analysed. All data are presented as mean ± SEM. *P < 0.05, **P < 0.01 compared to the OVA control group.

3.4. Phillyrin alleviated eosinophil infiltration and goblet cell hyperplasia

Compared to normal mice, OVA-sensitised mice exhibited greater eosinophil infiltration in the lungs, and phillyrin treatment decreased these eosinophil levels in the lungs (Figure 4). PAS staining was used to examine goblet cells in the airways. Compared to mice in the OVA group, phillyrin-treated OVA-sensitised mice exhibited reduced goblet cell hyperplasia of the airways (Figure 5).

Figure 4. Effects of phillyrin (Ph) on eosinophil infiltration in lung tissue. Lung sections were stained using haematoxylin/eosin (H&E) staining. N, Normal group; OVA, OVA-induced asthma group; Ph10, group treated with 10 mg/kg phillyrin; Ph30, group treated with 30 mg/kg phillyrin; P, group treated with 5 mg/kg prednisolone.

Figure 5. Effects of phillyrin (Ph) on goblet cell hyperplasia in lung tissue. Lung sections were stained with periodic acid-Schiff (PAS) stain. Arrows indicate goblet cells. N, Normal group; OVA, OVA-induced asthma group; Ph10, group treated with 10 mg/kg phillyrin; Ph30, group treated with 30 mg/kg phillyrin; P, group treated with 5 mg/kg prednisolone.

3.5. Phillyrin modulated antibody and cytokine levels in serum and splenocytes

Compared to mice in the OVA group, phillyrin treatment effectively reduced the OVA-IgE and OVA-IgG1 levels in serum (Figure 6(A, B)). Phillyrin also promoted greater OVA-IgG2a levels, compared in mice of the OVA group (Figure 6(C)). On the other hand, compared to mice in the OVA group, prednisolone-treated OVA-sensitive mice exhibited significantly decreased expressions of OVA-IgE and OVA-IgG1 antibodies, but no alteration of OVA-IgG2a level (Figure 6).

Figure 6. Effects of phillyrin (Ph) on OVA-specific antibodies in serum. Serum levels of OVA-IgE (A), OVA-IgG1 (B), and OVA-IgG2a (C) from mice without or with Ph treatment. Three independent experiments were analysed. All data are presented as mean ± SEM. *P < 0.05, **P < 0.01 compared to the OVA control group.

3.6. Phillyrin inhibited Th2-associated cytokine expression in OVA-activated

splenocytes

Compared to splenocytes from mice in the Normal group, the splenocytes from mice in the OVA group secreted more IL-4, IL-5, and IL-13. Moreover, compared to mice in the OVA group, phillyrin treatment (Ph30 group) decreased the IL-4, IL-5, and IL-13 levels, and increased IFN-γ levels. Additionally, compared to mice in the OVA group, prednisolone-treated mice exhibited significantly inhibited IL-4, IL-5, and IL-13 expressions, but not increased IFN-γ levels (Figure 7).

Figure 7. Effects of phillyrin (Ph) on cytokine production in OVA-activated spleen cells. (A) IL-4, (B) IL-5, (C) IL-13, and (D) IFN-γ levels were measured by ELISA. Three independent experiments were analysed. All data are presented as mean ± SEM. *P < 0.05, **P < 0.01 compared to the OVA control group.

4. Discussion

The prevalence of asthma is rising worldwide, thus increasing the clinical and public health problems related to asthma treatment and prevention (Stern et al., 2020). Asthma attacks can cause dry cough, wheezing, shortness of breath, and difficulty breathing (Koefoed et al., 2021). The pathological manifestations of asthma mainly include severe shortness of breath caused by deterioration of AHR, and the infiltration of large numbers of eosinophils in the lungs, which leads to aggravation of lung inflammation and oxidative damage (Carpaij et al., 2019). Additionally, hyperplasia of smooth muscle in the trachea causes airway narrowing, reducing the airflow rate. Hyperplasia of tracheal goblet cells also leads to excessive mucus secretion, which blocks the airway, exacerbates dyspnoea, and can even lead to death by suffocation during asthma attacks (Chen et al., 2020).

Steroids and bronchodilators are used for asthma treatment and prevention (Ramos-Ramírez & Tliba, 2022). However, steroids also reduce immune function; therefore, some asthma patients seek alternative therapies to attenuate and treat asthma (Guerau-de-Arellano & Britt, 2022). Common alternative treatments include traditional Chinese and herbal medicine, yoga, and homeopathy (Kohn & Paudyal, 2017). Traditional Chinese medicine includes the use of Ding Chuan Tang and Xiao-Qing-Long Tang to treat asthma (Liu et al., 2021). Double-blind experiments have confirmed that Ding Chuan Tang can regulate the AHR, and reduce the excessive expression of Th2 cells, in patients with asthma (Chan et al., 2006). In recent years, other Chinese herbal medicine formulas or pure compounds from medicinal plants have also been found to improve and treat asthma symptoms (Liu et al., 2021).

Phillyrin is a purified compound isolated from F. suspensa (Zhou et al., 2022). Cell and animal experiments demonstrate that phillyrin has many pharmacological effects, such as anti-inflammatory, anti-obesity, anti-tumor (Zhou et al., 2022). Previous studies demonstrate that phillyrin decreases norepinephrine-induced cardiac hypertrophy and inflammatory response through blocking MAPK and NF-κB pathways (Tang et al., 2022). Phillyrin can also reduce LPS-induced lung inflammation, and inhibit inflammatory cell infiltration in the lungs to improve symptoms of acute lung injury (Zhong et al., 2013). Moreover, phillyrin is found as a cyclic AMP phosphodiesterase 4 (PDE4) inhibitor (Nishibe et al., 2021). In recent years, many studies have found that PDE4 inhibitors can suppress inflammatory response in respiratory diseases like asthma and pneumonia (Phillips, 2020). We also previously discovered that viscolin, PDE4 inhibitor, could inhibit airway inflammation, AHR and reduced eosinophil infiltration of the lungs in OVA-sensitised mice (Shen et al., 2011). In our present study, we found that phillyrin treatment of asthmatic mice significantly reduced AHR and eosinophil infiltration in the lungs, and reduced goblet cell hyperplasia in the trachea. Our experiments also revealed that phillyrin treatment mitigated the secretion of the inflammatory cytokine IL-6, and of the chemokines IL-8, MCP-1, and eotaxin-1 (CCL11) in inflamed human tracheal epithelial cells. Additionally, phillyrin-treated asthmatic mice exhibited reduced expressions of Th2-related cytokines in BALF and spleen cells, and reduced OVA-IgE expression in serum. Overall, our results indicated that phillyrin can reduce lung inflammation and improve the pathological manifestations of asthma in mice.

The hypersecretion of cytokines by activated Th2 cells is a major factor promoting greater inflammatory cell infiltration in the lungs of patients with asthma (Lambrecht et al., 2019). Th2-cell-related cytokines are also important factors in exacerbating airway remodelling and asthma symptoms (Hammad & Lambrecht, 2021). OVA or house dust mite would induce more eosinophil infiltration in lung of asthmatic mice. OVA-sensitised mice can secret excessive IL-5 by Th2 cells to simulate bone marrow cells to differentiate more activated eosinophils, which can be attracted into inflamed tissues under the induction of eotaxins (Pitlick & Pongdee, 2022). Our present experiments revealed increase eotaxin-1 (CCL11) expression in the lungs of asthmatic mice and supernatants of inflamed tracheal epithelial cells, which will induce greater eosinophil infiltration into the lungs. These activated eosinophils secrete eosinophil peroxidase (EPO) and eosinophil cationic protein (ECP), thereby exacerbating severe inflammation and lung cell damage in patients with asthma (Oppenheimer et al., 2022). Our results showed that phillyrin administered intraperitoneally to asthmatic mice significantly reduced the IL-5 production in BALF and splenocytes, thus helping reduce eosinophil proliferation and differentiation in the bone marrow. We also demonstrated that phillyrin inhibited the secretion of CCL11 from tracheal epithelial cells, thereby reducing the infiltration of eosinophils into the lungs, and improving inflammation and oxidative damage in the lungs of asthmatic mice.

AHR is an indicator of respiratory deterioration, often caused irritation from allergens, dust, or external molecules, and causing respiratory symptoms, such as severe coughing and shortness of breath (Pincus et al., 2021). In asthma patients, the respiratory tract is highly sensitive to allergens, which also hinders the flow rate of exhalation and inhalation, resulting in whistling inhalation and shortness of breath, to contraction during asthma attacks (Chen et al., 2020). When researchers attempted to use mites to induce asthma in IL-13 knockout mice, they found that they were unable to worsen AHR in these mice (Verma et al., 2018). Obviously, excessive IL-13 secreted by Th2 cells is considered an important factor in aggravated AHR among asthmatic patients (Ntontsi et al., 2018). Here we found that phillyrin significantly reduced the IL-13 production in BALF and spleen cells, and effectively reduced the Penh value, indicating improved airway resistance and respiratory function.

Recent research has also confirmed that both IL-13 and IL-4 can stimulate the proliferation and activation of goblet cells of the trachea (Moran & Pavord, 2020; Pelaia et al., 2022). When the respiratory tract of asthmatic patients is stimulated by allergens, it can increase goblet cell hyperplasia and aggravate the secretion of additional mucus, thereby blocking the airways and causing suffocation (Chen et al., 2020). We performed PAS staining of lung tissue, and found that phillyrin treatment significantly reduced goblet cells hyperplasia in the trachea. Therefore, phillyrin can reduce and improve dyspnoea caused by asthmatic attacks in mice, which may be related to the reduced secretion of IL-13 and IL-4. Previous studies have confirmed that IL-4 secreted by Th2 cells can stimulate B-cell activation, and lead to excessive IgE production (Lambrecht et al., 2019). IgE can bind to mast cells, and the activated mast cells can release histamine and leukotrienes, thereby causing acute allergic reactions in the airways (Hammad & Lambrecht, 2021). We found that phillyrin treatment reduced serum OVA-IgE levels in asthmatic mice, and also inhibited expression of the Th2-related antibody OVA-IgG1 and increased expression of the Th1-related antibody OVA-IgG2a.

Overall, our present results indicated that phillyrin alleviated the pathological manifestations of AHR, goblet cell hyperplasia, and eosinophil infiltration of the lungs in asthmatic mice, by suppressing Th2 cell activity. Our experimental results suggest that phillyrin has excellent potential to ameliorate airways inflammation and AHR in asthma.

Disclosure statement

No potential conflict of interest was reported by the authors.

Data availability statement

The data used to support the findings of this study are included with the article.

Additional information

Funding

This study was supported in part by grants from the Chang Gung Memorial Hospital (CMRPF1L0032, CMRPF1M0121, CMRPF1K0042 and CMRPF1M0141), and the Chang Gung University of Science and Technology (ZRRPF3M0091-6 and ZRRPF3M0091-8).