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. 2023 Oct 11;39(12):1213–1221. doi: 10.1002/kjm2.12764

Mulberroside F improves airway hyperresponsiveness and inflammation in asthmatic mice

Wen‐Chung Huang 1,2,3, Shu‐Ju Wu 4,5, Feng‐Wen Hsu 1, Li‐Wen Fang 6, Chian‐Jiun Liou 3,7,✉
PMCID: PMC11895982  PMID: 37819590

Abstract

Mulberroside F is isolated from the leaves and roots of Morus alba L. Here, we investigated whether mulberroside F could alleviate airway inflammation and eosinophil infiltration in the lungs of asthmatic mice. We also examined whether mulberroside F attenuated inflammatory responses in human tracheal epithelial BEAS‐2B cells. Female BALB/c mice were sensitized and challenged with ovalbumin (OVA), and administered different doses of mulberroside F via intraperitoneal injection. Additionally, tumor necrosis factor (TNF)‐α‐stimulated BEAS‐2B cells were treated with various doses of mulberroside F, followed by detection of the expressions of inflammatory cytokines and chemokines. The results demonstrated that mulberroside F mitigated the levels of proinflammatory cytokines and chemokines, and CCL11, in inflammatory BEAS‐2B cells. Mulberroside F also suppressed reactive oxygen species (ROS) production and ICAM‐1 expression in TNF‐α‐stimulated BEAS‐2B cells, which effectively suppressed monocyte cell adherence. In an animal model of asthma, mulberroside F treatment attenuated airway hyperresponsiveness, eosinophil infiltration, and goblet cell hyperplasia. Mulberroside F treatment also decreased lung fibrosis and airway inflammation in OVA‐sensitized mice. Moreover, mulberroside F significantly reduced expressions of Th2‐associated cytokines (including interleukin(IL)‐4, IL‐5, and IL‐13) in bronchoalveolar lavage fluid compared to OVA‐sensitized mice. Our results confirmed that mulberroside F is a novel bioactive compound that can effectively reduce airway inflammation and eosinophil infiltration in asthmatic mice via inhibition of Th2‐cell activation.

Keywords: airway hyperresponsiveness, airway inflammation, asthma, eosinophil infiltration, mulberroside F

1. INTRODUCTION

Asthma is a chronic respiratory allergic and inflammatory disease that affects up to 8%–15% of children worldwide. 1 Climate warming and urbanization have caused an increase of airborne particulates, leading to a rising annual incidence of asthma worldwide. 2 During an asthma attack, the main clinical manifestations include recurrent shortness of breath, dry cough, and wheezing. Additionally, thickening of the smooth muscle of the trachea leads to narrowing of the trachea, thereby reducing respiratory airflow. 3 During an asthma attack, the secretion of mucus in the airway will block the narrowed airway, resulting in difficulty in breathing and even death from suffocation. 4 Pathological features of allergic asthma include airway inflammation, airway hyperresponsiveness (AHR), pulmonary eosinophilic infiltration, tracheal mucus hypersecretion, and elevated serum IgE concentrations. 3 Recent studies have demonstrated that the exacerbation of allergic asthma is related to excessive Th2‐cell activation, and that activated Th2 cells release more Th2‐related cytokines, including IL‐4, IL‐5, and IL‐13. 5 IL‐5 released by Th2 cells can induce pluripotent stem cells of bone marrow to differentiate to produce more eosinophils. 6 IL‐4 can induce the activation of B cells into IgE‐secreting plasma cells, which enhances the activation of mast cells and eosinophils. 7 Excessive IL‐13 secretion by airway Th2 cells stimulates AHR deterioration and increases airway mucus hypersecretion. 8 Therefore, in the respiratory system, reducing Th2‐cell activation and increasing Th1‐cell activation can potentially improve asthma attacks and relieve asthma symptoms.

In patients with allergic asthma, allergens stimulate the tracheal epithelial cells to secrete inflammatory cytokines and chemokines to aggravate airway inflammation, deteriorate alveolar epithelial function, and induce pulmonary fibrosis. 9 Furthermore, there are many activated macrophages in the bronchoalveolar lavage fluid (BALF) of asthmatic patients, and activated macrophages will contribute to release more inflammatory cytokines to cause inflammation and damage of tracheal epithelial cells. 5 Goblet cells of the airways release mucin to suppress microbes or allergens into the airways and lung. However, allergens can stimulate the goblet cell hyperplasia of the airways. Activated goblet cells will release excessive mucin to block the airways and cause breathing difficulties in patients with asthma. 10

Drugs for asthma treatment are mainly divided into two categories. The first category of drugs is used to control, prevent, and reduce the frequency of asthma attacks, mainly by relieving respiratory tract inflammation. 11 Clinical drugs include oral and inhaled steroids, leukotriene receptor antagonists, and long‐acting beta 2 agonists. 12 The second category of asthma drugs is used to relieve and improve asthma attacks, and mainly comprises bronchodilators, including short‐acting beta‐agonists and anticholinergics. 8 Long‐term use of oral or injectable steroids will produce a variety of side effects, and suppress immune system function, reducing the activities of Th1 cells and Th2 cells. 11 In recent years, many studies have shown that some natural products can regulate the activation of Th1 cells and Th2 cells, and improve the development of asthma symptoms. 13 Therefore, to screen for and investigate natural products that may improve asthma symptoms, research has focused on regulating the activities of Th1 and Th2 cells.

Mulberry trees (Morus alba L.) are widely distributed in China, Taiwan, Southeast Asia, and South Asia. 14 The fruit of M. alba, the mulberry, can be eaten or made into jam. 15 In traditional Chinese medicine, the bark of the mulberry tree root is used to relieve coughs, bronchitis, and asthma attacks. 16 Mulberroside F has been isolated from the roots and leaves of the mulberry tree. 17 In the present study, we investigated whether mulberroside F could attenuate AHR and modulate Th2‐related cytokine expression in an ovalbumin (OVA)‐induced asthma mouse model.

2. MATERIALS AND METHODS

2.1. Animals

Mulberroside F (≥98% purity) was purchased from ChemFaces Co. (Wuhan, China), and was dissolved in dimethyl sulfoxide (DMSO)DA solution. A total of 48 female BALB/c mice (age, 6–8 weeks; weight, 20–22 g) were purchased from the National Laboratory Animal Center (Taiwan). Mice were housed in a standard animal room, and experimental procedures were approved by the Animal Ethics Committee of the Chang Gung University of Science and Technology (IACUC approval number: 2019‐011). The mice were randomly divided into four groups (each group n = 12): the normal control (N) group; OVA group; and OVA‐sensitized mice treated with intraperitoneal injections of 10 or 20 mg/kg mulberroside F (MuF10 and MuF20 groups, respectively).

2.2. Sensitization and mulberroside F treatment

Mice were intraperitoneally injected with 50 μg OVA and 0.8 mg aluminum hydroxide adjuvant in 0.2‐mL sterile saline solution on days 1, 2, 3, and 14, as previously described. 18 Subsequently, mice were challenged using inhaled 2% atomized OVA on days 14, 17, 20, 23, and 27. Mice were administered mulberroside F (or DMSO as a negative control) by intraperitoneal injection, at 1 h before OVA challenge. On day 29, mice were anesthetized and sacrificed to assay lung pathology and immunomodulatory effects (Figure 1).

FIGURE 1.

FIGURE 1

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Ovalbumin (OVA) allergen sensitization and challenge procedures. Mice were injected intraperitoneally (IP) with OVA on days 1–3 and 14. Mice were then challenged by inhalation (IH) OVA allergen on days 14, 17, 20, 23, and 27. On day 28, all mice inhaled aerosolized methacholine (0–30 mg/mL) to detect airway hyperresponsiveness (AHR). On day 29, mice sacrificed to assay lung pathology and immunomodulatory effects. Mice were administered mulberroside F (or DMSO as a negative control) by IP, at 1 h before OVA challenge.

2.3. Pulmonary function test

On day 28, all mice inhaled aerosolized methacholine (0–30 mg/mL) to detect pulmonary function (Figure 1), as previously described. 19 Briefly, mice were put in an airtight chamber, and a whole‐body plethysmograph (Buxco Electronics, Troy, NY, USA) was used to record the enhanced pause (Penh) for AHR evaluation.

2.4. Serum collection and splenocyte cultures

Mice were anesthetized for blood collection. Blood samples were centrifuged to obtain serum and stored at −80°C. Serum antibodies were detected using specific ELISA kits as previously described. 20 Additionally, the mouse spleens were removed, single splenocytes were isolated, and the splenocytes were cultured with 100 μg/mL OVA for five continuous days. Next, we collected the culture medium and detected the levels of cytokines using specific ELISA kits.

2.5. BALF collection

Mice were sacrificed, and indwelling needles were inserted into the trachea. The lungs were lavaged three times with normal saline to collect fluid, which was defined as BALF. In the collected BALF, we measured cytokine and chemokine concentrations using specific ELISA kits. Additionally, immune cells in BALF were stained with Giemsa stain (Sigma).

2.6. Immunohistochemistry

Lung tissues were embedded in paraffin, and 6‐μm lung sections were incubated with COX‐2 antibody (1:100 dilution; Cell Signaling Technology, MA, USA). The tissue slides were then treated with secondary antibody and DAB substrate solution to detect COX‐2 expression.

2.7. Immunofluorescence

Lung tissue was incubated overnight with NF‐κB p65 antibody (1:50 dilution; Cell Signaling Technology). Subsequently, the samples were incubated with fluorescent antibody, and the nuclei were stained with 4',6‐diamidino‐2‐phenylindole stain. We observed NF‐κB p65 expression under a fluorescence microscope (Olympus, Tokyo, Japan).

2.8. Histologic analysis

Lung tissue samples (6‐μm sections) were stained with hematoxylin/eosin to observe eosinophil infiltration, as previously described. 19 The inflammatory pathology scores were evaluated using a five‐point grading system described previously. 21 Goblet cell hyperplasia of the trachea was visualized using periodic acid‐Schiff (PAS) staining, and the goblet cells were examined using a light microscope (Olympus). Moreover, lung tissue was stained with Masson's trichrome solution to detect collagen expression, as previously described. 20

2.9. Western blot analysis

Lung tissues were homogenized, and the extracted proteins were separated using sodium dodecyl‐sulfatepolyacrylamide gel electrophoresis. Subsequently, the proteins were transferred to polyvinylidene difluoride membranes, which were incubated with specific antibodies, followed by incubation with secondary antibodies. Specific proteins were visualized using luminol/enhancer solution with the UVP Biospectrum Imaging System (UVP, Upland, CA, USA). Specific antibodies included COX‐2 and iNOS (Cell Signaling), HO‐1 (Santa Cruz, CA, USA), and β‐actin (Sigma).

2.10. Cell viability assay

The BEAS‐2B bronchial epithelial cell line was purchased from the American Type Culture Collection (Manassas, VA, USA). BEAS‐2B cells were maintained in DMEM/F‐12 medium containing 10% fetal bovine serum. BEAS‐2B cells were treated with mulberroside F (0–30 μM) for 24 h, and cell viability was detected using CCK‐8 assay reagent (Enzo Life Sciences, NY, USA).

2.11. Mulberroside F treatment of BEAS‐2B cells

BEAS‐2B cells were treated with mulberroside F (0–15 μM) for 1 h. Subsequently, the cells were incubated with 10 ng/mL TNF‐α or 10 ng/mL IL‐4/TNF‐α for 24 h. Finally, we collected the supernatant, and detected the chemokine and cytokine levels using specific ELISA kits.

2.12. ROS analysis

TNF‐α‐induced BEAS‐2B cells were treated with mulberroside F for 1 h, and then incubated with 10 ng/mL TNF‐α for 6 h. Subsequently, the cells were incubated with 2′,7′‐dichlorofluorescin diacetate (DCFH‐DA), and ROS production was detected using a fluorescence microscope (Olympus).

2.13. Cell–cell adhesion analysis

BEAS‐2B cells were treated with mulberroside F for 1 h, and then stimulated with 10 ng/mL TNF‐α for 24 h. Calcein‐AM (Sigma) was used to stain THP‐1 human monocytes, and THP‐1 cells were co‐cultured with BEAS‐2B cells. The effect on THP cell attachment was observed using a fluorescence microscope (Olympus).

2.14. ELISA

Serum antibodies were calculated using specific antibody ELISA kits (BD Biosciences, Franklin Lakes, NJ, USA). Cytokine and chemokine levels were detected using specific ELISA kits (R&D Systems, Minneapolis, MN, USA). The levels of antibodies, chemokines, and cytokines were assayed using a microplate reader (Thermo Fisher Scientific, Grand Island, NY, USA).

2.15. Statistical analysis

All experiments were performed as at least three independent experiments. The experimental data were analyzed 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. Mulberroside F suppressed inflammatory mediator expression in BEAS‐2B cells

We evaluated the cytotoxicity of mulberroside F in BEAS‐2B cells using a CCK‐8 kit. Mulberroside F did not exert significant cytotoxic effects at concentrations of ≤30 μM (Figure 2A), and all cell experiments in this study used mulberroside F concentrations of 0–15 μM. Next, we found that mulberroside F treatment significantly decreased IL‐6, IL‐8, and MCP‐1 levels in a concentration‐dependent manner, compared to TNF‐α stimulated BEAS‐2B cells (Figure 2B–D). Mulberroside F also decreased CCL11 secretion in IL‐4/TNF‐α‐stimulated BEAS‐2B cells (Figure 2E). Furthermore, mulberroside F treatment decreased the adherence of THP‐1 cells to TNF‐α‐stimulated BEAS‐2B cells (Figure 3A,B). Mulberroside F also inhibited the release of ICAM‐1 from TNF‐α‐activated BEAS‐2B cells (Figure 3C).

FIGURE 2.

FIGURE 2

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Effects of mulberroside F (MuF) on cytokine and chemokine production in BEAS‐2B cells. (A) Cell viability of MuF‐treated BEAS‐2B cells. (B–E) ELISA results show the levels of (B) IL‐6, (C) IL‐8, (D) MCP‐1, and (E) CCL11 in BEAS‐2B cells. Data are presented as mean ± SEM from three independent experiments. *p < 0.05; **p < 0.01, compared to BEAS‐2B cells stimulated with TNF‐α or TNF‐α/IL‐4.

FIGURE 3.

FIGURE 3

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Mulberroside F (MuF) inhibits THP‐1 cell adherence to activated BEAS‐2B cells. (A) Fluorescence microscopy images show THP‐1 cells adhered to BEAS‐2B cells under various conditions. (B) Quantitation of THP‐1‐cell adhesion under various conditions. (C) ICAM‐1 levels detected with ELISA. (D) MuF effects on ROS production in activated BEAS‐2B cells. Fluorescence microscopy images show ROS labeled with DCFH‐DA. Values are mean ± SEM from three independent experiments. *p < 0.05; **p < 0.01 versus TNF‐α‐activated BEAS‐2B cells.

3.2. Mulberroside F affected ROS production in BEAS‐2B cells

Compared to unactivated BEAS‐2B cells, TNF‐α‐activated BEAS‐2B cells exhibited increased ROS expression. Treatment with mulberroside F effectively reduced ROS expression in TNF‐α‐stimulated BEAS‐2B cells (Figure 3D).

3.3. Mulberroside F attenuated AHR and inflammatory cells in BALF

Methacholine stimulation of the airway can be used to evaluate airway function in patients with asthma. 22 We found that Penh values were significantly increased in OVA‐sensitized mice compared to normal mice, with gradual inhalation of increasing doses of nebulized methacholine. At inhalation of 30 mg/mL methacholine, asthmatic mice treated with mulberroside F exhibited significantly attenuated Penh values compared to the OVA group (Figure 4A). This indicated that mulberroside F improved AHR in asthmatic mice.

FIGURE 4.

FIGURE 4

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Effects of mulberroside F (MuF) on airway hyperresponsiveness (AHR) and percentage of eosinophils in bronchoalveolar lavage fluid (BALF) of asthmatic mice. (A) Mice inhaled increasing doses of methacholine, and AHR was assessed (shown as Penh values). (B) Percentage of eosinophils in BALF. Cytokine and chemokine levels in BALF, including (C) IL‐4, (D) IL‐5, (E) IL‐13, and (F) CCL11. Data are presented as mean ± SEM of three independent experiments. *p < 0.05; **p < 0.01 compared to the ovalbumin (OVA) control group.

Next, we found that mulberroside F‐treated OVA‐sensitized mice exhibited a reduced percentage of eosinophils in BALF, compared to mice in the OVA group (Figure 4B). We also found that mulberroside F‐treatment significantly suppressed IL‐4, IL‐5, IL‐13, and CCL11 levels in asthmatic mice, compared to OVA‐sensitized mice (Figure 4C–F).

3.4. Mulberroside F alleviated eosinophil infiltration and goblet cell hyperplasia

Mulberroside F treatment suppressed eosinophil infiltration into the lungs compared to untreated asthmatic mice (Figure 5A). Therefore, mulberroside F‐treated asthmatic mice had a reduced inflammatory pathology score (Figure 5B). PAS staining revealed that mulberroside F effectively reduced goblet cell hyperplasia, compared to the OVA group (Figure 5C,D). Additionally, Masson's trichrome stain demonstrated that mulberroside F attenuated collagen deposition in the lungs of asthmatic mice (Figure 5E,F).

FIGURE 5.

FIGURE 5

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Mulberroside F (MuF) affected eosinophil infiltration and goblet cell hyperplasia in mouse lungs. MuF reduced (A) eosinophil infiltration (HE stain; 200× magnification). Eosinophils are indicated by arrows. (B) The inflammatory score. (C) Periodic acid‐Schiff (PAS)‐stained lung sections show goblet cell hyperplasia (200× magnification). Goblet cells are indicated by arrows. (D) The number of PAS‐positive cells per 100 μm basement membrane. Lung sections were stained with Masson's trichrome stain to detect collagen expression (200× magnification) (E) for quantitative analysis of collagen (F). Data are presented as mean ± SEM of three independent experiments. *p < 0.05; **p < 0.01 compared to the ovalbumin (OVA) control group.

3.5. Mulberroside F improved airway inflammation

Immunohistochemical staining and western immunoblot protein analysis demonstrated that mulberroside F treatment decreased COX‐2 expression in the lungs, compared to mice of the OVA group (Figure 6A–C). We also found that mulberroside F decreased iNOS expression and increased HO‐1 expression compared to mice in the OVA group (Figure 6B,D,E). Furthermore, immunofluorescence staining revealed that mulberroside F‐treated asthmatic mice exhibited attenuated NF‐κB expression, compared with OVA‐sensitized mice (Figure 6F).

FIGURE 6.

FIGURE 6

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Mulberroside F (MuF) affects lung inflammation in mice. (A) Immunohistochemistry staining to detect COX‐2 expression. (B) Lung protein expression detected by western blot, and quantitative analyses of (C) COX‐2, (D) iNOS, and (E) HO‐1 expression. The results are presented as fold‐change in comparison with the ovalbumin (OVA) group. (F) Immunofluorescence staining to detect NF‐κB expression. Data are presented as mean ± SEM of three independent experiments. *p < 0.05; **p < 0.01 compared to the OVA control group.

3.6. Mulberroside F modulated antibody and cytokine levels in serum and splenocytes

Compared to mice in the OVA group, mulberroside F‐treated asthmatic mice exhibited reduced OVA‐IgE and IgG1 levels in serum (Figure 7A,B).

FIGURE 7.

FIGURE 7

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Mulberroside F (MuF) effects on antibodies in serum. Serum levels of (A) ovalbumin (OVA)‐IgE, (B) IgG1 are shown for normal (N) and OVA‐stimulated (OVA) mice treated without or with mulberroside F (MuF10 and MuF20). MuF regulated the levels of (C) IL‐4, (D) IL‐5, and (E) IL‐13 produced by OVA‐activated splenocytes. Data are presented as mean ± SEM of three independent experiments. *p < 0.05; **p < 0.01 compared to the OVA control group.

Allergens can stimulate specific T cells to accumulate in spleen cells. 23 Hence, splenocytes are incubated with OVA to detect cytokine expression using specific ELISA kits. Compared to splenocytes of mice in the normal group, the splenocytes of mice in the OVA group secreted more IL‐4, IL‐5, and IL‐13. Mulberroside F treatment (MuF30 group) decreased the IL‐4, IL‐5, and IL‐13 levels, compared to OVA group mice (Figure 7C–E).

4. DISCUSSION

The mulberry tree (M. alba L.) is a perennial shrub widely grown in East and South Asia. 14 The leaves, fruit, and seeds of the mulberry tree all have nutritional and medicinal properties, and mulberry is used as a medicinal plant in traditional Chinese medicine and Ayurvedic medicine. 15 Mulberroside F is a natural product isolated from the roots and leaves of the mulberry tree. 24 Previous studies have found that mulberroside F can inhibit melanin biosynthesis, 24 and mulberroside F reportedly exhibits binding affinity against SARS‐CoV‐2. 25 However, it is unclear whether mulberroside F can improve lung inflammation and AHR in asthmatic mice. Here we conducted a 7‐day animal toxicity test for mulberroside F, finding a 100% survival rate following administration of 30 mg/kg mulberroside F (data not shown). We also found that treatment of mice with 30 mg/kg mulberroside F via intraperitoneal injection did not significantly reduce their mobility or appetite compared to normal mice (data not shown). Therefore, we conducted experiments using 10 or 20 mg/kg mulberroside F, to explore whether these treatments could improve the pathological manifestations of asthma in OVA‐sensitized asthmatic mice. Our results showed that asthmatic mice treated with mulberroside F exhibited significantly reduced pulmonary AHR and eosinophil infiltration, inhibited tracheal goblet cell hyperplasia, and reduced pulmonary fibrosis. Mulberroside F also reduced the expression of pulmonary inflammation media COX‐2 and NF‐κB, and alleviated the expression of Th2‐related cytokines in BALF and splenocytes. Asthmatic mice treated with mulberroside F also showed reduced expression of OVA‐IgE in serum. Furthermore, our experiments demonstrated that mulberroside F reduced the secretion of inflammatory cytokines, chemokines, and eotaxin‐1 (CCL11) from inflamed tracheal epithelial cells, reduced immune cell attachment to inflamed tracheal epithelial cells, and reduced ROS expression. Collectively, our results suggested that mulberroside F could attenuate lung inflammation and ameliorate the pathogenic symptoms in mice with OVA‐induced asthma.

In patients with allergic asthma, the lungs will be infiltrated with a large number of eosinophils, which play an important role in the development of asthma symptoms. 26 These activated eosinophils release large amounts of inflammatory mediators, such as eosinophil cationic protein, which aggravate airway inflammation and damage lung cells. 27 IL‐5 stimulates the differentiation of pluripotent stem cells in bone marrow to form eosinophils, which then migrate to the lungs, driven by CCL11, and induce severe respiratory inflammation. 7 , 27 We found that mulberroside F reduced the secretion of IL‐5 in BALF and spleen cells, and also inhibited CCL11 production in tracheal epithelial cells, which would help reduce the ability of the bone marrow to produce eosinophils, and thereby reduce eosinophil migration into the lungs. Therefore, mulberroside F could reduce the ratio of eosinophils in the BALF of asthmatic mice, and suppress eosinophil infiltration into the lungs. Inflamed tracheal epithelial cells expressed high levels of ICAM‐1, enabling the attachment of more inflammatory cells. 28 We found that mulberroside F reduced the adhesion of immune cells to inflamed tracheal epithelial cells, which would reduce the adhesion and infiltration of inflamed immune cells in the lungs of asthmatic mice.

AHR is an important index for evaluating deterioration of the respiratory system. 22 Respiratory tract stimulation by allergens or foreign microorganisms will not only induce acute exacerbations of asthma but also cause cough and shortness of breath. 29 Methacholine is often clinically used as a stimulating molecule to evaluate respiratory function. 30 In asthmatic patients, the respiratory tract is highly sensitive to methacholine, and excessive inhalation of methacholine will cause shortness of breath and respiratory obstruction. 31 AHR deterioration is related to the excessive release of IL‐13 by Th2 cells in the lungs. 32 Researchers tried to use mites to induce asthma in IL‐13‐knockout mice, and found that AHR could not be induced in IL‐13‐knockout mice. 33 IL‐13 is clearly an important factor that induces AHR deterioration in patients with asthma. 34 In our present experimental study, we found that mulberroside F significantly reduced IL‐13 production in BALF and splenocytes, and effectively reduced the Penh value. These findings indicate that mulberroside F could reduce IL‐13 secretion from Th2 cells and contribute to the reduction of AHR.

In recent years, many studies have confirmed that excessive secretion of IL‐13 and IL‐4 by Th2 cells in the airway stimulates hyperplasia of tracheal goblet cells. 35 During an acute asthma attack, allergens stimulate the goblet cells of the tracheal epithelium, causing them to secrete excessive mucus that blocks the airway, resulting in airflow obstruction and dyspnea in asthmatic patients. 4 Mulberroside F was able to reduce the secretion of IL‐13 and IL‐4 in the respiratory tract and spleen cells of asthmatic mice. The mouse lungs were stained with PAS, which revealed that mulberroside F treatment significantly reduced the hyperplasia of tracheal goblet cells in asthmatic mice, which reduced the excessive mucus secretion in the respiratory tract. Additionally, mulberroside F reduced the expression of OVA‐IgE in serum. IgE can bind to mast cells, thereby activating mast cells to release histamine and leukotrienes, which cause acute respiratory allergic reactions. 7 Since IL‐4‐stimulated B cells can secrete excessive IgE, we thought that mulberroside F might reduce the IgE production by B cells, and reduce mast‐cell activation by inhibiting the production of IL‐4.

Allergens or inflammatory cytokines can stimulate the inflammatory response of airway epithelial cells, and induce epithelial cells to release more inflammatory cytokines, causing airway and lung inflammation. 36 Inflamed tracheal epithelial cells can also release chemokines to attract macrophages, neutrophils, and eosinophils to infiltrate the lungs. 36 , 37 Moreover, these inflamed immune cells can release inflammatory mediators to exacerbate lung cell damage and induce lung cell fibrosis. 7 Inflammation of lung cells will increase the collagen deposition in the lungs, increase lung fibrosis, and reduce the elasticity of alveolar cells, thus reducing the vital capacity and tidal volume of asthmatic patients. 38 Our immunohistochemical staining experiments revealed that mulberroside F reduced lung fibrosis in asthmatic mice, indicating that mulberroside F can reduce lung collagen deposition and pulmonary fibrosis by reducing lung inflammation in asthmatic mice.

Overall, our present experimental results confirmed that mulberroside F inhibited Th2‐cell activation, and thereby reduced pulmonary eosinophilic infiltration, tracheal goblet cell hyperplasia, and AHR in asthmatic mice. Moreover, mulberroside F reduced the effect of pulmonary fibrosis caused by inflamed lungs. Our findings support that mulberroside F is an effective natural product for improving asthma.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

Huang W‐C, Wu S‐J, Hsu F‐W, Fang L‐W, Liou C‐J. Mulberroside F improves airway hyperresponsiveness and inflammation in asthmatic mice. Kaohsiung J Med Sci. 2023;39(12):1213–1221. 10.1002/kjm2.12764

Wen‐Chung Huang and Shu‐Ju Wu contributed equally as first authors.

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