Introduction: Clinical management of asthma remains as a prevalent challenge. Monotropein (MON) is a naturally occurring cyclic enol ether terpene glycoside with medical application potential. This study aims to evaluate the potential therapeutic effects of MON in the mouse model of chronic asthma. Methods: An ovalbumin (OVA)-induced asthmatic mouse model was established to evaluate the therapeutic effect of MON at different doses (20, 40, and 80 mg/kg). The potential involvement of protein kinase B (AKT)/nuclear factor kappa B (NF-κB) pathway in the effect of MON was investigated by the administration of an AKT activator SC79. Histological changes in pulmonary tissues were examined by hematoxylin and eosin staining. The profiles of inflammatory cytokines (interleukin [IL]-4, IL-5, IL-13, and tumor necrosis factor [TNF]-α) in bronchoalveolar lavage fluid (BALF), and OVA-specific IgE in blood samples were analyzed by enzyme-linked immunosorbent assay (ELISA). The oxidative stress in the lung tissues was determined by measuring malondialdehyde level. The phosphorylation activation of AKT and NF-κB was examined by immunoblotting in the lung tissues. Results: MON treatment suppressed the infiltration of inflammatory cells in the airways of OVA-induced asthma mice and reduced the thickness of the bronchial wall and smooth muscle layer in a dose-dependent manner. MON treatment also reduced the levels of OVA-specific IgE in serum and cytokines in BALF in asthma-induced mice, and attenuated the oxidative stress in the lung tissues. OVA induced the phosphorylation of AKT and NF-κB proteins in the lung tissues of asthmatic mice, which was significantly suppressed by MON treatment. The co-administration of AKT activator SC79 impaired the therapeutic effect of MON on asthma-induced mice. Conclusion: Our data demonstrated the potential therapeutic effect of MON on asthmatic mouse model, suggesting that MON attenuated the inflammatory and oxidative damages in ling tissues by dampening the AKT/NF-κB signaling pathway.

Asthma has emerged as a chronic allergy in the lung tissues, which is characterized by coughing, wheezing, and dyspnea due to inflammation and hyperresponsiveness in the airways [1]. Global epidemiological statistics indicate that over 300 million people are affected by asthma, and there is an increasing tendency of asthma and its complications worldwide [2]. Owing to the variability in symptoms, pathological manifestations, and airflow limitation, the successful treatment of asthma requires long-term assessment and continuous monitoring to optimize the treatment strategy while minimizing the side effects [3]. In this context, the maintenance therapy with long-term and short-term inhaled corticosteroids for asthmatic symptom relief has been widely implemented in the clinical management of asthma [4]. Considering the unavoidable adverse effects of inhaled corticosteroids, the economic burden on patients, and medication-related complications, the Global Initiative for Asthma (GINA) guidelines recommend that oral-administered corticosteroids should be only used with the shortest possible period and the lowest possible dosage for patients with uncontrolled severe asthma [5]. Nevertheless, short-term corticosteroid administration can also lead to side effects in asthma patients [6]. Therefore, there is an urgent need to develop a safer and more effective drug for asthma treatment.

Monotropein (MON) is a major cyclic enol ether terpene glycoside extracted from the roots of Morinda officinalis [7]. The extract of Morinda officinalis has been widely used in traditional Chinese medicine, although its medical usage has not been approved in many other countries. Previous studies indicated that MON possesses anti-inflammatory and antipyretic activities [7, 8]. Specifically, the anti-inflammatory effect of MON alleviated acute colitis in a dextran sodium sulfate-induced mouse model by inhibiting nuclear factor kappa B (NF-κB) activation in the colonic mucosa. These findings suggest the potential therapeutic potential of MON in inflammatory bowel disease. Besides, MON has been widely reported to ameliorate the inflammation and oxidative stress in different pathophysiological conditions, such as acute kidney injury, muscle atrophy, and secondary liver injury [9‒12]. However, whether MON administration could alleviate the inflammation and symptoms in asthma has not been explored.

The current study explored the therapeutic effect of MON in an ovalbumin (OVA)-induced experimental mouse model of asthma. We systematically examined the effect of MON on the histological damages, inflammatory response, and oxidative stress in pulmonary tissues in asthma model. Besides, we also investigated the mechanism of MON in asthma protection through AKT/NF-κB pathway. Our data demonstrated the therapeutic potential of MON against the inflammatory and oxidative damages in the lung tissues, highlighted the inhibition of AKT/NF-κB signaling as an underlying mechanism for ameliorating asthma.

Animal Model of Asthma

BALB/c mice (female, 6-8-week-old) were purchased from Junke Animal Inc. (Nanjing, China) and housed under specific pathogen-free and temperature-controlled conditions with a 12 h light-dark cycle. The animals were allowed to access food and water ad libitum. To establish a chronic asthma model, mice were intraperitoneally injected with 200 μL solution containing 100 μg of OVA emulsified in 1.5 mg of aluminum hydroxide on days 0 and 7. From day 21, OVA-sensitized mice were exposed to aerosolized 5% OVA (grade II, Sigma-Aldrich, St. Loius, USA) for 30 min using a nebulizer three times per week, for a total period of 8 weeks. To further investigate the therapeutic effect of MON, mice were randomly divided into six experimental groups with 10 mice in each group: sham (without OVA induction and exposure), MON (80 mg/kg/week administration only), OVA, OVA + MON (20 mg/kg/week), OVA + Mon (40 mg/kg/week), and OVA + MON (80 mg/kg/week) groups. MON was administrated by gavaging for 8 weeks during the exposure to aerosolized OVA (see Fig. 1a). To investigate the mechanism of action of MON, four experimental groups were included in the study (n = 10 animals in each group): sham group, OVA group, OVA + MON (80 mg/kg/week) group, and OVA + MON + SC79 (AKT/NF-κB activator, 10 mg/kg/week) group (see Fig. 1b). The mice were euthanized at the end of experiments by intraperitoneal injection of excessive pentobarbital sodium. The blood samples, bronchoalveolar lavage fluid (BALF), and lung tissues were collected for further analysis. The experimental procedures were approved by the Animal Care and Use Committee of Yantai Yuhuangding Hospital.

Fig. 1.

Animal study design. a Schematics of OVA-induced asthma model and the administration of different MON doses (20, 40, 80 mg/kg/week). b Schematics of OVA-induced asthma model and the administration of MON (80 mg/kg/week) or the administration of MON together with SC79 (AKT/NF-κB activator, 10 mg/kg/week). N = 10 animals per group.

Fig. 1.

Animal study design. a Schematics of OVA-induced asthma model and the administration of different MON doses (20, 40, 80 mg/kg/week). b Schematics of OVA-induced asthma model and the administration of MON (80 mg/kg/week) or the administration of MON together with SC79 (AKT/NF-κB activator, 10 mg/kg/week). N = 10 animals per group.

Close modal

Measurement of Wet/Dry Weight Ratio of Pulmonary Tissues

Immediately after collection, the left lobe of lung tissues was accurately weighed as wet weight. The same tissue sample was then dried at 56°C for 18 h, and then the dry weight of the lung tissue was recorded. The ratio of the wet weight and dry weight of the same tissue sample was calculated as the Wet/Dry (W/D) ratio.

Histopathological Examinations

The histopathological examination of the lung tissues was conducted by hematoxylin and eosin (H&E) staining. The left upper lobe of the lung tissue was fixed in 4% formaldehyde for 18–24 h and then embedded in paraffin. The paraffin-embedded lung sections sliced into sections with a thickness of 4 μm and stained using a H&E-staining kit (P0173, Beyotime, Beijing, China) to assess inflammatory cell infiltration, mucus secretion, and pulmonary fibrosis. Notably, sections with well-defined bronchi (n = 20) of an internal diameter at 100–200 μm were randomly selected and evaluated by light microscopy (×200 magnification). All quantitative analyses related to the lung tissues were performed by two researchers using Image-Pro Plus v6.0 software (Media Cybernetics, Rockville, USA). The inflammation score was determined based on a 5-point scale: normal = 0; few inflammatory cells = 1; one layer of cell rings = 2; two to four layers of cell rings = 3; and >4 layers of cell rings = 4. The thicknesses of the airway wall and the smooth muscle layer (μm) were determined against basement membrane length (mm). Further, the mucus scoring was calculated based on a 5-point scale according to the percentage of cup cells in the epithelium: no cup cells = 0; <25% of cup cells = 1; 25–50% of cup cells = 2; 51–75% of cup cells = 3; >75% of cup cells = 4.

BALF Collection and Cell Counting

After euthanization tracheal intubation was performed in the terminally dead mice, and the lungs were washed thrice with 0.8 mL of cold phosphate-buffered saline (PBS) to collect BALF. Afterward, BALF samples were centrifuged, and the supernatant was collected and stored at −80°C for cytokine measurement. The resulting cell pellets were suspended in PBS for cell counting, and the cell samples were spread onto slides to determine the number of eosinophils, neutrophils, macrophages, and lymphocytes in BALF by Wright-Giemsa staining (C0133, Beyotime, Beijing, China).

Enzyme-Linked Immunosorbent Assay

The relative levels of interleukin (IL)-4, IL-5, IL-13, and TNF-α were determined in BALF supernatant using corresponding ELISA kits (Sigma-Aldrich, Shanghai, China) based on the manufacturer’s instructions. After antibody incubation and signal development, the optical density (OD) value of each sample was recorded at 450 nm using the Synergy H1 microplate reader (BioTek Instruments, Winooski, VT, USA). A series of diluted standards were used to plot the curve, with the concentration of the standards as the X axis and the OD value as the Y axis. The concentration of cytokine in the sample was calculated based on the standard curve and the OD value of each sample.

Determination of Serum OVA-Specific IgE

The collected blood samples were kept at room temperature for 1 h and then were subjected to centrifugation at 3,000 × g for 15 min at room temperature. Further, the supernatant was collected as the serum sample for OVA-specific IgE level determination using an OVA-IgE ELISA kit (JL23405-48T, Jianglai Biotechnology, Shanghai, China) according to the manufacturer’s instructions. The OD values of serum sample and the standards were measured at 450 nm, and the concentration of OVA-specific IgE in each sample was determined by the linear regression of the standard curve.

Detection of Oxidative Stress Indicators

The collected the lung tissues from sacrificed mice were homogenized in normal saline and lysed on ice for 15 min using the tissue lysis buffer (S3062, Beyotime, Beijing, China). The protein content in the homogenates was measured by a bicinchoninic acid kit (P0011, Beyotime, Beijing, China), using bovine serum albumin as the standard.

Superoxide dismutase (SOD) enzyme activity was determined by preparing a 3 mL reaction mixture containing 2.8 mL of potassium phosphate buffer (0.1 M, pH 7.4), tissue homogenate (0.1 mL), and o-toluene trizol solution (0.1 mL). The mixture was incubated at room temperature for 30 min and the absorbance values were recorded at 325 nm using a microplate reader. In addition, the catalase (CAT) activity was measured by preparing a solution of 1.95 mL of PBS (pH 7.0), 1 mL of hydrogen peroxide (30 mM), and 0.05 mL of tissue homogenate. The solution was incubated at room temperature for 30 min before OD reading at 325 nm. The output of SOD and CAT activity was normalized against the protein content in each sample.

Lipid peroxidation was indirectly measured by detecting malondialdehyde (MDA) levels in the tissue homogenate. Briefly, 1 mL of tissue homogenate and 3 mL of thiobarbituric acid were mixed with shaking on ice for 15 min. Afterward, the solution was centrifuged at 3,000 rpm for 10 min at 4°C. The absorbance of the supernatant was measured at 532 nm, and the MDA levels were calculated using the following formula: MDA concentration = (ABS × 100 × VT)/(1.56 × 105 × W × VU); ABS refers to absorbance; VT indicates the total volume of the mixture; VU represents the volume of the liquid; WT is the weight of the glycerol, and 1.56 × 105 denotes the molar exhalation coefficient.

To detect the reduced glutathione (GSH) levels, 1 mL of potassium chloride and 1 mL of tissue homogenate were mixed in 4 mL of cold distilled water. Afterward, 1 mL of trichloroacetic acid was added to the mixture and the solution was centrifuged at 3,000 rpm for 30 min at 4°C. Next, 4 mL of Tris buffer (0.4 M) and 0.1 mL of 0.001 M DTNB were mixed with 2 mL of supernatant. Finally, the absorbance values of the mixture were recorded at 412 nm. GSH content was determined using the following formula: (absorbance × dilution factor)/extinction coefficient, here extinction coefficient = 13,000 M−1 cm−1.

Immunoblot Analysis

The right lobe of the lung tissues was collected for protein level detection using Western blotting. About 40 mg of the lung tissues were mixed with 2 mL of using the tissue lysis buffer (S3062, Beyotime, Beijing, China) for 15 min on ice. After centrifugation at 15,000 rpm for 10 min, the protein content in the supernatant was measured by a bicinchoninic acid kit (P0011, Beyotime, Beijing, China). After homogenization and protein content quantification, 60 μg of protein sample was mixed with 5 × sodium dodecyl sulfate sampling buffer (volume ratio of 4:1) and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis to separate the protein bands, followed by the transfer onto the poly(vinylidene fluoride) membrane. The membrane was then sealed with 5% skimmed milk for 1 h and incubated with the following primary antibodies (p-AKT [ab183556; 1:1,000], AKT [ab227385; 1:1,000], p-NF-κB [ab76302; 1:1,000], and NF-κB [ab16502; 1:1,000]) at 4°C overnight (Abcam, Cambridge, USA). After washing, the blots were incubated with horseradish peroxidase-labeled secondary antibody (ab6721; 1:5,000) at 37°C room temperature for 1 h. Signal development was conducted using the BeyoECL Pus chemiluminescence (ECL) kit (P0018M, Beyotime, Beijing, China), and protein band intensity was quantitatively analyzed using Image J software (NIH, Bethesda, MA, USA). The results of the protein phosphorylation levels were expressed as the ratio of p-Akt/Akt and p-NF-κB/NF-κB.

Statistical Analysis

Quantitative data were expressed as mean ± standard deviation (SD). The statistical analyses were performed using GraphPad Prism v9.0.0 software (GraphPad, San Diego, CA, USA) based on one-way analysis of variance and Turkey’s post hoc test. The results of lung histopathology scores were compared by the Kruskal-Wallis H test. The differences were considered statistically significant when the p value is lesser than 0.05.

MON Attenuates Histological Damages of the Lung Tissues in OVA-Sensitized Mice

Initially, the W/D ratio of the lung tissues was determined as an indicator of edema in each group of animals since pulmonary edema is often caused by lung inflammation and tissue damages in asthma [2, 3]. It was observed that the W/D ratio was significantly higher in the OVA model group when compared with the sham group. MON treatment dose-dependently decreased the W/D ratio of the lung tissues in asthma-induced mice (Fig. 2a). Further, H&E staining in the lung tissue sections showed an increased peribronchial inflammatory cell infiltration in the lung parenchyma of OVA-induced mice compared to the sham group or the group with MON treatment alone. The inflammatory cell infiltration was significantly reduced after MON treatment in the OVA-induced mice (Fig. 2b). Concomitantly, the peribronchial inflammatory cell score was significantly lower in the OVA + MON groups than that of OVA-induced model group. In addition, the repeated exposure to OVA substantially increased the thickness of the bronchial wall and smooth muscle layer, which was significantly reduced after MON treatment in a dose-dependent manner. Notably, the highest dose of MON (80 mg/kg) alone showed no apparent detrimental effects on normal mouse trachea. These results suggest that airway inflammation and remodeling were induced by OVA exposure in the mouse model of asthma, and MON administration could effectively alleviate the pathological changes in the lung tissues upon asthma induction.

Fig. 2.

MON treatment inhibits inflammatory infiltration of immune cells in lungs of OVA-sensitized mice. a W/D ratio of the lung tissues in each experimental group. b Representative images of H&E-staining in lung tissue sections from each group. Blinded inflammation score, airway wall thicknesses and smooth muscle layer thicknesses were determined from lung tissue sections using Image-Pro Plus software. Data were presented as mean ± SD (n = 10 animals, 20 bronchioles/3 sections of an animal, each bronchiole with 100 ∼ 200 μm of internal diameter). All images are at ×200 magnification. Scale bar = 100 μm. ***p < 0.001 versus sham group; ++p < 0.01, +++p < 0.001 versus OVA model group.

Fig. 2.

MON treatment inhibits inflammatory infiltration of immune cells in lungs of OVA-sensitized mice. a W/D ratio of the lung tissues in each experimental group. b Representative images of H&E-staining in lung tissue sections from each group. Blinded inflammation score, airway wall thicknesses and smooth muscle layer thicknesses were determined from lung tissue sections using Image-Pro Plus software. Data were presented as mean ± SD (n = 10 animals, 20 bronchioles/3 sections of an animal, each bronchiole with 100 ∼ 200 μm of internal diameter). All images are at ×200 magnification. Scale bar = 100 μm. ***p < 0.001 versus sham group; ++p < 0.01, +++p < 0.001 versus OVA model group.

Close modal

MON Alleviates Pulmonary Inflammation in OVA-Sensitized Mice

Next, the relative levels of inflammatory cells and cytokines were determined in BALF. The total number of cells, eosinophils, neutrophils, and macrophages was significantly increased in the BALF of OVA-sensitized mice, and MON administration reduced the quantity of inflammatory cells in BALF in a dose-dependent manner, suggesting that MON treatment attenuated OVA-induced infiltration of inflammatory cells in the lung airways (Fig. 3a). Allergic airway inflammation in asthma is associated with Th2-related responses and IgE production. ELISA analysis in serum samples showed that the production of OVA-specific IgE was significantly suppressed by MON treatment in OVA-induced mice (Fig. 3b). Subsequent analyses of inflammatory cytokines showed that IL-4, IL-5, IL-13, and TNF-α levels were significantly increased in the BALF of OVA-induced mice, while MON administration attenuated the production of these cytokines in BALF (Fig. 3c–f). These data collectively indicate that MON administration alleviates pulmonary inflammation in OVA-sensitized mice.

Fig. 3.

MON treatment reduces immune cell counts and the production of inflammatory cytokines in BALF from the OVA-sensitized mice. (a) Quantification of different leukocytes (105/mL) in the BALF from different groups of mice. The concentrations of (b) serum OVA-specific IgE and (b–f) inflammatory cytokine (IL-4, IL-5, IL-13, and TNF-α) in the BALF were determined by ELISA. The values are expressed as the mean ± SD (n = 10). ***p < 0.001 versus sham group; +p < 0.05, ++p < 0.01, +++p < 0.001 versus OVA model group.

Fig. 3.

MON treatment reduces immune cell counts and the production of inflammatory cytokines in BALF from the OVA-sensitized mice. (a) Quantification of different leukocytes (105/mL) in the BALF from different groups of mice. The concentrations of (b) serum OVA-specific IgE and (b–f) inflammatory cytokine (IL-4, IL-5, IL-13, and TNF-α) in the BALF were determined by ELISA. The values are expressed as the mean ± SD (n = 10). ***p < 0.001 versus sham group; +p < 0.05, ++p < 0.01, +++p < 0.001 versus OVA model group.

Close modal

MON Attenuates Oxidative Stress in the Lung Tissues of OVA-Sensitized Mice

Considering the elevated levels of inflammatory cytokines in the BALF, we further determined the levels of various oxidative indicators in the lung tissues, such as MDA, SOD, CAT, and GSH. Compared with the sham group of mice or the mice treated with MON alone, there was a significant increase of MDA level in OVA-induced mice, which was mitigated upon MON administration (Fig. 4a). In contrast, the antioxidant factors such as SOD, CAT, and GSH showed a significant decrease in the lung tissues of the OVA-induced mice, and MON administration increased their levels in a dose-dependent manner (Fig. 4c, d). These data indicate the elevated oxidative stress and impaired antioxidant capacity in the lung tissues of asthma-induced mice. With MON intervention, the oxidative stress (MDA) level was mitigated and the levels of antioxidant factors (SOD, CAT, and GSH) were increased. Together, these data suggest that MON attenuates the oxidative stress in the lung tissues of OVA-sensitized mice.

Fig. 4.

MON treatment attenuates OVA-stimulated oxidative stress in the lung tissues. (a) The content of MDA in the lung tissues of each experimental group. (b) GSH, (c) SOD, and (d) CAT activities were determined in the lung tissues of each group, and the data were normalized against the protein content in each sample. The values are expressed as the mean ± SD (n = 10). ***p < 0.001 versus sham group; ++p < 0.01, +++p < 0.001 versus OVA model group.

Fig. 4.

MON treatment attenuates OVA-stimulated oxidative stress in the lung tissues. (a) The content of MDA in the lung tissues of each experimental group. (b) GSH, (c) SOD, and (d) CAT activities were determined in the lung tissues of each group, and the data were normalized against the protein content in each sample. The values are expressed as the mean ± SD (n = 10). ***p < 0.001 versus sham group; ++p < 0.01, +++p < 0.001 versus OVA model group.

Close modal

MON Inhibits AKT/NF-κB Signaling Pathway in the Lung Tissues of OVA-Induced Mice

We next attempted to explore the mechanism of action of MON in alleviating asthma in mice. The effect of MON on OVA-induced AKT and NF-κB phosphorylation levels was analyzed by protein blot analysis in the lung tissues. We found that the phosphorylation levels of AKT and NF-κB were significantly elevated in the lung tissues of OVA-sensitized mice when compared with the sham group. MON treatment significantly suppressed the phosphorylation of AKT and NF-κB proteins in the lung tissues of OVA-induced mice (Fig. 5). Of note, in the mice without OVA induction, the highest treatment dose of MON alone (80 mg/kg) showed no significant effect on the phosphorylation AKT and p-NF-κB proteins. These findings suggest that MON treatment may alleviate asthmatic inflammation through dampening AKT/NF-κB signaling pathway.

Fig. 5.

MON inhibits AKT/NF-κB signaling pathway in the lung tissues of OVA-sensitized mice. Western blotting and semiquantitative analysis of the relative phosphorylation levels of AKT and NF-κB in the lung tissues of each experimental group. The values are expressed as the mean ± SD (n = 6). ***p < 0.001 versus sham group; +++p < 0.001 versus OVA model group.

Fig. 5.

MON inhibits AKT/NF-κB signaling pathway in the lung tissues of OVA-sensitized mice. Western blotting and semiquantitative analysis of the relative phosphorylation levels of AKT and NF-κB in the lung tissues of each experimental group. The values are expressed as the mean ± SD (n = 6). ***p < 0.001 versus sham group; +++p < 0.001 versus OVA model group.

Close modal

AKT Activator SC79 Impairs the Therapeutic Effect of MON in Asthmatic Mice

To validate the involvement of AKT/NF-κB signaling in the therapeutic effect of MON in OVA-induced asthmatic mice, an AKT/NF-κB activator (SC79) was jointly administrated with MON in the asthma-induced mice. Histological analysis demonstrated that SC79 co-administration promoted peribronchial inflammatory cell infiltration and increased the thickness of the bronchial wall and smooth muscle layer in MON-treated asthmatic mice (Fig. 6a). Consistently, SC79 co-administration also curtailed the effects of MON treatment on suppressing OVA-specific IgE production (Fig. 6b) and the secretion of inflammatory cytokines (Fig. 6c–f), as well as on the amelioration of oxidative stress in the lung tissues of asthmatic mice (Fig. 6g). In addition, the analysis of AKT and NF-κB phosphorylation in the lung tissues showed that the phosphorylation levels of P-AKT and p-NF-κB were significantly elevated by SC79 in the MON-treated asthmatic mice (Fig. 6h). Together, our findings suggest that MON alleviates oxidative stress and inflammatory responses in OVA-induced asthmatic mouse model at least partially through the inhibition of AKT/NF-κB signaling.

Fig. 6.

MON alleviates OVA-induced asthma by suppressing AKT/NF-κB signaling pathway (a) Representative images of H&E-staining in lung tissue sections from sham, OVA, OVA + MON, and OVA + MON + SC79 (AKT/NF-κB activator) groups. Blinded inflammation score, airway wall thicknesses, and smooth muscle layer thicknesses were determined from the stained lung sections using Image-Pro Plus software. Data were presented as mean ± SD (n = 10 animals, 20 bronchioles/3 sections of an animal, each bronchiole with 100 ∼ 200 μm of internal diameter). All images are at ×200 magnification. Scale bar = 100 μm. b The concentration of OVA-specific IgE in serum, and relative concentrations of (c) IL-4, (d) IL-5, (e) IL-13, and (f) TNF-α in the BALF were examined by ELISA (n = 10 per group). g The content of MDA and the relative activities of GSH, SOD, and CAT were measured in the lung tissues of each group of mice (n = 10 per group). h Western blotting and semi-quantitative analysis of the relative phosphorylation levels of AKT and NF-κB in the lung tissues of each experimental group. The values are expressed as the mean ± SD (n = 6). ***p < 0.001 versus sham group; +++p < 0.001 versus OVA model group; ^ ^p < 0.001, ^ ^ ^p < 0.001 versus OVA + MON group.

Fig. 6.

MON alleviates OVA-induced asthma by suppressing AKT/NF-κB signaling pathway (a) Representative images of H&E-staining in lung tissue sections from sham, OVA, OVA + MON, and OVA + MON + SC79 (AKT/NF-κB activator) groups. Blinded inflammation score, airway wall thicknesses, and smooth muscle layer thicknesses were determined from the stained lung sections using Image-Pro Plus software. Data were presented as mean ± SD (n = 10 animals, 20 bronchioles/3 sections of an animal, each bronchiole with 100 ∼ 200 μm of internal diameter). All images are at ×200 magnification. Scale bar = 100 μm. b The concentration of OVA-specific IgE in serum, and relative concentrations of (c) IL-4, (d) IL-5, (e) IL-13, and (f) TNF-α in the BALF were examined by ELISA (n = 10 per group). g The content of MDA and the relative activities of GSH, SOD, and CAT were measured in the lung tissues of each group of mice (n = 10 per group). h Western blotting and semi-quantitative analysis of the relative phosphorylation levels of AKT and NF-κB in the lung tissues of each experimental group. The values are expressed as the mean ± SD (n = 6). ***p < 0.001 versus sham group; +++p < 0.001 versus OVA model group; ^ ^p < 0.001, ^ ^ ^p < 0.001 versus OVA + MON group.

Close modal

Asthma has emerged as a chronic inflammatory disease with an increasing thread of prevalence. Different risk factors including changes in climate patterns, air pollution, urbanization, and crowded cohabitation could contribute to the development of asthma [13, 14]. Several medications have been applied to treat asthma, including corticosteroids, TNF-α antibody (infliximab), which predominantly suppresses macrophage-mediated inflammation. Other medications such as leukotriene inhibitors, β Receptor agonists, H1 receptor antagonists, theophylline drugs, are used to temporarily relieve asthmatic symptoms [2, 3]. However, long-term usage of these drugs can lead to serious side effects, such as steroid dependence and impaired immunity [15, 16]. Developing novel therapy for asthma management is an urgent need. Here, we reported the therapeutic effect of MON in OVA-induced asthmatic mouse model. MON administration significantly suppressed bronchopathological changes, infiltration of inflammatory cells, production of OVA-specific IgE, inflammatory cytokines levels, and oxidative stress in the lung tissues of OVA-induced asthma mice.

Asthma is typically characterized by Th2-type immune responses in the airways [17]. OVA exposure in the airways also provokes a Th2-dominant response in asthma-induced animal model. Previous studies indicate that the production of Th2 cytokines promotes the infiltration of inflammatory cells in both the patients with asthma and OVA-induced asthmatic mice [18, 19]. Thus, Th2 cytokines, such as IL-4, IL-5, and IL-13, as well as the cytokine from innate immune cells (TNF-α) play critical roles in driving the allergic pathogenesis of airway [20]. In addition, the airway epithelial cells secrete mucus to trap the inhaled microorganisms and allergens, regulating the allergic and inflammatory responses caused by exposure to these environmental agents. The activated epithelial cells also release cytokines and chemokines to recruit immune cells and exacerbate respiratory inflammation in the lung tissues [21]. Inflamed airway due to inflammation can trigger the production of reactive oxygen species by inducing the expression of various oxidative enzymes, leading to oxidative damages [21]. Among various cytokines, IL-4 has been recognized as the most important cytokine to mediate Th2 inflammatory response, inducing the maturation of B-cells and the isotype switch from IgG to IgE [22]. On the other hand, IL-5 triggers eosinophil differentiation and maturation to mount allergen-induced eosinophilic airway inflammation. Another key cytokine implicated in asthma is IL-13, which could promote airway hyperresponsiveness to the foreign antigens [23]. A growing body of evidence suggests that neutralizing Th2 cytokines could attenuate allergic asthma [24]. In line with this, IL-4, IL-5, IL-13, and TNF-α levels were significantly increased in the BALF of OVA-sensitized mice, whereas the secretion of these cytokines was significantly suppressed after MON treatment. Our data also showed that MON administration attenuated the oxidative stress in the lung tissues of OVA-sensitized mice. MON treatment also dampened the production of OVA-specific IgE. Together, these findings indicate that MON could alleviate the pulmonary damages in asthmatic mice by suppressing Th2-mediated inflammation and the oxidative stress in the lung tissues.

The recruitment of eosinophils plays an important role in the pathogenesis of allergic inflammation in asthma [25], which is mediated by the secretion of Th2 cytokines [26]. Accordingly, our findings suggest that MON inhibited the infiltration of inflammatory cells (such as macrophages and eosinophils) into the airway of asthmatic mouse model. Moreover, MON reduced the overall immune cell infiltration and ameliorated the pathological signs of inflammation in the histological analysis of the lung tissues of asthmatic mouse model. These findings further demonstrated that MON treatment could suppress immune cell infiltration in the lung tissues to prevent the progression of asthma.

NF-κB is an essential transcription factor involved in a variety of cellular events, including cancer progression and immune regulation [27‒29]. NF-κB dependent transcription is required for the proliferation and inflammatory activation of different immune cell types. Upon allergen stimulation in innate immune cells, IkB, the inhibitor of NF-κB, becomes phosphorylated and dissociates from NF-κB. This allows NF-κB to translocate into the nucleus and mediate the expression of pro-inflammatory cytokines [30, 31]. Since NF-κB mediates the inflammatory activation of innate immune cells, targeting NF-κB has been proposed as a promising strategy for the treatment of asthma [32, 33]. Here, we showed the activation of AKT and NF-κB signaling in the lung tissues of OVA-induced asthmatic mice, and MON treatment was able to suppress OVA-induced phosphorylation of NF-κB. These data imply that MON alleviates OVA-induced asthma possibly by dampening AKT/NF-κB signaling pathway.

Importantly, no detrimental effects of MON administration were observed on the lung tissues and bronchioles in mice without OVA-induced asthma. These data indicate that MON can be developed as a safe therapeutic agent in the clinical management of asthma. Since we observed that MON administration was able to suppress inflammation and the production of allergy-related IgE, we surmise that MON can be used to mitigate both the inflammation and pulmonary allergy in critically ill patients with asthma. Besides, since MON could also suppress the oxidative stress and immune cell infiltration in the lung tissues of asthmatic mice, MON could be jointly applied with other medications with asthma-relieving effect to suppress the progression of asthma-related lung tissue damages.

The major limitation in our study is that the conclusion comes from the animal model of experimentally induced asthma. Whether the similar therapeutic effect could be recapitulated in other asthmatic model (such as transgenic mouse model of chronic asthma and airway remodeling) remains to be explored. Future efforts are warranted to investigate the therapeutic effect of MON intervention in the clinical trials of asthmatic patients. Besides, our study only focused on the overall effects of MON treatment in the lung tissues. The potentially divergent effects of MON on different cellular components in the lung tissues need to be systematically elucidated.

In summary, our study demonstrated that MON showed potential therapeutic effects in OVA-sensitized asthmatic mouse model. MON administration suppressed oxidative stress and inflammatory responses in the lung tissues of asthmatic mice by dampening the activity of AKT/NF-κB signaling pathway. These data suggest the application potential of MON in the clinical management of asthma.

All animal experiments were approved by the Animal Care and Use Committee of Yantai Yuhuangding Hospital (China) (No. 2022011602) and conducted in accordance with the national and institutional guide for the care and application of laboratory animals.

All authors have declared no competing interests.

This study received no funding.

Xin Guo conducted experiments, collected dat, and drafted paper; Wenjie Sun analyzed data, modified the figures, and collected references; Bingbing Zhang conceived the study, supervised the experiments and revision, and edited the manuscript.

Additional Information

Edited by: H.-U. Simon, Bern.

Data are not publicly available due to ethical reasons. Further inquiries can be directed to the corresponding author via email.

1.
Wu
TD
,
Brigham
EP
,
McCormack
MC
.
Asthma in the primary care setting
.
Med Clin
.
2019
;
103
(
3
):
435
52
. .
2.
Dave
PH
,
Preetha
.
Pathogenesis and novel drug for treatment of asthma: a review
.
Res J Pharm Technol
.
2016
;
9
(
9
):
1519
. .
3.
Chung
KF
.
Managing severe asthma in adults: lessons from the ERS/ATS guidelines
.
Curr Opin Pulm Med
.
2015
;
21
(
1
):
8
15
. .
4.
Barnes
PJ
.
Glucocorticosteroids: current and future directions
.
Br J Pharmacol
.
2011
;
163
(
1
):
29
43
. .
5.
Rothe
T
,
Spagnolo
P
,
Bridevaux
PO
,
Clarenbach
C
,
Eich-Wanger
C
,
Meyer
F
, et al
.
Diagnosis and management of asthma: the Swiss guidelines
.
Respiration
.
2018
;
95
(
5
):
364
80
. .
6.
Volmer
T
,
Effenberger
T
,
Trautner
C
,
Buhl
R
.
Consequences of long-term oral corticosteroid therapy and its side-effects in severe asthma in adults: a focused review of the impact data in the literature
.
Eur Respir J
.
2018
;
52
(
4
):
1800703
. .
7.
Choi
J
,
Lee
KT
,
Choi
MY
,
Nam
JH
,
Jung
HJ
,
Park
SK
, et al
.
Antinociceptive anti-inflammatory effect of monotropein isolated from the root of Morinda officinalis
.
Biol Pharm Bull
.
2005
;
28
(
10
):
1915
8
. .
8.
Shin
JS
,
Yun
KJ
,
Chung
KS
,
Seo
KH
,
Park
HJ
,
Cho
YW
, et al
.
Monotropein isolated from the roots of Morinda officinalis ameliorates proinflammatory mediators in RAW 264.7 macrophages and dextran sulfate sodium (DSS)-induced colitis via NF-κB inactivation
.
Food Chem Toxicol
.
2013
;
53
:
263
71
. .
9.
Zhang
Y
,
Chen
Y
,
Li
B
,
Ding
P
,
Jin
D
,
Hou
S
, et al
.
The effect of monotropein on alleviating cisplatin-induced acute kidney injury by inhibiting oxidative damage, inflammation and apoptosis
.
Biomed Pharmacoth
.
2020
;
129
:
110408
. .
10.
Wang
P
,
Kang
SY
,
Kim
SJ
,
Park
YK
,
Jung
HW
.
Monotropein improves dexamethasone-induced muscle atrophy via the AKT/mTOR/FOXO3a signaling pathways
.
Nutrients
.
2022
;
14
(
9
):
1859
. .
11.
Jiang
F
,
Xu
XR
,
Li
WM
,
Xia
K
,
Wang
LF
,
Yang
XC
.
Monotropein alleviates H2O2-induced inflammation, oxidative stress and apoptosis via NF-κB/AP-1 signaling
.
Mol Med Rep
.
2020
;
22
(
6
):
4828
36
. .
12.
Chen
Y
,
Lu
YY
,
Pei
CY
,
Liang
J
,
Ding
P
,
Chen
S
, et al
.
Monotropein alleviates secondary liver injury in chronic colitis by regulating TLR4/NF-κB signaling and NLRP3 inflammasome
.
Eur J Pharmacol
.
2020
;
883
:
173358
. .
13.
Braman
SS
.
The global burden of asthma
.
Chest
.
2006
;
130
(
1 Suppl
):
4S
12
. .
14.
Asher
I
,
Pearce
N
.
Global burden of asthma among children
.
Int J Tuberc Lung Dis
.
2014
;
18
(
11
):
1269
78
. .
15.
Lowther
AL
,
Somani
AK
,
Camouse
M
,
Florentino
FT
,
Somach
SC
.
Invasive Trichophyton rubrum infection occurring with infliximab and long-term prednisone treatment
.
J Cutan Med Surg
.
2007
;
11
(
2
):
84
88
. .
16.
Taillé
C
,
Poulet
C
,
Marchand-Adam
S
,
Borie
R
,
Dombret
MC
,
Crestani
B
, et al
.
Monoclonal anti-TNF-α antibodies for severe steroid-dependent asthma: a case series
.
Open Respir Med J
.
2013
;
7
(
1
):
21
5
. .
17.
Cohn
L
,
Elias
JA
,
Chupp
GL
.
Asthma: mechanisms of disease persistence and progression
.
Annu Rev Immunol
.
2004
;
22
:
789
815
. .
18.
Afshar
R
,
Medoff
BD
,
Luster
AD
.
Allergic asthma: a tale of many T cells
.
Clin Exp Allergy
.
2008
;
38
(
12
):
1847
57
. .
19.
Müller
T
,
Hüttel
A
,
Idzko
M
.
The serotoninergic receptor subtype 5-HTR1B contributes to the pathogenesis of allergic airway inflammation
.
Pneumologie
.
2012
;
66
(
11
):
P3740
. .
20.
Elias
JA
,
Lee
CG
,
Zheng
T
,
Ma
B
,
Homer
RJ
,
Zhu
Z
.
New insights into the pathogenesis of asthma
.
J Clin Invest
.
2003
;
111
(
3
):
291
7
. .
21.
Adam
P
.
Clinical and Sweet talking-cellular carbohydrates and epithelial Experimental repair Allergy
.
Clin Exp Allergy
.
2006
;
36
(
5
):
560
2
.
22.
Renz
H
,
Enssle
K
,
Lauffer
L
,
Kurrle
R
,
Gelfand
EW
.
Inhibition of Allergen-Induced IgE and IgG1 Production by Soluble IL-4 Receptor
.
Int Arch Allergy Immunol
.
1995
;
106
(
1
):
46
54
. .
23.
Halim
TYF
,
Krauss
RH
,
Sun
A
,
Takei
F
.
Lung Natural Helper Cells Are a Critical Source of Th2 Cell-Type Cytokines in Protease Allergen-Induced Airway Inflammation
.
Immunity
.
2012
;
36
(
3
):
451
63
. .
24.
Barnes
PJ
.
Cytokine-directed therapies for asthma
.
J Allergy Clin Immunol
.
2001
;
108
(
2 Suppl
):
S72
6
. .
25.
Hogan
SP
,
Rosenberg
HF
,
Moqbel
R
,
Phipps
S
,
Foster
PS
,
Lacy
P
, et al
.
Eosinophils: Biological Properties and Role in Health and Disease
.
Clin Exp Allergy
.
2008
;
38
(
5
):
709
50
. .
26.
Liang
Z
,
Nie
H
,
Xu
Y
,
Peng
J
,
Zeng
Y
,
Wei
Y
, et al
.
Therapeutic effects of rosmarinic acid on airway responses in a murine model of asthma
.
Int Immunopharmacol
.
2016
;
41
:
90
7
. .
27.
Van Antwerp
DJ
,
Martin
SJ
,
Kafri
T
,
Green
DR
,
Verma
IM
.
Suppression of TNF-alpha-induced apoptosis by NF-kappaB
.
Science
.
1996
;
274
(
5288
):
787
9
. .
28.
Beg
AA
,
Sha
WC
,
Bronson
RT
,
Ghosh
S
,
Baltimore
D
.
Embryonic lethality and liver degeneration in mice lacking the RelA component of NF-kappa B
.
Nature
.
1995
;
376
(
6536
):
167
70
. .
29.
Li
Q
,
Verma
IM
.
NF-kappaB regulation in the immune system
.
Nat Rev Immunol
.
2002
;
2
(
10
):
725
34
. .
30.
Xia
YF
,
Ye
BQ
,
Li
YD
,
Wang
JG
,
He
XJ
,
Lin
X
, et al
.
Andrographolide attenuates inflammation by inhibition of NF-kappa B activation through covalent modification of reduced cysteine 62 of p50
.
J Immunol
.
2004
;
173
(
6
):
4207
17
. .
31.
Das
J
,
Chen
C
,
Yang
L
,
Cohn
L
,
Ray
P
,
Ray
A
.
A critical role for NF-kappa B in GATA3 expression and TH2 differentiation in allergic airway inflammation
.
Nat Immunol
.
2001
;
2
(
1
):
45
50
. .
32.
Mishra
V
,
Baranwal
V
,
Mishra
RK
,
Sharma
S
,
Paul
B
,
Pandey
AC
.
Titanium dioxide nanoparticles augment allergic airway inflammation and Socs3 expression via NF-kappa B pathway in murine model of asthma
.
Biomaterials
.
2016
;
92
:
90
102
. .
33.
Lim
JW
,
Goh
FY
,
Sagineedu
SR
,
Yong
AH
,
Sidik
S
,
Lajis
N
, et al
.
A semisynthetic diterpenoid lactone inhibits NF-κB signalling to ameliorate inflammation and airway hyperresponsiveness in a mouse asthma model
.
Toxicol Appl Pharmacol
.
2016
;
302
:
10
22
. .