Allergic asthma is a chronic inflammatory disease of the airway that is characterized by general pathological alteration, severe eosinophilia, lymphocyte infiltration, fibrosis deposition, and mucus overproduction (
Nuclear factor-κB (NF-κB) plays a pivotal role in the production of Th2 cytokines and recruitment of inflammatory cells in the airways of murine asthma models (
Traditional herbal medicines are generally recognized to be safe and exhibit various therapeutic effects (
The aim of the present study was to investigate the anti-asthmatic action of DC
HMC-1 cells were generously provided by Professor Hyun-Ja Jeong (Department of Food Science and Technology and Research Institute for Basic Science, Hoseo University, Republic of Korea). HMC-1 cells were grown in Iscove's modified Dulbecco's medium (IMDM), supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 10 µM monothioglycerol, and 10% heat-inactivated FBS at 37°C, in 5% CO2 and 95% humidity.
DC (KFRI-SL-2021) was provided by the Division of Nutrition and Metabolism Research, Korea Food Research Institute. DC used in this study was purchased from the Kyeong-Dong Oriental Pharmacy Market. DC underwent reflux extraction twice in 95% ethanol. The ethanol extract was dried under vacuum in a rotary evaporator. The concentrated extract was lyophilized, yielding a dried powder that was kept at 4°C until needed and dissolved in saline prior to use.
To identify the chemical constituents of DC, the ethanolic extracts of DC were analyzed using UPLC/Q-TOF MS (Waters Corp.). The extract was injected into an Acquity UPLC BEH C18 column (2.1×100 mm, 1.7 µm; Waters Corp.) at a column temperature of 40°C. The mobile phase consisted of water with 0.1% formic acid and acetonitrile with 0.1% formic acid, at a flow rate of 0.35 ml/min for 9 min. The capillary voltage was set at 3 or 2.5 kV, for positive or negative mode, respectively, while the sample cone voltage was 40 V. The desolvation flow rate was 900 l/h at 400°C and source temperature was set at 100°C. Leucine enkephalin [(M+H)=m/z 556.2771] was used as a reference for lock mass at a frequency of 10 sec. The MS/MS spectra were obtained using collision energy ramps from 20 to 45 eV. Metabolites were identified by Unifi software using various LC/MS databases.
Pathogen-free 5-week-old male BALB/c mice, weighing approximately 20 g, were purchased from Damool Science. Five mice were housed per cage in a laminar air-flow cabinet, maintained at 23±2°C at a relative humidity of 55±10%, with a 12 h dark/light cycle, throughout the study period. All animal experiments were performed in compliance with the NIH guidelines for the care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committees of Chonbuk National University Laboratory Animal Center (CBNU 2016-37 and CBNU 2019-071).
In this study, the mice were randomly divided into four groups (n=6 per group), namely control, OVA, DC, and Dex. The first sensitization was performed on day 1 by intraperitoneally injecting 50 µg of OVA (Grade V, Sigma, St. Louis, MO, USA), emulsified in 1 mg of alum (Imject Alum; Pierce), in a total volume of 200 µl. The control group received saline alone (NaCl 0.9%; B. Braun Medical BV, Oss, The Netherlands). On day 14, the second sensitization was performed by intraperitoneally injecting 50 µg of OVA in saline. From days 15 to 26, mice of the DC group and Dex group received DC (20 mg/ml) and Dex (2.5 mg/ml) by oral gavage. Mice belonging to the control and OVA groups received sham saline. On days 27, 28, and 29, the OVA, Dex, and DC groups were challenged with inhalation of ultrasonically nebulized 5% OVA solution in saline, for 20 min. Animals were sacrificed 24 h after day 29, to evaluate for airway inflammation and the production of allergen-specific cytokines (
The airway lumina of the sacrificed animals were washed using a tracheal cannula with 1 ml of saline twice. The BALF so obtained, was centrifuged and supernatants were stored at −80°C and subjected to ELISA assay using kits. Total and differential cell numbers were counted double-blind, using a hemocytometer. Cytospin cell preparations were made by placing the cells onto glass slides, centrifuging at 4°C for 10 min at 1,000 × g, and staining with Diff-Quik. Lung tissues were homogenized in saline to a concentration of 100 mg/ml with the complete, Mini, EDTA-free Protease Inhibitor (Roche Applied Science) and the debris-free supernatant was used for cytokine measurement.
After collecting the BALF, the lobes of the lung were removed for histological examination, fixed in 10% paraformaldehyde, and embedded in paraffin, using standard methods.
Hematoxylin and eosin staining to assess the general morphological structure of lung tissue. Periodic acid-Schiff (PAS) staining for visualizing goblet cell hyperplasia. PAS staining was performed on the lung tissue sections to visualize the development of goblet cell hyperplasia. Congo red staining for visualizing eosinophilic infiltration of the nasal mucosa. Eosinophils were morphologically defined by the presence of granules in the cytoplasm and a two-lobed nucleus and counted under a microscope. Masson's trichrome staining was used to reveal the sub-epithelial deposition of collagen in the lung tissue. Positive trichrome-stained areas to assess the degree of sub-epithelial fibrosis.
For assessing the Th1 response, the level of anti-inflammatory (Th1-associated) cytokines, such as IFN-γ and IL-10, in BALF and/or lung homogenates was assayed using ELISA kits (R&D Systems), following the manufacturer's instructions.
For assessing the Th2 response, we evaluated the secretion of inflammatory (Th2-associated) cytokines, such as IL-4, IL-5, and IL-13 and proinflammatory cytokines, including IL-6, in BALF and lung homogenates using ELISA kits (R&D Systems), as per the manufacturer's instructions.
Blood was collected from the orbital venous plexus of anesthetized mice 24 h after the last challenge. The samples were centrifuged (1,000 × g, 10 min, 4°C) to isolate the serum and stored at −80°C until further analysis. Thereafter, the serum was separated and levels of total IgE and OVA-specific IgE, IgG1, and IgG2a were measured in each group using ELISA (Chondrex), following the manufacturer's instruction.
The expression of NF-κB p65, IκB, phosphorylation of NF-κB p65 (p-NF-κB p65) and p-IκB in BALF and lung homogenate were examined by using ELISA kits (eBioscience Inc.), according to the manufacturer's instructions. The optical density was measured in 96-well plates using an ELISA reader, at 450 nm. We evaluated the activation of NF-κB p65 and p-NF-κB p65 and the nuclear translocalization of NF-κB in lung tissues, using ProteinSimple capillary immunoassay (Wes) method a gel- and blot-free method requiring less sample, antibody, and time to run than conventional western blot assays.
HMC-1 cells were pretreated with various concentrations of DC (0.1, 1.0, and 10 mg/ml) for 30 min and stimulated with 10 µM of A23187 and 200 nM of PMA overnight. The samples were centrifuged (1,000 × g, 10 min, 4°C) and the supernatants were used to evaluate the concentration of cytokines, including IL-6 and TNF-α. The activation of NF-κB and p-NF-κB was quantified using ELISA, according to the manufacturer's instructions (R&D Systems).
Each experiment was repeated three times with six mice per group. Data are expressed as mean ± SEMs. Statistical comparisons were performed using one-way ANOVA, followed by Fisher's test. P<0.05 was considered to indicate a statistically significant difference.
To examine the anti-inflammatory effect of DC on allergic asthma, we used the OVA-induced allergic asthma mouse model (
To evaluate histological changes after DC treatment, in the OVA-induced asthma model, the levels of general pathological changes, eosinophil infiltration, mucus production, goblet cell hyperplasia, and bronchial subepithelial fibrosis were examined in the lung tissue. Pulmonary histopathology was found to be normal in the control group. Mice from the OVA-challenged group exhibited a severe infiltration of inflammatory cells around the respiratory tract and blood vessels (
To investigate the therapeutic impact of DC on OVA-specific immune responses, we determined the serum immunoglobulin levels. As shown in
To elucidate the anti-allergic mechanism of DC on the Th1/Th2-mediated allergic response in the OVA-induced asthma mice models, the levels of secreted cytokines in BALF and lung homogenates were examined. First, we examined the secretion of the Th2 cytokines and proinflammatory cytokines, including IL-4, IL-5, IL-6, and IL-13, using ELISA. The OVA group showed increased levels of both in BALF and lung homogenates compared with the control group. However, DC treatment significantly decreased the levels of cytokines, including IL-4, IL-5, IL-6, and IL-13. In addition, the levels of anti-inflammatory cytokines (Th1 cytokines), such as IFN-γ and IL-10, were assayed. The DC-treated group showed a significant increase in the levels of IL-10 in BALF and IFN-γ in lung homogenates, and a decrease in IL-10 in lung homogenates and IFN-γ in BALF, as compared to the OVA group; however, no statistically significant differences were observed (
To understand the inhibitory effect of DC on allergic asthma, activation of NF-κB signaling-that regulates the expression Th2 cytokines-was measured in OVA-challenged mice. The levels of NF-κB p65 and p-NF-κB p65 activation in the lung tissue were assessed via western blot analysis. As presented in
Next, we determined whether DC suppressed nuclear translocalization of NF-κB in the lung tissue. As shown in
We assessed the levels of total NF-κB and IκB, as markers of NF-κB activation, degradation levels of p-NF-κB, and p-IκB activation. NF-κB, p-NF-κB, IκB, and p-IκB levels were higher in the OVA group compared with the control group, both in BALF and lung homogenates. However, we found that DC treatment downregulated the levels of NF-κB, p-NF-κB, IκB, and p-IκB in BALF and p-NF-κB, IκB, and p-IκB in lung homogenates, compared with the OVA group. DC treatment had a tendency to decrease the levels of NF-κB signaling components in lung homogenates. In addition, the Dex group also showed inhibitory effects on NF-κB signaling (
These data combined with those presented in
Mast cells play a major role in allergic inflammation. Pro-inflammatory cytokines, such as TNF-α and IL-6, are potent multifunctional cytokines that play an important role in the pathogenesis of allergic disease (
Activated mast cells play a key role in allergy by releasing numerous mediators, such as cytokines and leukotrienes, via degranulation (
To further investigate whether the NF-κB pathway plays an important role in the DC-mediated regulation of allergic inflammation, we analyzed the activation of NF-κB and p-NF-κB in HMC-1 cells. In HMC-1 cells, NF-κB activation was inhibited dose-dependently by DC treatment compared to PMA and A2318 treatment alone (
To investigate the main component present in the ethanolic extract of DC, UPLC/Q-TOF MS was used. The results showed four negative and eight positive modes (
The aim of this study was to investigate the anti-allergic and anti-inflammatory effects of DC using the OVA-induced allergic asthma mouse model. OVA-challenged mice showed an increased number of inflammatory cells in BALF; elevated total IgE, anti-OVA IgG1 and IgE in the serum; increased Th2 cytokines, including IL-4, IL-5, IL-6, and IL-13 in BALF; goblet cell hyperplasia with excessive airway mucus and collagen deposition; eosinophil and mast cell infiltration in lung tissues; activated NF-κB signaling in BALF and lung homogenates; and NF-κB activation in PMA and A23187-stimulated HMC-1 cells. However, DC treatment increased the level of Th1 cytokines and decreased the Th2 cytokines; inhibited NF-κB signaling activation in BALF, lung homogenates, and PMA and A23187-stimulated HMC-1 cells; inhibited total IgE, anti-OVA IgG1, and IgE production in the serum; and lowered the number of inflammatory cells in BALF. The administration of DC also suppressed the infiltration of eosinophils and mast cells, mucus overproduction, and collagen deposition. In addition, DC treatment attenuated TNF-α and IL-6 in PMA and A23187-stimulated HMC-1 cells. Thus, we demonstrated that DC has anti-allergic and anti-inflammatory effects on an OVA-induced asthma mouse model.
Medicinal herbs have been used in traditional medicines in Asian countries, such as Korea and China, since ancient times (
Asthma is a multifactorial and complex disease in which repetitive allergen challenge leads to permanent airway remodeling (
Previous studies have shown that NF-κB plays an important role in cytokine production (
A23187 and PMA induced an increase in cytosolic calcium concentration, leading to mast cell degranulation (
In summary, our data demonstrated that DC exerts a pivotal role in OVA-induced airway inflammation, both
Not applicable.
This research was supported by the Korea Food Research Institute (grant no. E0170401-03).
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
OHC designed the experiments. CHP, TTB, TVN and YF performed the experiments. CHP and HSS analyzed the data and performed the biological analysis. CHS and HTK collected and analyzed data. CHP, YF and TVN sacrificed mice and performed the ELISA assay, DS took samples and performed western blot analysis. SYL also checked the quality of DC and performed western blot analysis. The manuscript was written by CHP and OHC. All authors read and approved the manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolved. All authors read and approved the final manuscript.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
Dryopteris crassirhizoma
dexamethasone
ovalbumin
control
bronchoalveolar lavage fluid
hematoxylin and eosin
periodic acid-schiff
intraperitoneal
peroral
human mast cell
T helper type 1
T helper type 2
Interferon
Phorbol myristate acetate
Experimental protocol for asthma mouse model and effect of
Effect of
Effect of
Effects of
Effect of
Effect of DC on the expression of TNF-α and IL-6 and NF-ĸB signaling pathway in HMC-1 cells, stimulated by a combination of PMA and A23187. and infiltration of mast cells in the lung tissue. HMC-1 cells were pretreated with DC (0.1, 1.0, and 10 mg/ml) and stimulated with 10 µM of A23187 and 200 nM of PMA, overnight. The secretion levels of (A) TNF-α and IL-6 were measured using ELISA. (B) Staining and localization of mast cells in pulmonary sections from the ovalbumin-induced asthma mice. Representative Giemsa staining of lung sections of different groups of mice. Arrows point to stained mast cells. ###P<0.001, ***P<0.001 vs. ovalbumin group. BR, bronchial; OVA, ovalbumin; DC, Dryopteris crassirhizoma; DEX, dexamethasone; IL, interleukin.
Chemical profiles of DC by UPLC/Q-TOF MS. (A) Negative and positive modes. (B) Schematic representation of the main components of DC. DC, Dryopteris crassirhizoma.