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Research article
Aromadendrin Ameliorates Airway Inflammation in Experimental Mice with Chronic Obstructive Pulmonary Disease
1Natural Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju 28116, Republic of Korea
2College of Pharmacy, Chungbuk National University, Cheongju 28160, Republic of Korea
3O2MEDi Inc. 50 UNIST-gil, Eonyang-eup, Ulju-gun, Ulsan 44919, Republic of Korea
4Office of Surveillance for Narcotics Abuse, Ministry of Food and Drug Safety, Osong Health Technology Administration Complex, Cheongju 28159, Republic of Korea
5Department of Pharmacology, College of Medicine, Kangwon National University, Chuncheon 24341, Republic of Korea
6Department of Biotechnology, University of Science and Technology (UST), Daejeon 34113, Republic of Korea
J. Microbiol. Biotechnol. 2025. 35: e2408022
Published January 15, 2025 https://doi.org/10.4014/jmb.2408.08022
Copyright © The Korean Society for Microbiology and Biotechnology.
Abstract
Keywords
Graphical Abstract

Introduction
Chronic obstructive pulmonary disease (COPD) is a serious respiratory ailment resulting in increasing mortality worldwide [1]. Meanwhile, the need for drugs or supplements to treat COPD has become urgent. Smoking and bacterial infection promote the development of COPD by inducing immune cell activation and bronchial inflammation [2]. In COPD, neutrophils induce lung damage by forming reactive oxygen species (ROS), myeloperoxidase (MPO), and elastase [3-5]. Elevated levels of cytokines and chemokines, such as interleukin (IL)-6 [6], IL-1β [7], and monocyte chemoattractant protein (MCP)-1 [8] are observed in patients with COPD. Macrophages produce IL-6/IL-1β/MCP-1 and influence neutrophil influx, promoting bronchial inflammation in COPD [9]. Mucus accumulation limits airflow and affects pulmonary function in COPD [10].
Activation of the mitogen-activated protein kinase (MAPK)/nuclear factor kappa B (NF-κB)/NOD-like receptor protein 3 (NLRP3) inflammasome promotes bronchial inflammation [11-13]. Cigarette smoke extract (CSE) induces the proinflammatory M1 phenotype in alveolar macrophages by activating JNK MAPK [14]. p38 MAPK activation in the lungs of patients with COPD is associated with the development of bronchial inflammation [15, 16]. In vitro and in vivo models of COPD have shown activation of ERK MAPK [17-19]. NF-κB activation is a critical event in COPD development and has been confirmed in the lungs of cigarette smoke (CS)/lipopolysaccharide (LPS)-induced COPD animal models [20]. Activation of the NLRP3 inflammasome promotes the expression of IL-1β and is closely associated with the progression of pulmonary inflammation in COPD [21].
The intranasal administration of LPS accelerated CS-induced bronchial inflammation, similar to that observed in patients with COPD, by promoting neutrophil/macrophage accumulation, ROS/cytokine/chemokine formation, and MAPK/NF-κB activation in experimental COPD mice [18, 22].
Phenolic compounds exhibit various biological effects, including anti-inflammatory activity, according to cumulative in vitro and in vivo studies [18, 23, 24]. A flavanonol, aromadendrin (ARO), also known as dihydrokaempferol, is present in the pulp of
Materials and Methods
Reagents
Aromadendrin (ARO) was purchased from the Natural Products Research & Development Enterprise (ChemFaces, China).
Experimental Mouse Model of COPD
Six-week-old male C57BL/6 mice were purchased from Koatech Co. Ltd. (Republic of Korea). The procedures for animal experiments were approved by the IACUC of KRIBB (KRIBB-AEC-23122).
To establish bronchial inflammation, similar to that observed in COPD, mice were exposed to CS and LPS as previously described [11]. Briefly, the mice were exposed to CS for 50 min/day (seven cigarettes/day) for 7 days using a smoking machine (SciTech Korea, Inc., Republic of Korea). LPS was intranasally injected into the mice on day 6 (5 μg in 40 μl/mouse). Oral gavage (o.g.) of aromadendrin (ARO) and roflumilast (ROF) were administered for 7 consecutive days.
Five experimental groups (
Analysis of Immune Cells and Molecules in Bronchoalveolar Lavage (BAL) Fluid
To count immune cells in the BAL fluid, the mice were anesthetized with a mixture of Zoletil (30 mg/kg) and xylazine (5 mg/kg) [31]. Cell morphology was distinguished by Diff-Quik staining and cell numbers were measured using a light microscope (400 × magnification).
The level of ROS in BAL fluid was estimated using 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA) based on previous protocols [17]. The levels of IL-6, IL-1β, and MCP-1 in BAL fluid supernatant were measured using specific ELISA kits.
Western Blotting Analysis
The harvesting of lung tissue lysate with lysis buffer and protein quantification with BCA assay were performed based on a previous study [31] to detect the expression levels of neutrophil elastase (NE) and phosphorylated (p)-CREB/p-JNK/p-p38/p-ERK/p-p65/p-IκBα/NLRP3/ASC/Caspase-1. Each sample was then loaded onto an SDS-PAGE gel and transferred onto a PVDF membrane. Subsequently, membranes were incubated in blocking reagent (1× TBST with 5% skim milk) and primary antibodies (Table 1). The membranes were washed with 1× TBST four times prior to incubation with the corresponding secondary antibodies. Finally, the membranes were exposed to an ECL solution to visualize the bands.
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Table 1 . List of antibodies.
NO Primary antibody Company Molecular weight Host Secondary antibody 1 NE (bs-6982R) Bioss Antibodies 26 Rabbit Goat anti rabbit-HRP 2 p-CREB (9198s) Cell Signaling 43 Rabbit Goat anti rabbit -HRP 3 CREB (9197s) Cell Signaling 43 Rabbit Goat anti rabbit -HRP 4 p-JNK (4668S) Cell Signaling 46, 54 Rabbit Goat anti rabbit -HRP 5 JNK (9252S) Cell Signaling 46, 54 Rabbit Goat anti rabbit -HRP 6 p-p38 (sc7973) Santa Cruz 38 Mouse Goat anti mouse-HRP 7 p38 (sc-7972) Santa Cruz 38 Mouse Goat anti mouse-HRP 8 p-ERK (9101s) Cell Signaling 42, 44 Rabbit Goat anti rabbit-HRP 9 ERK (9102s) Cell Signaling 42, 44 Rabbit Goat anti rabbit-HRP 10 p-p65 (3033S) Cell Signaling 65 Rabbit Goat anti rabbit-HRP 11 p65 (sc-8008) Santa Cruz 65 Mouse Goat anti mouse-HRP 12 p-IκBα (2859S) Cell Signaling 40 Rabbit Goat anti rabbit-HRP 13 NLRP3 (15101s) Cell Signaling 110 Rabbit Goat anti rabbit-HRP 14 ASC (sc-514414) Santa Cruz 24 Mouse Goat anti Mouse-HRP 15 Caspase-1 (sc56036) Santa Cruz 45 Mouse Goat anti Mouse-HRP 16 β-action (sc-47778) Santa Cruz 43 Mouse Goat anti mouse-HRP
Histological Analysis
Histological changes in the lungs of mice were determined using hematoxylin and eosin (H&E) and PAS staining. For each staining, the lung tissues dissected from the experimental mice were washed in ice-cold PBS, fixed in 10% formalin solution, and embedded in paraffin. Subsequently, lung tissue sections were stained with H&E and PAS [32].
Statistical Analysis
Values are expressed as the mean ± SD. One-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test was performed to analyze differences between groups. Data were analyzed using SPSS software (version 20.0; IBM Corp.).
Results
ARO Reduced the Influx of Immune Cells in Mice with COPD
Phenolic compounds that inhibit immune cell inflow ameliorate bronchial inflammation in mice with COPD [18]. Therefore, we first examined whether ARO exerts inhibitory effects on neutrophil/macrophage influx. As shown in Fig. 1A and 1B, the influx of these cells was upregulated in the BAL fluid of the COPD group, which was significantly decreased by ARO oral administration (5 and 10 mg/kg). The inhibition rates of ARO on neutrophil counts were 47.8% (5 mg/kg ROF), 29.5% (5 mg/kg ARO), and 39.6% (10 mg/kg ARO). The inhibition rates of ARO on macrophage counts were 51.2% (5 mg/kg ROF), 34.1% (5 mg/kg ARO), and 40.0% (10 mg/kg ARO). The inhibitory effect of ARO (10 mg/kg) on cell influx was comparable to that of 5 mg/kg ROF, which was used as a positive control.
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Fig. 1. ARO inhibits neutrophils/macrophages influx in experimental COPD mice. (A) The images of neutrophils/macrophages were obtained using Diff-Quik staining and microscopy (magnification, x400; scale bar, 25 μm). Number of (B) neutrophils and (C) macrophages in BAL fluid of mice (green arrows indicate the neutrophils and red arrows indicate the macrophages). Data are expressed as the mean ± SD (#
p < 0.05 for comparison with normal control; *p < 0.05 for comparison with COPD group). NC: normal control mice; COPD: cigarette smoke exposed/LPS-administered mice; ROF: 5 mg/kg ROF-treated COPD mice; ARO 5: 5 mg/kg ARO-treated COPD mice; and ARO 10: 10 mg/kg ARO-treated COPD mice.
ARO Decreased the Concentration of Molecules in Mice with COPD
Increased levels of ROS, MPO, IL-6, IL-1β, and MCP-1 in BAL fluid have been confirmed in COPD mice [11, 18, 19]. In this study, a notable upregulation of these molecules was observed in the BAL fluid of mice with COPD (Fig. 2A-2E). This tendency was mitigated by ARO. The inhibition rates of ARO on ROS production were 30.1%(5 mg/kg ROF), 27.4% (5 mg/kg ARO), and 35.5% (10 mg/kg ARO). The inhibition rates of ARO on MPO production were 42.8% (5 mg/kg ROF), 18.2% (5 mg/kg ARO), and 28.4% (10 mg/kg ARO). The inhibition rates of ARO on IL-6 production were 53.1% (5 mg/kg ROF), 38.8% (5 mg/kg ARO), and 57.4% (10 mg/kg ARO). The inhibition rates of ARO on IL-1β production were 50.9% (5 mg/kg ROF), 29.9% (5 mg/kg ARO), and 42.2%(10 mg/kg ARO). The inhibition rates of ARO on MCP-1 production were 56.3% (5 mg/kg ROF), 35.8% (5 mg/kg ARO), and 44.4% (10 mg/kg ARO). In general, the inhibitory effects of 10 mg/kg ARO on the formation of these molecules were comparable to those of 5 mg/kg ROF.
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Fig. 2. ARO reduces the concentration of inflammatory molecules in experimental COPD mice. (A) Total cellular ROS level in BAL fluid cells was determined using 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA). Levels of inflammatory molecules, such as (B) MPO (C) IL-6, (D) IL-1β, and (D) MCP-1 in BAL fluid, were detected by ELISA. Data are expressed as the mean ± SD (#
p < 0.05 for comparison with normal control; *p < 0.05 for comparison with COPD group). NC: normal control mice; COPD: cigarette smoke exposed/LPS-administered mice; ROF: 5 mg/kg ROF-treated COPD mice; ARO 5: 5 mg/kg ARO-treated COPD mice; and ARO 10: 10 mg/kg ARO-treated COPD mice.
Reduced Cell Accumulation and NE Expression by ARO in Mice with COPD
Next, histological changes were observed using H&E staining in the lungs of COPD mice. The results showed that the COPD group had higher cell accumulation compared to the normal control (NC) group (Fig. 3A). However, the ARO-treated group showed reduced cell accumulation compared to the COPD group. NE concentration was increased in an in vivo study of COPD [22]; thus, we measured the inhibitory effect of ARO on NE level using western blotting. NE expression in the lungs of the COPD group was significantly reduced by ARO treatment (Fig. 3B and 3C).
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Fig. 3. Cell accumulation and elastase expression were reduced by ARO in experimental COPD mice. (A) The histological changes in lungs of mice, which show cell accumulation, were assessed using H&E staining (left panel: magnification, 100×; scale bar, 100 μm; right panel: magnification, 400×; scale bar, 25 μm). (B) The expression of neutrophil elastase was analyzed using western blotting. Quantitative analysis of neutrophil elastase was performed using ImageJ software. Data are expressed as the mean ± SD (#
p < 0.05 for comparison with normal control; *p < 0.05 for comparison with COPD group). NC: normal control mice; COPD: cigarette smoke exposed/LPS-administered mice; ROF: 5 mg/kg ROF-treated COPD mice; ARO 5: 5 mg/kg ARO-treated COPD mice; and ARO 10: 10 mg/kg ARO-treated COPD mice.
ARO Reduced Mucus and CREB Activation in Mice with COPD
As shown in Fig. 4A, an increase in mucus formation in the airway epithelium was confirmed in the COPD group using PAS staining, and this increase was restrained in the ARO-treated COPD group. CREB activation is associated with mucus formation [33, 34]. To examine whether ARO could modulate CREB activation, the expression levels of phosphorylated (p)-CREB in the lungs of mice were examined. The results showed that the upregulation of p-CREB in the lungs of the COPD group was notably attenuated by ARO (Fig. 4B and 4C).
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Fig. 4. ARO attenuates mucus formation and CREB activation in experimental COPD mice. (A) The histological changes in lungs of mice, which indicate mucus formation, were assessed using PAS staining (magnification, 400×; scale bar, 25 μm). (B) The activation of CREB was analyzed using western blotting. Quantitative analysis of phosphorylated (p)-CREB was performed using ImageJ software. Data are expressed as the mean ± SD (#
p < 0.05 for comparison with normal control; *p < 0.05 for comparison with COPD group). NC: normal control mice; COPD: cigarette smoke exposed/LPSadministered mice; ROF: 5 mg/kg ROF-treated COPD mice; ARO 5: 5 mg/kg ARO-treated COPD mice; and ARO 10: 10 mg/kg ARO-treated COPD mice.
ARO Attenuated MAPK Activation in Mice with COPD
As described previously [14-19], activation of JNK, p38, and ERK is an important event in COPD development. Therefore, we explored whether ARO affects MAPK activation in a murine model of COPD. As shown in Fig. 5A-5D, the expressions of p-JNK, p-p38, and p-ERK were significantly increased in the lungs of the COPD group; however, they were reduced in the lungs of the ARO-treated COPD group. Particularly, the inhibitory effect of ARO on JNK and p38 activation was much stronger than that on ERK activation.
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Fig. 5. ARO inhibits MAPK activation in experimental COPD mice. (A) The activation of JNK, p38, and ERK in lungs of mice was analyzed using western blotting. Quantitative analysis of (B) p-JNK, (C) p-p38, and (D) p-ERK was performed using ImageJ software. Data are expressed as the mean ± SD (#
p < 0.05 for comparison with normal control; *p < 0.05 for comparison with COPD group). NC: normal control mice; COPD: cigarette smoke exposed/LPS-administered mice; ROF: 5 mg/kg ROF-treated COPD mice; ARO 5: 5 mg/kg ARO-treated COPD mice; and ARO 10: 10 mg/kg ARO-treated COPD mice.
ARO Reduced NF-κB Activation in Mice with COPD
NF-κB inactivation alleviates bronchial inflammation in COPD mice [20], and we therefore investigated the inhibitory effects of ARO on NF-κB activation in this study. As shown in Fig. 6A-6C, NF-κB p65 and IκBα were activated in the lungs of COPD mice; this activation was decreased by ARO administration.
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Fig. 6. ARO inhibits NF-κB activation in experimental COPD mice. (A) The activation of NF-κB p65 and IκBα was analyzed using western blotting. Quantitative analysis of (B) p- NF-κB p65 and (C) p-IκBα was performed using ImageJ software. Data are expressed as the mean ± SD (#
p < 0.05 for comparison with normal control; *p < 0.05 for comparison with COPD group). NC: normal control mice; COPD: cigarette smoke exposed/LPS-administered mice; ROF: 5 mg/kg ROF-treated COPD mice; ARO 5: 5 mg/kg ARO-treated COPD mice; and ARO 10: 10 mg/kg ARO-treated COPD mice.
ARO Diminished NLRP3 Activation in Mice with COPD
Next, we investigated the effects of ARO on NLRP3 inflammasome activation. As shown in Fig. 7A and 7B, the expression of NLRP3 in the lungs of the COPD mice was increased; however, this increase was suppressed by the oral administration of ARO. Furthermore, the increase in ASC and Caspase-1 expression in the COPD group was suppressed by ARO treatment (Fig. 7A, 7C, and 7D).
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Fig. 7. ARO inhibits NLRP3 activation in experimental COPD mice. (A) The expression of NLRP3, ASC, and caspase-1 was analyzed using western blotting. Quantitative analysis of (B) NLRP3, (C) ASC, and (D) caspase-1 was performed using ImageJ software. Data are expressed as the mean ± SD (#
p < 0.05 for comparison with normal control; *p < 0.05 for comparison with COPD group). NC: normal control mice; COPD: cigarette smoke exposed/LPS-administered mice; ROF: 5 mg/kg ROF-treated COPD mice; ARO 5: 5 mg/kg ARO-treated COPD mice; and ARO 10: 10 mg/kg ARO-treated COPD mice.
Discussion
Neutrophil- and macrophage-derived molecules, such as toxic molecules, cytokines, and chemokines, promote airway inflammation and lung damage in COPD. Thus, controlling the inflow of neutrophils/macrophages and the formation of these cell-mediated molecules are important for ameliorating bronchial inflammation and lung damage. In the present study, ARO excellently inhibited the influx of neutrophils/macrophages and the formation of ROS/MPO/NE/IL-6/IL-1β/MCP-1 in COPD mice. This ability was comparable to that of the positive control ROF. These observations indicated that ARO may exert anti-inflammatory effects on bronchial inflammation in COPD.
As unnecessary mucus generation impedes airflow and CREB activation is closely associated with mucus formation [33, 34], controlling CREB activation may improve airway flow restriction. ARO inhibited both mucus formation and CREB activation in experimental mice with COPD, indicating that ARO exerts a modulatory effect on mucus formation.
Previously, p38 was shown to be activated in the alveolar spaces of COPD patients [35]. Earlier clinical research demonstrated that NF-κB expression was upregulated in the bronchial epithelium of COPD patients [36]. Meanwhile, a recent clinical study confirmed a significant increase in NLRP3/caspase-1/IL-1β expression in COPD patients [37]. A preclinical study also showed that suppression of JNK/p38/ERK MAPK ameliorates inflammatory responses in a murine model of COPD [22]. Previous and current observations have indicated the usefulness of NF-κB inactivation in ameliorating airway inflammation in mice with COPD [11, 38]. Furthermore, inhibition of the NLRP3 inflammasome disrupts the expression of IL-1β, a therapeutic target for COPD treatment [21]. These observations have demonstrated that targeting MAPK/NF-κB/NLRP3 inflammasome pathways may be an effective therapeutic approach against COPD. In the present study, the inhibitory effects of ARO on MAPK (JNK and p38)/NF-κB/NLRP3 activation were notable, and its ability was comparable to that of ROF. These results highlight the potential of ARO as an adjuvant for COPD.
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Fig. 8. Ameliorative effects of ARO on bronchial inflammation in experimental COPD mice. Oral administration of ARO reduces neutrophil/macrophage inflow and ROS/MPO/elastase/IL-6/IL-1β/MCP-1 formation in COPD mice. These effects of ARO are accompanied by its modulatory effect on MAPK/NF-κB/NLRP3 activation.
Natural products and their bioactive compounds exhibit various biological activities, including anti-inflammatory and antioxidant effects [17, 39]. Accumulating studies have proven that bioactive compounds ameliorate lung inflammation in COPD mice by inducing MAPK/NF-κB/NLRP3 inactivation. Recently, ARO was shown to exert anti-asthmatic effects by suppressing NF-κB activation [30]. In addition, Ma
Collectively, our results demonstrate that ARO ameliorated bronchial inflammation by suppressing immune cell inflow, inflammatory molecule formation, and mucus formation. These effects were accompanied with suppressive effect on MAPK/NF-κB/NLRP3 activation. Overall, ARO showed excellent efficacy at a dose of 10 mg/kg, highlighting its potential for the development of anti-COPD adjuvants.
Acknowledgments
This study was supported by grants from the KRIBB Research Initiative Program (Grant No. KGM5522423), the Bio & Medical Technology Development Program of the National Research Foundation (NRF), and the Korean Government (MSIT) (Grant No. NRF–2020R1A2C2101228) of the Republic of Korea.
Conflict of Interests
The authors have no financial conflicts of interest to declare.
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Article
Research article
J. Microbiol. Biotechnol. 2025; 35():
Published online January 15, 2025 https://doi.org/10.4014/jmb.2408.08022
Copyright © The Korean Society for Microbiology and Biotechnology.
Aromadendrin Ameliorates Airway Inflammation in Experimental Mice with Chronic Obstructive Pulmonary Disease
Jinseon Choi1,2†, Seok Han Yun1,2†, Hyueyun Kim1†, Juhyun Lee1, Seong-Man Kim3, Mi-Hyeong Park4, Hee Jae Lee5, Wanjoo Chun5, Sang-Bae Han2*, Kyung-Seop Ahn1*, and Jae-Won Lee1,6*
1Natural Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju 28116, Republic of Korea
2College of Pharmacy, Chungbuk National University, Cheongju 28160, Republic of Korea
3O2MEDi Inc. 50 UNIST-gil, Eonyang-eup, Ulju-gun, Ulsan 44919, Republic of Korea
4Office of Surveillance for Narcotics Abuse, Ministry of Food and Drug Safety, Osong Health Technology Administration Complex, Cheongju 28159, Republic of Korea
5Department of Pharmacology, College of Medicine, Kangwon National University, Chuncheon 24341, Republic of Korea
6Department of Biotechnology, University of Science and Technology (UST), Daejeon 34113, Republic of Korea
Correspondence to:Sang-Bae Han, shan@chungbuk.ac.kr
Kyung-Seop Ahn, ksahn@kribb.re.kr
Jae-Won Lee, suc369@kribb.re.kr
†These authors contributed equally to this work.
Abstract
Aromadendrin (ARO) is an active plant compound that exerts anti-inflammatory effects. However, its ameliorative effects on chronic obstructive pulmonary disease (COPD) remain unclear. Therefore, we investigated the inhibitory effects of ARO on bronchial inflammation using an experimental model of COPD. In vivo analysis confirmed a notable increase in the number of neutrophils/macrophages and the formation of reactive oxygen species (ROS), myeloperoxidase (MPO), interleukin (IL)-6/IL-1β, and monocyte chemoattractant protein (MCP)-1 in the bronchoalveolar lavage (BAL) fluid of COPD mice, which was attenuated by oral gavage of ARO. In addition, hematoxylin and eosin staining showed a notable cell influx in the lungs of the COPD group, which was ameliorated by ARO. Western blotting revealed that ARO decreased the upregulation of neutrophil elastase expression in the lungs of the COPD group. Furthermore, periodic acid–Schiff staining showed that increased mucus formation in the lungs of the COPD group was downregulated by ARO. ARO also blocked CREB activation in the lungs of COPD mice. This in vivo, anti-inflammatory effect of ARO was accompanied by its modulatory effect on the activation of the MAPK/NF-κB/NLRP3 inflammasome. In summary, our study demonstrated that ARO has protective effects on bronchial inflammation by attenuating immune cell accumulation, toxic molecule/cytokine/chemokine formation, and MAPK/NF-κB/NLRP3 inflammasome activation, suggesting the potential development of ARO as an adjuvant for the prevention and treatment of COPD.
Keywords: COPD, aromadendrin, cytokines, NF-&kappa,B, NLRP3 inflammasome
Introduction
Chronic obstructive pulmonary disease (COPD) is a serious respiratory ailment resulting in increasing mortality worldwide [1]. Meanwhile, the need for drugs or supplements to treat COPD has become urgent. Smoking and bacterial infection promote the development of COPD by inducing immune cell activation and bronchial inflammation [2]. In COPD, neutrophils induce lung damage by forming reactive oxygen species (ROS), myeloperoxidase (MPO), and elastase [3-5]. Elevated levels of cytokines and chemokines, such as interleukin (IL)-6 [6], IL-1β [7], and monocyte chemoattractant protein (MCP)-1 [8] are observed in patients with COPD. Macrophages produce IL-6/IL-1β/MCP-1 and influence neutrophil influx, promoting bronchial inflammation in COPD [9]. Mucus accumulation limits airflow and affects pulmonary function in COPD [10].
Activation of the mitogen-activated protein kinase (MAPK)/nuclear factor kappa B (NF-κB)/NOD-like receptor protein 3 (NLRP3) inflammasome promotes bronchial inflammation [11-13]. Cigarette smoke extract (CSE) induces the proinflammatory M1 phenotype in alveolar macrophages by activating JNK MAPK [14]. p38 MAPK activation in the lungs of patients with COPD is associated with the development of bronchial inflammation [15, 16]. In vitro and in vivo models of COPD have shown activation of ERK MAPK [17-19]. NF-κB activation is a critical event in COPD development and has been confirmed in the lungs of cigarette smoke (CS)/lipopolysaccharide (LPS)-induced COPD animal models [20]. Activation of the NLRP3 inflammasome promotes the expression of IL-1β and is closely associated with the progression of pulmonary inflammation in COPD [21].
The intranasal administration of LPS accelerated CS-induced bronchial inflammation, similar to that observed in patients with COPD, by promoting neutrophil/macrophage accumulation, ROS/cytokine/chemokine formation, and MAPK/NF-κB activation in experimental COPD mice [18, 22].
Phenolic compounds exhibit various biological effects, including anti-inflammatory activity, according to cumulative in vitro and in vivo studies [18, 23, 24]. A flavanonol, aromadendrin (ARO), also known as dihydrokaempferol, is present in the pulp of
Materials and Methods
Reagents
Aromadendrin (ARO) was purchased from the Natural Products Research & Development Enterprise (ChemFaces, China).
Experimental Mouse Model of COPD
Six-week-old male C57BL/6 mice were purchased from Koatech Co. Ltd. (Republic of Korea). The procedures for animal experiments were approved by the IACUC of KRIBB (KRIBB-AEC-23122).
To establish bronchial inflammation, similar to that observed in COPD, mice were exposed to CS and LPS as previously described [11]. Briefly, the mice were exposed to CS for 50 min/day (seven cigarettes/day) for 7 days using a smoking machine (SciTech Korea, Inc., Republic of Korea). LPS was intranasally injected into the mice on day 6 (5 μg in 40 μl/mouse). Oral gavage (o.g.) of aromadendrin (ARO) and roflumilast (ROF) were administered for 7 consecutive days.
Five experimental groups (
Analysis of Immune Cells and Molecules in Bronchoalveolar Lavage (BAL) Fluid
To count immune cells in the BAL fluid, the mice were anesthetized with a mixture of Zoletil (30 mg/kg) and xylazine (5 mg/kg) [31]. Cell morphology was distinguished by Diff-Quik staining and cell numbers were measured using a light microscope (400 × magnification).
The level of ROS in BAL fluid was estimated using 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA) based on previous protocols [17]. The levels of IL-6, IL-1β, and MCP-1 in BAL fluid supernatant were measured using specific ELISA kits.
Western Blotting Analysis
The harvesting of lung tissue lysate with lysis buffer and protein quantification with BCA assay were performed based on a previous study [31] to detect the expression levels of neutrophil elastase (NE) and phosphorylated (p)-CREB/p-JNK/p-p38/p-ERK/p-p65/p-IκBα/NLRP3/ASC/Caspase-1. Each sample was then loaded onto an SDS-PAGE gel and transferred onto a PVDF membrane. Subsequently, membranes were incubated in blocking reagent (1× TBST with 5% skim milk) and primary antibodies (Table 1). The membranes were washed with 1× TBST four times prior to incubation with the corresponding secondary antibodies. Finally, the membranes were exposed to an ECL solution to visualize the bands.
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Table 1 . List of antibodies..
NO Primary antibody Company Molecular weight Host Secondary antibody 1 NE (bs-6982R) Bioss Antibodies 26 Rabbit Goat anti rabbit-HRP 2 p-CREB (9198s) Cell Signaling 43 Rabbit Goat anti rabbit -HRP 3 CREB (9197s) Cell Signaling 43 Rabbit Goat anti rabbit -HRP 4 p-JNK (4668S) Cell Signaling 46, 54 Rabbit Goat anti rabbit -HRP 5 JNK (9252S) Cell Signaling 46, 54 Rabbit Goat anti rabbit -HRP 6 p-p38 (sc7973) Santa Cruz 38 Mouse Goat anti mouse-HRP 7 p38 (sc-7972) Santa Cruz 38 Mouse Goat anti mouse-HRP 8 p-ERK (9101s) Cell Signaling 42, 44 Rabbit Goat anti rabbit-HRP 9 ERK (9102s) Cell Signaling 42, 44 Rabbit Goat anti rabbit-HRP 10 p-p65 (3033S) Cell Signaling 65 Rabbit Goat anti rabbit-HRP 11 p65 (sc-8008) Santa Cruz 65 Mouse Goat anti mouse-HRP 12 p-IκBα (2859S) Cell Signaling 40 Rabbit Goat anti rabbit-HRP 13 NLRP3 (15101s) Cell Signaling 110 Rabbit Goat anti rabbit-HRP 14 ASC (sc-514414) Santa Cruz 24 Mouse Goat anti Mouse-HRP 15 Caspase-1 (sc56036) Santa Cruz 45 Mouse Goat anti Mouse-HRP 16 β-action (sc-47778) Santa Cruz 43 Mouse Goat anti mouse-HRP
Histological Analysis
Histological changes in the lungs of mice were determined using hematoxylin and eosin (H&E) and PAS staining. For each staining, the lung tissues dissected from the experimental mice were washed in ice-cold PBS, fixed in 10% formalin solution, and embedded in paraffin. Subsequently, lung tissue sections were stained with H&E and PAS [32].
Statistical Analysis
Values are expressed as the mean ± SD. One-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test was performed to analyze differences between groups. Data were analyzed using SPSS software (version 20.0; IBM Corp.).
Results
ARO Reduced the Influx of Immune Cells in Mice with COPD
Phenolic compounds that inhibit immune cell inflow ameliorate bronchial inflammation in mice with COPD [18]. Therefore, we first examined whether ARO exerts inhibitory effects on neutrophil/macrophage influx. As shown in Fig. 1A and 1B, the influx of these cells was upregulated in the BAL fluid of the COPD group, which was significantly decreased by ARO oral administration (5 and 10 mg/kg). The inhibition rates of ARO on neutrophil counts were 47.8% (5 mg/kg ROF), 29.5% (5 mg/kg ARO), and 39.6% (10 mg/kg ARO). The inhibition rates of ARO on macrophage counts were 51.2% (5 mg/kg ROF), 34.1% (5 mg/kg ARO), and 40.0% (10 mg/kg ARO). The inhibitory effect of ARO (10 mg/kg) on cell influx was comparable to that of 5 mg/kg ROF, which was used as a positive control.
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Figure 1. ARO inhibits neutrophils/macrophages influx in experimental COPD mice. (A) The images of neutrophils/macrophages were obtained using Diff-Quik staining and microscopy (magnification, x400; scale bar, 25 μm). Number of (B) neutrophils and (C) macrophages in BAL fluid of mice (green arrows indicate the neutrophils and red arrows indicate the macrophages). Data are expressed as the mean ± SD (#
p < 0.05 for comparison with normal control; *p < 0.05 for comparison with COPD group). NC: normal control mice; COPD: cigarette smoke exposed/LPS-administered mice; ROF: 5 mg/kg ROF-treated COPD mice; ARO 5: 5 mg/kg ARO-treated COPD mice; and ARO 10: 10 mg/kg ARO-treated COPD mice.
ARO Decreased the Concentration of Molecules in Mice with COPD
Increased levels of ROS, MPO, IL-6, IL-1β, and MCP-1 in BAL fluid have been confirmed in COPD mice [11, 18, 19]. In this study, a notable upregulation of these molecules was observed in the BAL fluid of mice with COPD (Fig. 2A-2E). This tendency was mitigated by ARO. The inhibition rates of ARO on ROS production were 30.1%(5 mg/kg ROF), 27.4% (5 mg/kg ARO), and 35.5% (10 mg/kg ARO). The inhibition rates of ARO on MPO production were 42.8% (5 mg/kg ROF), 18.2% (5 mg/kg ARO), and 28.4% (10 mg/kg ARO). The inhibition rates of ARO on IL-6 production were 53.1% (5 mg/kg ROF), 38.8% (5 mg/kg ARO), and 57.4% (10 mg/kg ARO). The inhibition rates of ARO on IL-1β production were 50.9% (5 mg/kg ROF), 29.9% (5 mg/kg ARO), and 42.2%(10 mg/kg ARO). The inhibition rates of ARO on MCP-1 production were 56.3% (5 mg/kg ROF), 35.8% (5 mg/kg ARO), and 44.4% (10 mg/kg ARO). In general, the inhibitory effects of 10 mg/kg ARO on the formation of these molecules were comparable to those of 5 mg/kg ROF.
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Figure 2. ARO reduces the concentration of inflammatory molecules in experimental COPD mice. (A) Total cellular ROS level in BAL fluid cells was determined using 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA). Levels of inflammatory molecules, such as (B) MPO (C) IL-6, (D) IL-1β, and (D) MCP-1 in BAL fluid, were detected by ELISA. Data are expressed as the mean ± SD (#
p < 0.05 for comparison with normal control; *p < 0.05 for comparison with COPD group). NC: normal control mice; COPD: cigarette smoke exposed/LPS-administered mice; ROF: 5 mg/kg ROF-treated COPD mice; ARO 5: 5 mg/kg ARO-treated COPD mice; and ARO 10: 10 mg/kg ARO-treated COPD mice.
Reduced Cell Accumulation and NE Expression by ARO in Mice with COPD
Next, histological changes were observed using H&E staining in the lungs of COPD mice. The results showed that the COPD group had higher cell accumulation compared to the normal control (NC) group (Fig. 3A). However, the ARO-treated group showed reduced cell accumulation compared to the COPD group. NE concentration was increased in an in vivo study of COPD [22]; thus, we measured the inhibitory effect of ARO on NE level using western blotting. NE expression in the lungs of the COPD group was significantly reduced by ARO treatment (Fig. 3B and 3C).
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Figure 3. Cell accumulation and elastase expression were reduced by ARO in experimental COPD mice. (A) The histological changes in lungs of mice, which show cell accumulation, were assessed using H&E staining (left panel: magnification, 100×; scale bar, 100 μm; right panel: magnification, 400×; scale bar, 25 μm). (B) The expression of neutrophil elastase was analyzed using western blotting. Quantitative analysis of neutrophil elastase was performed using ImageJ software. Data are expressed as the mean ± SD (#
p < 0.05 for comparison with normal control; *p < 0.05 for comparison with COPD group). NC: normal control mice; COPD: cigarette smoke exposed/LPS-administered mice; ROF: 5 mg/kg ROF-treated COPD mice; ARO 5: 5 mg/kg ARO-treated COPD mice; and ARO 10: 10 mg/kg ARO-treated COPD mice.
ARO Reduced Mucus and CREB Activation in Mice with COPD
As shown in Fig. 4A, an increase in mucus formation in the airway epithelium was confirmed in the COPD group using PAS staining, and this increase was restrained in the ARO-treated COPD group. CREB activation is associated with mucus formation [33, 34]. To examine whether ARO could modulate CREB activation, the expression levels of phosphorylated (p)-CREB in the lungs of mice were examined. The results showed that the upregulation of p-CREB in the lungs of the COPD group was notably attenuated by ARO (Fig. 4B and 4C).
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Figure 4. ARO attenuates mucus formation and CREB activation in experimental COPD mice. (A) The histological changes in lungs of mice, which indicate mucus formation, were assessed using PAS staining (magnification, 400×; scale bar, 25 μm). (B) The activation of CREB was analyzed using western blotting. Quantitative analysis of phosphorylated (p)-CREB was performed using ImageJ software. Data are expressed as the mean ± SD (#
p < 0.05 for comparison with normal control; *p < 0.05 for comparison with COPD group). NC: normal control mice; COPD: cigarette smoke exposed/LPSadministered mice; ROF: 5 mg/kg ROF-treated COPD mice; ARO 5: 5 mg/kg ARO-treated COPD mice; and ARO 10: 10 mg/kg ARO-treated COPD mice.
ARO Attenuated MAPK Activation in Mice with COPD
As described previously [14-19], activation of JNK, p38, and ERK is an important event in COPD development. Therefore, we explored whether ARO affects MAPK activation in a murine model of COPD. As shown in Fig. 5A-5D, the expressions of p-JNK, p-p38, and p-ERK were significantly increased in the lungs of the COPD group; however, they were reduced in the lungs of the ARO-treated COPD group. Particularly, the inhibitory effect of ARO on JNK and p38 activation was much stronger than that on ERK activation.
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Figure 5. ARO inhibits MAPK activation in experimental COPD mice. (A) The activation of JNK, p38, and ERK in lungs of mice was analyzed using western blotting. Quantitative analysis of (B) p-JNK, (C) p-p38, and (D) p-ERK was performed using ImageJ software. Data are expressed as the mean ± SD (#
p < 0.05 for comparison with normal control; *p < 0.05 for comparison with COPD group). NC: normal control mice; COPD: cigarette smoke exposed/LPS-administered mice; ROF: 5 mg/kg ROF-treated COPD mice; ARO 5: 5 mg/kg ARO-treated COPD mice; and ARO 10: 10 mg/kg ARO-treated COPD mice.
ARO Reduced NF-κB Activation in Mice with COPD
NF-κB inactivation alleviates bronchial inflammation in COPD mice [20], and we therefore investigated the inhibitory effects of ARO on NF-κB activation in this study. As shown in Fig. 6A-6C, NF-κB p65 and IκBα were activated in the lungs of COPD mice; this activation was decreased by ARO administration.
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Figure 6. ARO inhibits NF-κB activation in experimental COPD mice. (A) The activation of NF-κB p65 and IκBα was analyzed using western blotting. Quantitative analysis of (B) p- NF-κB p65 and (C) p-IκBα was performed using ImageJ software. Data are expressed as the mean ± SD (#
p < 0.05 for comparison with normal control; *p < 0.05 for comparison with COPD group). NC: normal control mice; COPD: cigarette smoke exposed/LPS-administered mice; ROF: 5 mg/kg ROF-treated COPD mice; ARO 5: 5 mg/kg ARO-treated COPD mice; and ARO 10: 10 mg/kg ARO-treated COPD mice.
ARO Diminished NLRP3 Activation in Mice with COPD
Next, we investigated the effects of ARO on NLRP3 inflammasome activation. As shown in Fig. 7A and 7B, the expression of NLRP3 in the lungs of the COPD mice was increased; however, this increase was suppressed by the oral administration of ARO. Furthermore, the increase in ASC and Caspase-1 expression in the COPD group was suppressed by ARO treatment (Fig. 7A, 7C, and 7D).
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Figure 7. ARO inhibits NLRP3 activation in experimental COPD mice. (A) The expression of NLRP3, ASC, and caspase-1 was analyzed using western blotting. Quantitative analysis of (B) NLRP3, (C) ASC, and (D) caspase-1 was performed using ImageJ software. Data are expressed as the mean ± SD (#
p < 0.05 for comparison with normal control; *p < 0.05 for comparison with COPD group). NC: normal control mice; COPD: cigarette smoke exposed/LPS-administered mice; ROF: 5 mg/kg ROF-treated COPD mice; ARO 5: 5 mg/kg ARO-treated COPD mice; and ARO 10: 10 mg/kg ARO-treated COPD mice.
Discussion
Neutrophil- and macrophage-derived molecules, such as toxic molecules, cytokines, and chemokines, promote airway inflammation and lung damage in COPD. Thus, controlling the inflow of neutrophils/macrophages and the formation of these cell-mediated molecules are important for ameliorating bronchial inflammation and lung damage. In the present study, ARO excellently inhibited the influx of neutrophils/macrophages and the formation of ROS/MPO/NE/IL-6/IL-1β/MCP-1 in COPD mice. This ability was comparable to that of the positive control ROF. These observations indicated that ARO may exert anti-inflammatory effects on bronchial inflammation in COPD.
As unnecessary mucus generation impedes airflow and CREB activation is closely associated with mucus formation [33, 34], controlling CREB activation may improve airway flow restriction. ARO inhibited both mucus formation and CREB activation in experimental mice with COPD, indicating that ARO exerts a modulatory effect on mucus formation.
Previously, p38 was shown to be activated in the alveolar spaces of COPD patients [35]. Earlier clinical research demonstrated that NF-κB expression was upregulated in the bronchial epithelium of COPD patients [36]. Meanwhile, a recent clinical study confirmed a significant increase in NLRP3/caspase-1/IL-1β expression in COPD patients [37]. A preclinical study also showed that suppression of JNK/p38/ERK MAPK ameliorates inflammatory responses in a murine model of COPD [22]. Previous and current observations have indicated the usefulness of NF-κB inactivation in ameliorating airway inflammation in mice with COPD [11, 38]. Furthermore, inhibition of the NLRP3 inflammasome disrupts the expression of IL-1β, a therapeutic target for COPD treatment [21]. These observations have demonstrated that targeting MAPK/NF-κB/NLRP3 inflammasome pathways may be an effective therapeutic approach against COPD. In the present study, the inhibitory effects of ARO on MAPK (JNK and p38)/NF-κB/NLRP3 activation were notable, and its ability was comparable to that of ROF. These results highlight the potential of ARO as an adjuvant for COPD.
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Figure 8. Ameliorative effects of ARO on bronchial inflammation in experimental COPD mice. Oral administration of ARO reduces neutrophil/macrophage inflow and ROS/MPO/elastase/IL-6/IL-1β/MCP-1 formation in COPD mice. These effects of ARO are accompanied by its modulatory effect on MAPK/NF-κB/NLRP3 activation.
Natural products and their bioactive compounds exhibit various biological activities, including anti-inflammatory and antioxidant effects [17, 39]. Accumulating studies have proven that bioactive compounds ameliorate lung inflammation in COPD mice by inducing MAPK/NF-κB/NLRP3 inactivation. Recently, ARO was shown to exert anti-asthmatic effects by suppressing NF-κB activation [30]. In addition, Ma
Collectively, our results demonstrate that ARO ameliorated bronchial inflammation by suppressing immune cell inflow, inflammatory molecule formation, and mucus formation. These effects were accompanied with suppressive effect on MAPK/NF-κB/NLRP3 activation. Overall, ARO showed excellent efficacy at a dose of 10 mg/kg, highlighting its potential for the development of anti-COPD adjuvants.
Acknowledgments
This study was supported by grants from the KRIBB Research Initiative Program (Grant No. KGM5522423), the Bio & Medical Technology Development Program of the National Research Foundation (NRF), and the Korean Government (MSIT) (Grant No. NRF–2020R1A2C2101228) of the Republic of Korea.
Conflict of Interests
The authors have no financial conflicts of interest to declare.
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Table 1 . List of antibodies..
NO Primary antibody Company Molecular weight Host Secondary antibody 1 NE (bs-6982R) Bioss Antibodies 26 Rabbit Goat anti rabbit-HRP 2 p-CREB (9198s) Cell Signaling 43 Rabbit Goat anti rabbit -HRP 3 CREB (9197s) Cell Signaling 43 Rabbit Goat anti rabbit -HRP 4 p-JNK (4668S) Cell Signaling 46, 54 Rabbit Goat anti rabbit -HRP 5 JNK (9252S) Cell Signaling 46, 54 Rabbit Goat anti rabbit -HRP 6 p-p38 (sc7973) Santa Cruz 38 Mouse Goat anti mouse-HRP 7 p38 (sc-7972) Santa Cruz 38 Mouse Goat anti mouse-HRP 8 p-ERK (9101s) Cell Signaling 42, 44 Rabbit Goat anti rabbit-HRP 9 ERK (9102s) Cell Signaling 42, 44 Rabbit Goat anti rabbit-HRP 10 p-p65 (3033S) Cell Signaling 65 Rabbit Goat anti rabbit-HRP 11 p65 (sc-8008) Santa Cruz 65 Mouse Goat anti mouse-HRP 12 p-IκBα (2859S) Cell Signaling 40 Rabbit Goat anti rabbit-HRP 13 NLRP3 (15101s) Cell Signaling 110 Rabbit Goat anti rabbit-HRP 14 ASC (sc-514414) Santa Cruz 24 Mouse Goat anti Mouse-HRP 15 Caspase-1 (sc56036) Santa Cruz 45 Mouse Goat anti Mouse-HRP 16 β-action (sc-47778) Santa Cruz 43 Mouse Goat anti mouse-HRP
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