
==== Front
Biomol Ther (Seoul)
Biomol Ther (Seoul)
Biomolecules & Therapeutics
1976-9148
2005-4483
The Korean Society of Applied Pharmacology

39091099
10.4062/biomolther.2024.022
bt-32-5-546
Original Article
Aromadendrin Inhibits Lipopolysaccharide-Induced Inflammation in BEAS-2B Cells and Lungs of Mice
Lee Juhyun 12†
Park Ji-Won 3†
Choi Jinseon 14
Yun Seok Han 14
Rhee Bong Hyo 5
Jeong Hyeon Jeong 14
Kim Hyueyun 1
Lee Kihoon 6
Ahn Kyung-Seop 1
Jeong Hye-Gwang 2*
Lee Jae-Won 17*
1 Natural Medicine Research Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB), Cheongju 28116, Republic of Korea
2 College of Pharmacy, Chungnam National University, Daejeon 34134, Republic of Korea
3 Practical Research Division, Honam National Institute of Biological Resources (HNIBR), Mokpo 58762, Republic of Korea
4 College of Pharmacy, Chungbuk National University, Cheongju 28160, Republic of Korea
5 Department of Biology Education, Korea National University of Education, Cheongju 28173, Republic of Korea
6 Laboratory Animal Resource Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju 28116, Republic of Korea
7 Department of Biotechnology, University of Science and Technology (UST), Daejeon 34113, Republic of Korea
* Corresponding Authors E-mail: suc369@kribb.re.kr (Lee JW), hgjeong@cnu.ac.kr (Jeong HG), Tel: +82-43-240-6135 (Lee JW), +82-42-821-5936 (Jeong HG), Fax: +82-43-240-6129 (Lee JW), +82-42-823-6566 (Jeong HG)
† The first two authors contributed equally to this work.

1 9 2024
2 8 2024
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24 1 2024
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Copyright © 2024, The Korean Society of Applied Pharmacology
2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Aromadendrin is a phenolic compound with various biological effects such as anti-inflammatory properties. However, its protective effects against acute lung injury (ALI) remain unclear. Therefore, this study aimed to explore the ameliorative effects of aromadendrin in an experimental model of lipopolysaccharide (LPS)-induced ALI. In vitro analysis revealed a notable increase in the levels of cytokine/chemokine formation, nuclear factor kappa B (NF-κB) activation, and myeloid differentiation primary response 88 (MyD88)/toll-like receptor (TLR4) expression in LPS-stimulated BEAS-2B lung epithelial cell lines that was ameliorated by aromadendrin pretreatment. In LPS-induced ALI mice, the remarkable upregulation of immune cells and IL-1β/IL-6/TNF-α levels in the bronchoalveolar lavage fluid and inducible nitric oxide synthase/cyclooxygenase-2/CD68 expression in lung was decreased by the oral administration of aromadendrin. Histological analysis revealed the presence of cells in the lungs of ALI mice, which was alleviated by aromadendrin. In addition, aromadendrin ameliorated lung edema. This in vivo effect of aromadendrin was accompanied by its inhibitory effect on LPS-induced NF-κB activation, MyD88/TLR4 expression, and signal transducer and activator of transcription 3 activation. Furthermore, aromadendrin increased the expression of heme oxygenase-1/ NAD(P)H quinone dehydrogenase 1 in the lungs of ALI mice. In summary, the in vitro and in vivo studies demonstrated that aromadendrin ameliorated endotoxin-induced pulmonary inflammation by suppressing cytokine formation and NF-κB activation, suggesting that aromadendrin could be a useful adjuvant in the treatment of ALI.

Acute lung injury
Aromadendrin
LPS
Cytokines
NF-κB
HO-1
==== Body
pmcINTRODUCTION

Acute lung injury (ALI) is an uncontrolled inflammatory disease caused by various factors, including respiratory infection (Mokrá, 2020). The coronavirus disease 2019 (COVID-19) pandemic increased the severity of ALI worldwide (Gibson et al., 2020). Pulmonary epithelial cell-derived cytokines and chemokines contribute to the influx of immune cells (ICs) in ALI (Manicone, 2009; Kim et al., 2022a). The influx of ICs, such as macrophages, has been confirmed during ALI development (Huang et al., 2018; Laskin et al., 2019). Increases in interleukin (IL)-1β/IL-6/tumor necrosis factor (TNF)-α levels have been demonstrated in ALI studies; these elevated levels promote pulmonary inflammation (Al-Harbi et al., 2016; Kim et al., 2022a). Increased inducible nitric oxide synthase/cyclooxygenase-2 (iNOS/COX-2) expression promotes lung damage in ALI (Numata et al., 1998; Fukunaga et al., 2005; Mehta, 2005; Al-Harbi et al., 2016), with macrophages being the main source of these molecules (Lee et al., 2021). Thus, modulating IC influx and IC-released molecules may be an important therapeutic approach for ALI.

Nuclear factor kappa B (NF-κB) activation promotes the formation of IC-released molecules (IL-1β/IL-6/TNF-α/iNOS/COX-2) (Liu et al., 2017), and NF-κB activation-derived IL-6 leads to STAT3 activation, which promotes an inflammatory response (Kim et al., 2020). Thus, modulating NF-κB activation is emphasized in ALI therapy. Both, in vitro and in vivo studies have proved the usefulness of modulating NF-κB activation in ALI (Kang and Hyun, 2020; Park et al., 2020; Luo et al., 2022; Yang et al., 2022).

The upregulation of antioxidant protein, heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase 1 (NQO1) ameliorates inflammatory response in inflammatory lung diseases, such as ALI, and Nrf2 activation causes upregulation of HO-1/NQO1 expression (Kang et al., 2022). A recent study showed that Nrf2/HO-1 activation ameliorates NF-κB-dependent inflammatory response (Rahman et al., 2021).

Previous studies on ALI have demonstrated the anti-inflammatory effects of phenolic compounds (PCs) (Peng et al., 2015; Song et al., 2021; Hu et al., 2022). Aromadendrin (ARO; also known as dihydrokaempferol) is a PC found in Olea europaea (Venditti et al., 2013), Smilax glabra (Lu et al., 2015), Bauhinia championii (Zhang et al., 2018), and Euterpe oleracea (de Oliveira et al., 2021). Aromadendrin has recently been reported to exert inhibitory effects on IL-6 secretion in TNFα-stimulated HaCaT cells (Codo Toafode et al., 2022). Aromadendrin is known to exert anti-inflammatory effects in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages by suppressing iNOS/COX-2 expression and NF-κB activation (Lee et al., 2013). Considering this, it was hypothesized that aromadendrin may exert protective effects in ALI as well. Thus, the study aimed to examine whether aromadendrin ameliorates LPS-induced pulmonary inflammation using in vitro and in vivo models.

MATERIALS AND METHODS

Cell culture

BEAS-2B cells were obtained from American Type Culture Collection (Manassas, VA, USA) and grown in Dulbecco’s Modified Eagle Medium containing 10% fetal bovine serum (HyClone; GE Healthcare Life Sciences, Chicago, IL, USA) and a 1% antibiotic–antimycotic solution (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C in 5% CO2 condition. The cells were then incubated with aromadendrin (ChemFaces Biochemical Co, Ltd, Wuhan, China) for 1 h. Subsequently, the cells were incubated for 24 h with 1 μg/mL LPS (Sigma-Aldrich, St. Louis, MO, USA, #8274) or without LPS. Finally, cell viability was determined using a cell viability kit (CytoX, LPS solution, Inc., Daejeon, Korea).

To determine the levels of cytokines/chemokine generated in BEAS-2B cells, the cells (2.5×105 cells/mL) were incubated with 10, 25, 50, 100, and 200 μM aromadendrin for 1 h. The cells were then maintained for 24 h with LPS. Finally, the levels of IL-1β, IL-6, TNF-α, and MCP-1 were determined using the respective enzyme-linked immunosorbent assay (ELISA) kits (R&D systems, Inc., Minneapolis, MN, USA), based on the manufacturer’s instructions.

Establishment of ALI mice

Laboratory animals (6-week-old C57BL/6 male mice; n=30) were obtained from the Koatech Laboratory Animal Center (Peongtack, Korea), and the experimental protocol was approved by the Institutional Animal Care and Use Committee of the Korea Research Institute of Bioscience and Biotechnology (KRIBB-AEC-21111). To establish the ALI animal model, LPS (0.5 mg/kg in 35 μL phosphate-buffered saline (PBS)) was administered intranasally to the mice on day 1 (Min et al., 2021). To evaluate the protective effect of aromadendrin (ARO)/dexamethasone(DEX) on LPS-induced pulmonary inflammation, ARO/DEX was orally administered on days 0–1.

The experimental groups (n=6, each group) used in the in vivo study were as follows: normal control (NC), LPS (intranasal administration of lipopolysaccharide only), DEX (LPS+1 mg/kg dexamethasone, administered orally (p.o.)), ARO 15 (LPS+15 mg/kg aromadendrin, p.o.), and ARD 30 (LPS+30 mg/kg aromadendrin, p.o.).

IC and cytokine analyses

To quantify ICs and cytokines, the mice were anesthetized with Zoletil 50 and xylazine on day 3, as previously described (Min et al., 2021). Bronchoalveolar lavage fluid (BALF) was acquired based on previous methods (Park et al., 2020). BALF cells were stained with the Diff-Quik staining kit (Sysmex Corporation, Kobe, Japan) to confirm the morphology of ICs and counted using light microscopy (magnification, ×400). BALF supernatants were analyzed to detect cytokine formation using the respective ELISA kits (R&D systems, Inc.), according to the manufacturer’s instructions.

Western blot analysis

The expression of phosphorylated (p)-NF-κB p65/p-IκBα/MyD88/TLR4 in vitro and that of iNOS/COX-2/CD68/p-NF-κB p65/p-IκBα/MyD88/TLR4/p-STAT3/HO-1/NQO1 in vivo were determined using western blotting. Cell cultures and lung tissue lysates were prepared in a lysis buffer containing protease/phosphatase inhibitors (Roche Diagnostics, Basel, Switzerland), and the protein concentration in each lysate was analyzed using the bicinchoninic acid assay (Thermo Fisher Scientific, Inc.). Proteins were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. Subsequently, the membranes were maintained in 5% skim milk at room temperature for 1 h and incubated with primary antibodies (Table 1) at 4°C for 24 h. Subsequently, the membranes were maintained with corresponding secondary antibodies and finally exposed to the ECL detection system (Thermo Fisher Scientific, Inc.) to visualize proteins.

Histological analysis

To perform histological analysis, lung tissues excised from mice were washed with ice-cold PBS, embedded in paraffin wax, and cut into 4-μm sections using a microtome. Subsequently, the paraffin-embedded lung sections were stained with hematoxylin and eosin (H&E) solution.

Statistical analyses

Values are presented as mean ± standard deviation. Analysis of variance with Tukey’s multiple comparison test were performed for multiple comparisons among the groups using SPSS 20.0 (IBM Corp., Armonk, NY, USA). p<0.05 was considered statistically significant.

RESULTS

Aromadendrin inhibits the LPS-induced inflammatory response in BEAS-2B lung epithelial cells

We determined the optimum aromadendrin concentration to evaluate its anti-inflammatory effects using a CytoX assay. Remarkable cytotoxicity was not observed up to 200 μM aromadendrin in BEAS-2B or LPS-stimulated BEAS-2B cells (Fig. 1A). Thus, 10, 25, 50, 100, and 200 μM aromadendrin concentrations were used to examine the inhibitory ability of aromadendrin on cytokine/chemokine secretion in vitro. ELISA results revealed a significant increase (p<0.01) in pro-inflammatory cytokine (IL-1β, IL-6, and TNF-α) levels in the cell culture supernatants (CCS) of LPS-stimulated BEAS-2B cells compared to those in the CCS of the NC group (Fig. 1B-1D). However, these levels were significantly reduced (p<0.05) in the LPS group by 50, 100, or 200 μM aromadendrin pretreatment. The results also indicated a significant increase (p<0.01) in chemokine (MCP-1) levels in the CCS of LPS-stimulated BEAS-2B cells (Fig. 1E). This tendency was ameliorated in the LPS group by 50, 100, or 200 μM aromadendrin treatment (p<0.05). The inhibition rates of IL-1β, IL-6, TNF-α, and MCP-1 were 45.8, 60.2, 54.1, and 43.9% (200 μM aromadendrin).

Aromadendrin suppresses the LPS-induced NF-κB activation in BEAS-2B cells

We examined the ability of aromadendrin to mitigate LPS-induced NF-κB activation in BEAS-2B cells. The levels of NF-κB p65 activation in whole cell lysates (WCL) were analyzed using western blotting. NF-κB p65 activation was significantly upregulated (p<0.01) in the WCL of LPS-stimulated BEAS-2B cells compared to those in the WCL of the NC group (Fig. 2A, 2B), whereas 100 or 200 μM aromadendrin pretreatment suppressed NF-κB p65 activation in the LPS group (p<0.05). Similar to the findings for NF-κB p65, 50, 100, or 200 μM aromadendrin induced IκBα inactivation in the LPS group (NC vs. LPS group, p<0.01; LPS group vs. LPS+ARO group, p<0.05) (Fig. 2A, 2C). In addition, the upregulation of MyD88/TLR4 expression in WCL of LPS group (NC vs. LPS group, p<0.01) was reduced in the LPS group by 50, 100, or 200 μM aromadendrin pretreatment (LPS group vs. LPS+ARO group, p<0.05) (Fig. 2D, 2E). The in vitro study findings (Fig. 1, 2) led us to investigate whether aromadendrin had an ameliorative effect on LPS-induced ALI in mice.

Aromadendrin inhibits the upregulation of ICs and cytokines in LPS-induced ALI mice

ALI mice were used to evaluate the ameliorative effects of aromadendrin on LPS-induced ALI (Fig. 3). The number of neutrophils/macrophages was significantly increased (p<0.01) in the BALF of LPS-induced ALI mice (Fig. 4A), and this increase was mitigated in the ALI group by dexamethasone and aromadendrin administration (LPS group vs. LPS+1 mg/kg DEX group, p<0.05; LPS group vs. LPS+30 mg/kg ARO group, p<0.05). The neutrophil inhibition rates were 69.8% (1 mg/kg DEX), 23.4% (15 mg/kg ARO), and 59.7% (30 mg/kg ARO). The macrophage inhibition rates were 73.3% (1 mg/kg DEX), 45.2% (15 mg/kg ARO), and 64.7% (30 mg/kg ARO).

The ELISA findings revealed significant generation (p<0.01) of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) in the BALF of mice in the ALI group compared to those in the BALF of mice in the NC group (Fig. 4B-4D). However, these levels were decreased in the BALF of ALI mice treated with dexamethasone or aromadendrin (LPS group vs. LPS+1 mg/kg DEX group, p<0.05; LPS group vs. LPS+30 mg/kg ARO group, p<0.05). The IL-1β inhibition rates were 56.5% (1 mg/kg DEX), 3.7% (15 mg/kg ARO), and 46.2% (30 mg/kg ARO). The IL-6 inhibition rates were 60.7% (1 mg/kg DEX), -3.9% (15 mg/kg ARO), and 54.1% (30 mg/kg ARO). The TNF-α inhibition rates were 62.2% (1 mg/kg DEX), 15.4% (15 mg/kg ARO), and 55.0% (30 mg/kg ARO).

Aromadendrin decreases the expression of iNOS and COX-2 in LPS-induced ALI mice

As shown in Fig. 5A-5C, upregulated iNOS expression was confirmed in the lung tissue lysates (LTL) of ALI mice (NC vs. LPS group, p<0.01). However, this upregulation was markedly inhibited in the LTL of the ALI mice by dexamethasone and aromadendrin (LPS group vs. LPS+1 mg/kg DEX group, p<0.05; LPS group vs. LPS+30 mg/kg ARO group, p<0.05). Similarly, a notable increase in COX-2 expression in the LTL of the ALI mice was attenuated by aromadendrin and dexamethasone (NC vs. LPS group, p<0.01; LPS group vs. LPS+1 mg/kg DEX group, p<0.05; LPS group vs. LPS+30 mg/kg ARO group, p<0.05).

Aromadendrin mitigates the influx of cells and lung edema in LPS-induced ALI mice

H&E staining was used to detect cell influx in the lungs of mice. A notable influx of cells was detected around the airway epithelium in the lungs of ALI mice, whereas this characteristic was abrogated in the ALI group treated with dexamethasone and aromadendrin (Fig. 6A). A significant increase in CD68 expression was observed in the LTL of the ALI group compared to that in the LTL of the NC group (p<0.01) (Fig. 6B), and this upregulation was attenuated in the ALI group by dexamethasone and aromadendrin (LPS group vs. LPS+1 mg/kg DEX group, p<0.05; LPS group vs. LPS+30 mg/kg ARO group, p<0.05). Furthermore, lung damage in the ALI group was ameliorated by aromadendrin (Fig. 6C).

Aromadendrin suppresses the activation of NF-κB in LPS-induced ALI mice

In this study, aromadendrin suppressed LPS-induced cytokine production both in vitro (Fig. 1) and in vivo (Fig. 4). Furthermore, aromadendrin inhibited LPS-induced NF-κB activation in vitro (Fig. 2). Based on these effects, the impact of aromadendrin on NF-κB inactivation was evaluated in vivo; the marked activation of NF-κB p65/IκBα was confirmed in the LTL of the ALI group compared to that in the LTL of the NC group (NC vs. LPS group, p<0.01) (Fig. 7A-7C). Interestingly, this activation was significantly attenuated in the ALI group by dexamethasone and aromadendrin (LPS group vs. LPS+1 mg/kg DEX group, p<0.05; LPS group vs. LPS+30 mg/kg ARO group, p<0.05). Next, we examined the expression levels of MyD88 and TLR4 in the LTL of mice. The results showed the upregulation of MyD88/TLR4 expressions in LTL of LPS group (NC vs. LPS group, p<0.01). This upregulation was inhibited in the LPS group by aromadendrin administration (LPS group vs. LPS+1 mg/kg DEX group, p<0.05; LPS group vs. LPS+15 mg/kg ARO group, p<0.05; LPS group vs. LPS+30 mg/kg ARO group, p<0.05) (Fig. 7A, 7D, 7E).

Aromadendrin attenuates the activation of STAT3 in LPS-induced ALI mice

Similar to the results of NF-κB p65/IκBα activation, STAT3 activation was confirmed in the LTL of ALI mice (NC vs. LPS group, p<0.01); this was suppressed in the ALI mice by dexamethasone and aromadendrin (LPS group vs. LPS+1 mg/kg DEX group, p<0.05; LPS group vs. LPS+30 mg/kg ARO group, p<0.05) (Fig. 8).

Aromadendrin upregulates the expression of HO-1/NQO1 in LPS-induced ALI mice

A noticeable increase in HO-1/NQO1 expression was demonstrated in the LTL of 30 mg/kg aromadendrin-treated LPS group compared to that in the LTL of LPS group or dexamethasone-treated LPS group (LPS group vs. LPS+30 mg/kg ARO group, p<0.05) (Fig. 9A-9C).

DISCUSSION

In this study, we showed that aromadendrin exerts anti-inflammatory effects on lung epithelial cell lines by suppressing LPS-induced cytokine formation and NF-κB activation (Fig. 10). In addition, aromadendrin ameliorated the LPS-induced pulmonary inflammation in mice by impeding IL-1β, IL-6, and TNF-α secretion and NF-κB activation.

Lung epithelial cells, such as BEAS-2B cells, produce pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α in LPS-induced ALI (Meng et al., 2018). Lung epithelial cell-derived MCP-1 affects IC recruitment during pulmonary inflammation (Kwon et al., 2020; Kim et al., 2022a; Lee et al., 2022). The increase in macrophage recruitment and cell-derived molecules, including IL-1β/IL-6/TNF-α/iNOS/COX-2, promotes ALI development (Lee et al., 2021). Thus, the suppression of inflammatory molecules may ameliorate endotoxin-induced ALI. In the present study, in vitro analysis revealed that IL-1β/IL-6/TNF-α/MCP-1 secretions from LPS-stimulated BEAS-2B cells were effectively reduced by aromadendrin pretreatment. In vivo findings also indicated that the upregulation of IL-1β/IL-6/TNF-α formation and macrophage/neutrophil numbers in the ALI group were mitigated by aromadendrin administration. The inhibitory effect of aromadendrin on inflammatory cell influx was confirmed using histological analyses. These results indicated that aromadendrin exerts anti-inflammatory effects against endotoxin-induced ALI by suppressing cytokine secretion and IC influx.

iNOS contributes to peroxynitrite formation, which causes pulmonary injury (Beckman et al., 2000; Kim et al., 2022a; Park et al., 2022). COX-2 upregulation leads to acute pain (Giuliano and Warner, 2002; Lee et al., 2005). Macrophages overexpress iNOS/COX-2 in LPS-induced ALI (Lee et al., 2021; Park et al., 2022). Aromadendrin has been shown to decrease LPS-induced iNOS/COX-2 expression in RAW264.7 macrophages (Lee et al., 2013). Thus, aromadendrin was expected to inhibit iNOS/COX-2 upregulation in the ALI animal model. Inevitably, ARO suppressed the expression of these molecules in vivo. These results demonstrate the usefulness of ARO for iNOS/COX-2 inhibition in ALI.

Experimental results in ALI studies indicate that NF-κB inactivation ameliorates LPS-induced pulmonary inflammation by reducing the generation of inflammatory molecules and IC influx (Kim et al., 2022b). NF-κB activation-derived STAT3 activation promotes inflammatory response leading to the formation of inflammatory molecules (Kim et al., 2020). Therefore, the NF-κB signaling pathway is a therapeutic target in ALI. Interestingly, aromadendrin significantly inhibited NF-κB activation both in vitro and in vivo. Furthermore, aromadendrin exerted a suppressive effect on LPS-induced STAT3 activation in ALI mice. Thus, the anti-inflammatory effect of aromadendrin may be related to its inhibitory effects on LPS-induced NF-κB activation.

Based on importance of HO-1/NQO1 induction for amelioration of LPS-induced ALI (Kang et al., 2022), the effect of aromadendrin on the induction of these molecules was examined. Surprisingly, a remarkable upregulation of HO-1/NQO1 expression was induced by aromadendrin, indicating that aromadendrin has strong antioxidant activity.

PCs exert therapeutic effects in ALI by modulating cytokine formation and NF-κB activation (Lee et al., 2018; Kim et al., 2022a). In this study, the inhibitory effect of aromadendrin on LPS-induced cytokine/chemokine production was confirmed in vitro. In addition, our in vivo study showed remarkable inhibitory abilities of aromadendrin on IL-1β/IL-6/TNF-α formation, iNOS/COX-2 expression, and macrophage/neutrophil influx. Interestingly, these effects of aromadendrin were confirmed by its ability to suppress TLR4/MyD88/NF-κB activation, both in vitro and in vivo. Furthermore, aromadendrin inhibited STAT3 activation and increased HO-1/NQO1 expression in vivo. Generally, the in vivo anti-inflammatory effect of aromadendrin was comparable to that of DEX, a positive control. Collectively, the protective effect of aromadendrin against endotoxin-induced pulmonary inflammation was exceptional. Thus, aromadendrin has potential therapeutic implications in the prevention or treatment of ALI.

Despite the ALI improvement effect, there are some limitations to the use of aromadendrin as an adjuvant in the treatment of ALI. Previous (Lee et al., 2013) and present observations proved that aromadendrin had an anti-inflammatory effect in RAW264.7 and BEAS-2B cells against LPS stimulation. Although RAW264.7 and BEASE-2B cells were continuously adapted in the in vitro study of ALI (Wu et al., 2016; Xu et al., 2022) for evaluating the anti-inflammatory effects and its underlying mechanisms of compounds, the use of primary macrophages or bronchial cells from ALI patients will help confirm the anti-ALI effect of compounds including ARO. In addition, for application as an ALI adjuvant, pharmacokinetics or bioavailability testing must be considered.

ACKNOWLEDGMENTS

This research was supported by grants from the KRIBB Research Initiative Program (grant No. KGM5522423), the Honam National Institute of Biological Resources (HNIBR), funded by the Ministry of Environment (MOE) of the Republic of Korea (grant no. HNIBR202302113), and the Bio & Medical Technology Development Program of the National Research Foundation (NRF) and the Korean government (MSIT) (Grant. No. NRF–2020R1A2C2101228).

Fig. 1 Anti-inflammatory ability of aromadendrin (ARO) on lipopolysaccharide (LPS)-induced inflammatory response in BEAS-2B cells. (A) Cell viability results. BEAS-2B cells were incubated with ARO (10–200 μM) for 1 h, incubated in the absence or presence of LPS (1 μg/mL) for 24 h, and subjected to CytoX analysis. The (B) IL-1β, (C) IL-6, (D) TNF-α, and (E) MCP-1 levels in cell culture supernatants were detected using enzyme-linked immunosorbent assay (ELISA) kits. Data are expressed as the mean ± standard deviation (SD; #p<0.01 for comparison with the control; *p<0.05 and **p<0.01 for comparison with the LPS group). ARO stands for aromadendrin.

Fig. 2 Inhibitory ability of ARO on LPS-induced NF-κB activation in BEAS-2B cells. (A) The expression levels of p-NF-κB, p-IκBα, MyD88 and TLR4 in whole cell lysates (WCL) detected using western blotting. BEAS-2B cells were maintained ARO (25–200 μM) for 1 h and incubated in the presence of LPS (1 μg/mL) for 1 h. Quantitative analysis of (B) p-p65, (C) p-IқB, (D) MyD88, and (E) TLR4 was performed using the ImageJ software (version 1.50e; National Institute of Health, MD, USA). Data are expressed as the mean ± SD (#p<0.01 for comparison with the control; *p<0.05 for comparison with the LPS group). ARO stands for aromadendrin.

Fig. 3 Acute lung injury (ALI) mice establishment and ARO administration. ALI in mice was induced by the intranasal administration of 0.5 mg/kg LPS on day 1. The oral administration of ARO and dexamethasone (DEX) was performed on days 0 and 1. Finally, ALI mice were sacrificed on day 3 and the separation of bronchoalveolar lavage fluid (BALF)/lung tissues was performed for analysis. ARO stands for aromadendrin.

Fig. 4 Inhibitory ability of ARO on the upregulation of immune cells and molecules in ALI mice. (A) Immune cell counts in BALF (magnification, ×400). (B) IL-1β, (C) IL-6, and (D) TNF-α levels in BALF analyzed using ELISA (n=6 mice per group). Data are expressed as the mean ± SD (#p<0.01 for comparison with the normal control; *p<0.05 for comparison with the LPS group). NC: normal control mice; LPS: lipopolysaccharide-treated mice; DEX: 1 mg/kg dexamethasone-administered LPS mice; ARO 15: 15 mg/kg aromadendrin-administered LPS mice and ARO 30: 30 mg/kg aromadendrin-administered LPS mice. ARO stands for aromadendrin.

Fig. 5 Inhibitory ability of ARO on iNOS and COX-2 expression in ALI mice. (A) The expressions of iNOS and COX-2 in the lung tissue lysates (LTL) of mice detected using western blotting (n=6 mice per group). (B, C) Quantitative analysis performed using the ImageJ software. Data are expressed as the mean ± SD (#p<0.01 for comparison with the normal control; *p<0.05 for comparison with the LPS group). ARO stands for aromadendrin.

Fig. 6 Inhibitory ability of ARO on cell influx and lung edema in ALI mice. (A) Cell existence in the lungs of mice detected using hematoxylin and eosin staining and marked with an arrow (top, magnification ×100, scale bar 100 μM; bottom, magnification ×200, scale bar, 50 μM). (B) CD68 expression in the LTL of mice detected using western blotting (n=6 mice per group). (C) Image showing lung edema. Data are expressed as the mean ± SD (#p<0.01 for comparison with the normal control; *p<0.05 for comparison with the LPS group). ARO stands for aromadendrin.

Fig. 7 Inhibitory ability of ARO on NF-κB activation in ALI mice. (A) The expressions of p-NF-κB, p-IκBα, MyD88 and TLR4 in the LTL of mice detected using western blotting (n=6 mice per group). (B-E) Quantitative analysis performed using the ImageJ software. Data are expressed as the mean ± SD (#p<0.01 for comparison with the normal control; *p<0.05 for comparison with the LPS group). ARO stands for aromadendrin.

Fig. 8 Inhibitory ability of ARO on STAT3 activation in ALI mice. The expressions of p-STAT3 in the LTL of mice detected using western blotting (n=6 mice per group). Quantitative analysis performed using the ImageJ software. Data are expressed as the mean ± SD (#p<0.01 for comparison with the normal control; *p<0.05 for comparison with the LPS group). ARO stands for aromadendrin.

Fig. 9 Effect of ARO on HO-1/NQO1 induction in ALI mice. (A) The expressions of HO-1 and NQO1 in the LTL of mice detected using western blotting (n=6 mice per group). (B, C) Quantitative analysis performed using the ImageJ software. Data are expressed as the mean ± SD (†p<0.05 for comparison with the LPS group). ARO stands for aromadendrin.

Fig. 10 Protective effects of ARO on pulmonary inflammation in an experimental model of ALI. ARO Pretreatment negatively regulates the inflammatory response in LPS-stimulated epithelial cells. Furthermore, ARO administration inhibits macrophage influx, cytokine formation, and iNOS/COX-2 expression and upregulates HO-1/NQO1 expression in LPS-induced ALI mice. These effects of ARO in vitro and in vivo are accompanied by an inhibitory ability on NF-κB activation. ARO stands for aromadendrin.

Table 1 List of antibodies

Primary antibody	Company	Molecular weight	Dilution	Host	Secondary antibody	
β-action (sc-47778)	Santa Cruz (Dallas, TX, USA)	43	1:2000	Mouse	Goat anti mouse-HRP	
CD68 (sc-20060)	Santa Cruz	75-110	1:1000	Mouse	Goat anti mouse-HRP	
COX-2 (sc-376861)	Santa Cruz	70-72	1:1000	Mouse	Goat anti mouse-HRP	
HO-1 (PA5-27338)	Invitrogen	32	1:1000	Rabbit	Goat anti rabbit-HRP	
IκB-α (MA5-15132)	Invitrogen	36	1:1000	Mouse	Goat anti mouse-HRP	
iNOS (ab136918)	Abcam (Cambridge, UK)	130	1:1000	Rabbit	Goat anti rabbit-HRP	
MyD88 (sc-74532)	Santa Cruz	33	1:1000	Mouse	Goat anti mouse-HRP	
NQO1 (N5288)	Sigma (St. Louis, MO, USA)	28	1:1000	Rabbit	Goat anti rabbit-HRP	
p65 (sc-8008)	Santa Cruz	65	1:1000	Mouse	Goat anti mouse-HRP	
p-IκBα (2859S)	Cell Signaling (Danvers, MA, USA)	40	1:1000	Rabbit	Goat anti rabbit-HRP	
p-p65 (3033S)	Cell Signaling	65	1:1000	Rabbit	Goat anti rabbit-HRP	
p-STAT3 (9145S)	Cell Signaling	79-86	1:1000	Rabbit	Goat anti rabbit-HRP	
STAT3 (12640S)	Cell Signaling	79-86	1:1000	Rabbit	Goat anti rabbit-HRP	
TLR4 (sc-293072)	Santa Cruz	95-120	1:1000	Mouse	Goat anti mouse-HRP	

CONFLICT OF INTEREST

The authors declare that there is no conflict of interest.
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