Duchesnea indica Extract Attenuates Coal Fly Ash-Induced Inflammation in Murine Alveolar Macrophages through the NF-Kappa B Pathway

Duchesnea indica is known as false strawberry, is found in East Asia, and has numerous biological properties. The aim of this study was to investigate the anti-inflammatory effect of Duchesnea indica extract (DIE) on coal fly ash- (CFA-) induced inflammation in murine alveolar macrophages (MH-S). Following the induction of inflammation in MH-S cells by CFA, nitric oxide (NO) was measured to evaluate the anti-inflammatory property of DIE. Cell viability and inflammatory gene expression were analyzed using polymerase chain reaction (PCR). The inflammatory pathway in MH-S cells was determined via western blotting and immunofluorescence (IF) analysis. Finally, the major components of the DIE were identified and separated through ultra-performance liquid chromatography (UPLC) and gas chromatography-mass spectrometry (GC-MS) analysis. Our results showed that the DIE dose-dependently inhibited the CFA-induced NO production in MH-S cells. Moreover, the DIE could suppress the CFA-induced proinflammatory mediators, such as cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). In addition, the inhibitory effect of the DIE on proinflammatory cytokines, including interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-α (TNF-α), was detected with PCR. Moreover, the effect of the DIE on the nuclear factor-κB (NF-κB) pathway in CFA-activated MH-S cells was measured via western blotting. Furthermore, the inhibition of the phosphorylated NF-κB (p-NF-κB) translocation was analyzed using IF assay. The findings of this study indicated that the DIE potentially inhibited the CFA-induced inflammation by blocking the NF-κB inflammatory signaling pathway in MH-S cells and that the DIE might contain favorable anti-inflammatory compounds which may be effective in attenuating lung inflammation.


Introduction
Coal fly ash (CFA) is a major environmental factor of air pollution, which is produced regularly due to industrial activities and the use of motor vehicles in the urban areas [1]. CFA consists of liquid and solid particulate matters that vary in size and origin, including coal, combustion particles, and asbestos [1,2]. Nanoparticles can retain for a long period of time in the atmosphere and easily move in the air. Chronic exposures of CFA can cause accumulation of sediments in the lungs and lead to serious adverse effects, such as inflammation in the alveolar cells [3,4]. Moreover, nanoparticles are responsible for many respiratory diseases, such as asthma, bronchitis, chronic obstructive pulmonary diseases (COPD), and lung carcinoma [5].
Inflammation is an important biological response to harmful stimuli and the first line of defense in the immune system [6]. Alveolar macrophages (AM) are considered one of the defensive cell populations in the lungs and facilitate regulating lung inflammation and host defense protection against airborne nanoparticles [7]. After CFA invades the airway of the lungs, AM are recruited, resulting in the production of inflammatory mediators and cytokines, which is the initial response of the immune system [8]. CFA-induced excessive proinflammatory cytokines are considered one of the major causes of lung diseases. ey can be regulated by the activation of the transcription factors, such as nuclear factor kappa-B (NF-κB) signaling cascade [9,10]. Duchesnea indica is a perennial plant that belongs to the Rosaceae family and is widely distributed in Asian countries especially in Bangladesh, China, Japan, and Korea [11]. It has been extensively used as a folk medicine in Asia for its unique biological properties including antioxidative, antiinflammatory, antimutagenic, and anticancerous properties [12,13]. Several studies have demonstrated that some phenolic compounds, such as phenolic acid, flavonoids, and ellagic acid, are the main pharmacologically bioactive constituents in Duchesnea indica [11]. It has been shown that the Duchesnea indica extract (DIE) downregulates cervical cancer through apoptosis and cell cycle arrest [14]. Moreover, previous studies have reported that the DIE protected the skin fibroblast of humans against hydrogen peroxideinduced cytotoxicity and inhibited the growth of human cancer cells in vitro [15]. us, traditional medicines have been considered by the patients due to their therapeutic effects and bioactive ingredients [16].
In this study, we aimed to investigate the anti-inflammatory effects of the DIE on CFA-induced inflammation in MH-S cells. Taken together, our results demonstrated that the DIE can prevent the inflammation in CFA-activated MH-S cells, and such activity is mediated by inhibiting the expression of proinflammatory mediators and cytokines.

Preparation of the Duchesnea indica Ethanol Extract
2.3. UPLC-QTOF-MS Analysis of DIE. UPLC analysis of the DIE was performed using ACQUITY UPLC TM (Waters Corp., Milford, MA, USA), equipped with a binary solvent delivery system and coupled to the quadrupole time-of-flight mass spectrometer (Q-TOF Premier TM , Waters Corp., Milford, MA, USA) equipped with an auto-sampler and a UV detector. Briefly, the DIE (2 μL) was injected into the ACQUITY UPLC BEH C18 chromatography column (2.1 × 100 mm × 1.7 μm). e column temperature was fixed at 35°C, and the flow rate was 0.4 mL/min. e chromatographic gradient consisted of mobile phases: (A) water with 1% formic acid and (B) acetonitrile with 1% formic acid. e gradient duration was optimized: 0 min, 10% B; 0-11 min, 10%-90% B; 11-11.5 min, 90%-100% B; 11.5-13.5 min, 100% B; and 13.5-15 min, back to 10% B. e mass spectrometer condition was a negative ion mode with the capillary and cone voltages being 2.3 kV and 50 V, respectively. N 2 was used as a desolvation gas. e source temperature was 100°C, and the desolvation temperature was 350°C. Leucine-enkephalin was used as a reference compound (m/z 554.2615) in the form of a spray.

GC-MS Analysis of DIE.
GC-MS analysis of the DIE was performed using the Agilent 7890A GC (Agilent Technologies, Santa Clara, CA, USA). e GC-MS device was equipped with a 30 m × 0.25 mm (i.d. DB-5MS) chromatography column and the Agilent 5975C mass selective detector to separate and quantify the compounds of DIE. e extract was injected at 250°C. e temperatures for the transfer line and source were 280°C and 230°C, respectively. e column temperature was set at 70°C as an initial temperature for 1 min and raised to 300°C at a rate of 5°C/ min, with duration at a final temperature of 300°C for 30 min. e mass spectrometry was acquired via electron ionization and scan modes. e helium gas was used as a carrier gas with a constant flow rate (1 mL/min).

Cell Culture.
e MH-S cell line, originating from the American Type Culture Collection, was cultured in RPMI supplemented with 10% FBS, 100 IU/mL penicillin, and 100 µg/mL streptomycin sulfate (Welgene, South Korea). e incubation conditions were maintained at 37°C and 5% CO 2 during culture and treatment conditions. 2.6. Nitric Oxide (NO) Assay. NO was measured using the Griess reagent A (0.2% N-ethylenediamine dihydrochloride) and Griess reagent B (2% sulfanilamide in 5% phosphoric acid) reaction methods. Briefly, the MH-S macrophages were seeded in a 24-well plate and incubated with or without CFA (2.5 μg/mL) in the absence or presence of the DIE (12.5, 25, 50, and 100 μg/mL) at indicated concentrations for 18 h. e Griess reagents (100 µL) were added with cell culture supernatants (100 µL) and incubated for 5 min at normal condition. en, absorbance was measured in a microplate reader at 540 nm (Versamax, Microplate Reader, Molecular devices, CA, USA).

RNA Extraction, Reverse Transcription-Polymerase Chain Reaction (RT-PCR), and Real-Time qRT-PCR.
PCR analysis was conducted in accordance with the previously reported method [18]. e murine macrophages (MH-S) were treated with or without the DIE (12.5, 25, 50, and 100 μg/mL) for 30 min at indicated concentrations, followed by the CFA stimulation (2.5 μg/mL) for 18 h. RNA was collected from cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Two micrograms of total RNA was annealed with Oligo-dT (Bioneer, South Korea) for 10 min at 70°C, cooled for 5 min in ice, and then reverse-transcribed using reverse transcriptase premix (Bioneer, South Korea) in 20 µL of reaction mixture for 90 min at 42.5°C on a thermocycler. To inactivate the reverse transcriptase, the reaction was terminated at 95°C for 5 min. RT-PCR was performed using cDNA obtained from RT reaction in a PCR premix (Bioneer, South Korea). Moreover, the PCR products were electrophoresed on agarose gel (1%) stained with ethidium bromide. e band was visualized using Image-Quant LAS 500 (GE Health Care Life Sciences, South Korea). e intensity of the band density was normalized to GAPDH. Real-time PCR was performed using SYBR green. Table 1 shows the RT-PCR and real-time PCR primer sequences.

Western Blot Analysis.
Western blot analysis was conducted in accordance with the previously reported method [10]. MH-S cells were untreated (control group), treated with only CFA (2.5 μg/mL), and treated with the DIE (12.5, 25, 50, and 100 μg/mL) in the presence of CFA (2.5 μg/mL). e proteins were extracted from the cells with the protein extraction solution, Pro-Prep (iNtRON biotechnology, South Korea). en, the proteins were determined using the protein measurement solution, PRO-MEASURE assay kit (iNtRON biotechnology, South Korea). e cell lysates were then subjected to SDS-PAGE (10%) and transferred onto the PVDF membranes (Millipore, Immobilion-P, Billerica MA, USA). Membranes were blocked for 1 h in 5% (w/v) skim milk and 0.1% (v/v) Tween-20 in TBS. en membranes were incubated with different primary antibodies overnight at 4°C. After washing, 1 h of incubation with horse radish peroxidase-(HRP-) labelled secondary antibody (1 : 3000 dilution, Cell Signaling) at room temperature was performed. Proteins were detected using enhanced chemiluminescence (ECL) solution (Supex, Daegu, Korea). Immunoblots were quantified using the ImageJ software.
2.10. Immunofluorescence Staining. Immunofluorescence (IF) assay was done as described [19]. MH-S cells were washed with DPBS and fixed with 4% paraformaldehyde (PFA) for 10 minutes. Moreover, the cells were permeabilized with 0.2% triton X-100 in TBS (TBST) for 10 minutes and washed with TBST for 5 minutes every three times. Using 2% BSA, the samples were blocked for 1 h, and primary antibody rabbit anti p-NF-κB (Cell Signaling Technology, Danvers, MA, USA) was applied to the cells at 4°C overnight. e cells were washed with TBST for 5 minutes every three times. e samples were incubated with secondary antibody (anti-rabbit IgG Fab2, Alexa Fluor 555, Molecular Probes) for 1 hour in the dark, and after washing with TBST three times, the samples were mounted using ProLong Gold Antifade Reagent with DAPI to visualize the nuclei (Cell Signaling Technology, Danvers, MA, USA) and analyzed via confocal microscopy (ZEISS).

Statistical
Analysis. Data were analyzed by one-way ANOVA or unpaired Student's t-test followed by Dunnett's multiple comparison tests using the GraphPad Prism software. Data are presented as mean ± SEM. e statistical significance is denoted as * p < 0.05, * * p < 0.01, and * * * p < 0.001 or ns � not significant.

DIE Protects CFA-Induced Nitric Oxide Production and
Cell Death in MH-S Macrophages. Nitric oxide (NO) is an important mediator in the inflammatory condition, and excessive production of NO contributes to inflammatory Evidence-Based Complementary and Alternative Medicine diseases [23,24]. In this study, the levels of NO in response to CFA in MH-S murine AM were measured using the Griess reaction method. e DIE potently inhibited the NO induction in a dose-dependent manner (Figure 2(a)). MTT assay was performed to determine the cell viability assay, and the results showed that the DIE did not affect the cell viability at the used concentration (Figure 2(b)). ese results demonstrated that the DIE inhibited the NO production dose-dependently, and the used concentrations did not show cytotoxicity.

Preventive Effect of the DIE on CFA-Induced Proinflammatory
Cytokines in MH-S Macrophages. Pretreatment of MH-S with the DIE for 30 min decreased the levels of CFA-induced proinflammatory mediators and cytokines. e mRNA levels of proinflammatory mediators such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and proinflammatory cytokines including interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-α (TNF-α) were analyzed using RT-PCR to investigate the anti-inflammatory properties of the DIE. e results showed that proinflammatory mediators and cytokines were dose-dependently reduced at the mRNA level (Figures 3(a)-3(f )). is finding suggested that the DIE downregulated the CFA-induced inflammatory cytokines and significantly reduced the mRNA levels mainly at concentrations of 50 and 100 μg/ml.

DIE Ameliorates the CFA-Induced mRNA Expression of Proinflammatory Cytokines in MH-S Macrophages.
To confirm the results of the RT-PCR on the proinflammatory mediators and cytokines in MH-S cells, the mRNA expressions of the proinflammatory mediators were investigated and the proinflammatory cytokines were analyzed with real-time PCR. e mRNA expressions of the proinflammatory mediators and cytokines were noticeably dosedependently downregulated with the DIE treatment (Figures 4(a)-4(e)). Taken together, the real-time PCR results suggested that the DIE reduced the inflammation in a concentration-dependent manner.

DIE Inhibits the Activation of NF-κB Signaling in CFA-Treated MH-S Macrophages.
e effects of the DIE on nuclear factor-κB (NF-κB) signaling were measured as a transcription factor has an important role in inflammation. e activation with CFA leads to the inflammatory pathway, wherein NF-κB signaling is a key axis in the inflammatory mechanism [1,9]. Treatment with CFA significantly induced the phosphorylation of NF-κB and inhibitor of kappa-B (IκB), whereas the pretreatment of the DIE significantly inhibited the phosphorylation of NF-κB and IκB in MH-S cells (Figures 5(d) and 5(e)). is result suggested that the pretreatment with the DIE markedly downregulated the phosphorylation of the CFA-induced NF-κB and IκB activation in MH-S cells.

DIE Inhibits the Translocation of NF-κB in CFA-Treated MH-S Macrophages.
To find out whether the anti-inflammatory activities of the DIE are mediated by the NF-κB signal transduction pathway in CFA-activated MH-S cells, the translocation of the activated p-NF-κB from the cytoplasm to the nucleus was determined via immunofluorescence (IF) assay. e treatment with CFA increased the translocation of NF-κB from the cytoplasm to the nucleus, whereas the treatment with highest dose of DIE (100 μg/mL) significantly suppressed the nuclear translocation of p-NF-κB in activated MH-S cells ( Figure 6). Bay-11 was used as an NF-κB inhibitor. e results of the IF assay indicated that the anti-inflammatory properties of the DIE were associated with its inhibitory effects on the phosphorylation of the NF-κB signal pathway (Figure 7).

Discussion
Duchesnea indica has been widely used as a natural medicine in Asia especially in Bangladesh, China, Korea, and Japan for the prevention and treatment of numerous diseases, such as tissue inflammation, leprosy, congenital fever, and mainly cancer [12,14]. Earlier studies have shown that its antiinflammatory agents can suppress transcription factors, such as nuclear factor-κB (NF-κB) and function as a key regulator in inflammatory cascade [2,7,9]  Evidence-Based Complementary and Alternative Medicine DIE [11]. Ellagic acid was indicated as the main compound of the DIE via the UPLC-QTOF-MS analysis. It is a phenolic compound and has been reported to exert a variety of biological properties, such as antioxidant, anti-inflammatory, and anticoagulant effects [25,26]. Moreover, ellagic acid has also been shown to reverse hepatic damage by inhibiting the NF-κB signaling pathway [27]. Previous findings are consistent with our study and suggest that ellagic acid could be one of the major active components responsible for any antiinflammatory activity. e major compounds in the DIE were determined via GC-MS analysis: 2, 3-dihydro-3, 5dihydroxy-6-methyl-(4H)-pyran-4-one; hexadecanoic acid; (b-f ) Densitometric analysis of relative mRNA expression levels which were quantified using the ImageJ program. e cells were seeded in a 6-well plate, and the doses of DIE were used as 12.5, 25, 50, and 100 μg/mL. All values were expressed as standard error of mean ± (SEM) from three independent experiments. ### p < 0.001 when compared with the basal group; * p < 0.05, * * p < 0.01, and * * * p < 0.001 when compared with the only CFA-treated group. Densitometric analysis of protein expression levels which were measured using the ImageJ software. e cells were seeded in a six-well plate, and the doses of DIE were used as 12.5, 25, 50, and 100 μg/mL. All values were expressed as standard error of mean ± (SEM) from three independent experiments. * p < 0.05, * * p < 0.01, and * * * p < 0.001 when compared with the only CFA-treated group. 8 Evidence-Based Complementary and Alternative Medicine 9,12,15-octadecatrienoic acid; gamma-sitosterol; octadecanoic acid; and linoelaidic acid. Although the DIE has antiinflammatory effects on CFA-induced inflammation in MH-S cells, further research is required to investigate the immune modulatory properties of the DIE in an animal model. Particulate matter (PM) found in polluted air is becoming a major cause for health problems [28]. It has been reported that chronic exposure of PM is related to chronic inflammatory diseases especially severe lung diseases including chronic respiratory diseases, COPD, asthma, and mainly lung cancer [1,29]. Inflammatory disorders cause excessive production of proinflammatory mediators and cytokines, which are the critical factors of many pathological and clinical manifestations [30]. Macrophages are the immune cells found in the immune regulatory system and responsible for upregulating the generation of inflammatory mediators.
Nitric oxide (NO) is generated by the iNOS in activated macrophages. NO is a small signaling molecule with essential roles in numerous body functions, but unregulated NO production can cause pathophysiological activities in the airway system [31,32]. Such disturbances can lead to airway narrowing and lung hypersensitivity. e results showed that the DIE reduced NO production in CFA-induced MH-S cells, whereas NO production was markedly elevated in the only CFA-treated group. COX-2, which is another important proinflammatory mediator, plays a critical role in diverse inflammatory diseases [33]. In this study, it was found that the DIE suppressed the mRNA and protein expression levels of inflammatory mediators COX-2 and iNOS in CFA-activated MH-S cells.
Activated macrophages also generated other important proinflammatory cytokines, such as TNF-α, IL-6, and IL-1β, which are responsible for chronic inflammatory diseases and especially respiratory pathology [34]. IL-6 and TNF-α are the critical mediators of sepsis, and their uncontrolled regulation can cause cell death and DNA damage depending on the severity of cytokine production [9]. IL-1β increased the infiltration of inflammatory cells and mediated the activation of inflammatory cascade [35]. In the present study, the DIE inhibited the production of the IL-6, TNF-α, and IL-1β mRNA expressions in CFA-activated MH-S cells. In this study, the DIE also suppressed the protein expression of iNOS and COX-2, suggesting that the DIE has anti-inflammatory properties.
It is well established that the transcription factor, NF-κB, is a key regulator for inflammation in stimulated macrophages. In normal conditions, NF-κB is stable in the cytoplasm; however, upon CFA stimulation, activated cells result in the phosphorylation of IκB. Hence, the NF-κB is disrupted to induce the phosphorylation, and activated NF-κB translocates into the nucleus from the cytoplasm [36,37]. us, the p-NF-κB then promotes the expression of proinflammatory mediators and cytokines. It has been previously shown that phosphorylated NF-κB increases the transcription of inflammatory genes [38]. e western blot results demonstrated that the DIE dose-dependently reduced the CFA-stimulated IκB and NF-κB phosphorylation in the cytoplasm. Moreover, the results of the immunofluorescence staining indicated that CFA increased the p-NF-κB translocation in the nucleus, where the DIE (100 μg/mL) and Bay-11 (10 μM) markedly inhibited the p-NF-κB translocation from the cytoplasm to the nucleus. Based on our study, we speculated that the DIE might inhibit the phosphorylation of IκB and NF-κB resulting in the suppressive activity of the proinflammatory cytokines. In our study, only in vitro experiment was carried out, but in vivo excrement should be done to determine the exact mechanism of action of DIE as an anti-inflammatory agent.

Conclusion
In summary, the current study demonstrated the anti-inflammatory properties of the DIE in the CFA-induced MH-S cell line. e obtained data provide important information about CFA-activated inflammatory responses and suggest that the DIE could inhibit the expression of proinflammatory mediators and cytokines in MH-S cells.
ese findings demonstrate that the DIE could be used as a promising bioactive functional food for immune modulation, especially in the treatment of lung inflammatory diseases.

Data Availability
All data generated during this study are included within this manuscript.