Inflammation is one of the most crucial aspects of the host defense against invading pathogens.1 During the inflammatory process, large amounts of pro-inflammatory mediators such as nitric oxide (NO) and prostaglandin E2 (PGE2) are generated by the inducible isoforms of NO synthase (iNOS) and cyclooxygenase-2 (COX-2), respectively.2
Nuclear transcription factor kappa-B (NF-κB) is one of the most important transcription factors and is found in cell types that express cytokines, chemokines, growth factors, cell adhesion molecules, and some acute-phase proteins in healthy and diseased tissues.3,4 The activation of NF-κB involves the phosphorylation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitors (IκBs) via the IκB kinase (IKK) signalosome complex. Once IκBs have been phosphorylated, they are ubiquitinated and degraded by 26S proteasome. The resulting free NF-κB is then translocated to the nucleus, where it binds to κB binding sites in the promoter regions of target genes and induces the transcription of pro-inflammatory mediators.5 Inflammatory mediators and cytokines are responsible for the pathogenesis of a vast number of human diseases.6 Therefore, much attention is devoted to attenuating the pro-inflammatory mechanisms of cytokines. Owing to the resistance of such diseases to conventional treatment as well as the side effects of currently available anti-inflammatory drugs, there is a pressing need for the development of novel anti-inflammatory drugs. Recent efforts are focused on finding natural products that exhibit anti-inflammatory properties.7
Aster yomena is an edible vegetable and a perennial herb found in Korea, China, Japan, and Siberia and is used as a type of folk medicine to treat cough, bronchial asthma, and insect bites.8,9 Recently, A. yomena was shown to have antioxidant and anti-asthmatic activities.10–12 However, studies have not yet considered the anti-inflammatory effects of the various solvent fractions of A. yomena.
A. yomena contains various compounds which are flavonoids, phenolic acids such as asteryomenin, esculetin, 4-O-b-d-glucopyranoside-3-hydroxy methyl benzoate, caffeic acid, isoquercitrin, isorhamnetin-3-O-glucoside, and apigenin that provide a great assortment of biological properties.13 Although several compounds of A. yomena have been confirmed, there is little literature available regarding anti-inflammatory activities of A. yomena. In the present study, we examined the mechanism of action involved in the anti-inflammatory effects of A. yomena on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. The results of the current study clearly indicate that A. yomena has anti-inflammatory effects and that these effects appear to be mediated, at least in part, by the inhibition of NF-κB signaling.
Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and penicillin–streptomycin (PS), dichlorofluorescein diacetate (DCFH-DA) were obtained from Invitrogen (Carlsbad, CA, USA). LPS from Escherichia coli O55:B5 was purchased from Sigma–Aldrich (St. Louis, MO, USA). Antibodies against iNOS, COX-2, and β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). Monoclonal antibodies against p65 and p50 were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). NF-κB-luciferase vector was purchased from Promega (Madison, WI, USA). All other chemicals were purchased from Sigma unless otherwise specified.
2.2. Preparation of extract
A. yomena was obtained from the agricultural technology center, Gurye, Jellanam-do, Korea. Voucher specimens (SCHAY150722) were deposited in the Department of Life Science and Biotechnology herbarium, Soonchunhyang University, Korea. A. yomena species were cleaned, dried and weighed, then extracted two times with 50% ethanol. The 50% ethanol extract of A. yomena was concentrated. The extract was submitted to liquid–liquid fractionation using solvents of increasing polarity. Briefly, the dried extract was suspended in distilled water and sequentially partitioned with equal volumes of n-hexane, dichloromethane, ethyl acetate, and n-butanol. Subsequently, the fractions were combined and evaporated under vacuum (EYELA N-1000, Tokyo Riakikai Co., Ltd. Japan) and then lyophilized with a Bondiro Lyophpride freeze dryer (Ilshine Lab Co., Ltd., Korea) at −70 °C under reduced pressure (<20 Pa). The dry residue was stored at −20 °C. As preparation for further analysis, the dry residue was reconstituted with DMSO and diluted with PBS (pH 7.4) to the desired final concentration and filtered through a 0.45-μm syringe filter (Advanced MFS, Inc., Dublin, CA, USA) before use.
2.3. Cell culture and viability
The RAW 264.7 macrophage cell line was purchased from the Korean Cell Line Bank (Seoul, Korea). RAW 264.7 cells were cultured in DMEM containing 10% FBS and 1% PS at 37 °C in 5% CO2. To investigate the effects of A. yomena on cell viability, cells (1 × 104 cells/well) were plated in duplicate 48-well plates and treated with fractions of A. yomena at various concentrations (0, 5, 10, 25, and 50 μg/mL) and then co-incubated with LPS (1 μg/mL) for 24 h.14–16 Cell viability was measured with CellTiter Glo (Promega, Madison, WI, USA). Cell viability is presented as the percentage of live cells in each well.
2.4. Assessment of intracellular ROS
The intracellular ROS levels were measured by flow cytometry using DCFH-DA. After a 24-h incubation following LPS treatment, cells were stained with 10 μM DCFH-DA, and incubated for 30 min at 37 °C in the dark. Stained cells were then injected into a flow cytometer for analysis.
2.5. Nitrite oxide (NO) measurement
The nitrite accumulated in culture medium was measured as an indicator of NO production based on the Griess reaction. Briefly, 100 μL of cell culture medium was mixed with 100 μL of Griess reagent (Promega) and incubated at room temperature for 10 min, and then the absorbance at 540 nm was measured in a microplate reader (Molecular Devices, Sunnyvale, CA, USA). Fresh culture medium was used as the blank in all experiments. The amount of nitrite in the samples was measured from the sodium nitrite serial dilution standard curve, and nitrite production was measured.
2.6. Prostaglandin E2 (PGE2) measurement
After 24 h of sample fractions treatment and LPS stimulation, the culture supernatant was collected. PGE2 level was measured using a PGE2 enzyme-linked immunosorbent assay (ELISA) kit following the manufacturer's instructions (Abcam, Cambridge, MA, USA). Briefly, the diluted cell supernatant (100 μL) was placed in a 96-well goat anti-mouse IgG-coated plate and incubated for 2 h. After incubation, the plate was washed using the provided washing buffer, and the color was developed by adding PNPP (200 μL) substrate after 45 min. The amount of PGE2 was calculated using a PGE2 standard curve.
2.7. Real-time reverse transcription–polymerase chain reaction (RT–PCR) analyses
To determine the expression levels of iNOS and COX-2, real-time RT-PCR was performed using a real-time thermal cycler Qiagen rotorgene Q (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. The cells were treated with A. yomena fractions and LPS for 24 h. Thereafter, cDNA was synthesized from the total RNA isolated from cells. The real-time PCR reaction was performed using 2 × SYBR Green mix (Qiagen). All results were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. The following primer sequences were used for the real-time RT-PCR: GAPDH, 5′-GAG CCA AAA GGG TCA TCA TC-3′ (forward), 5′-TAA GCA GTT GGT GGT GCA GG-3′ (reverse); iNOS, 5′-AAT GGC AAC ATC AGG TCG GCC ATC ACT-3′ (forward), 5′-GCT GTG TGT CAC AGA AGT CTC GAA CTC-3′ (reverse); COX-2, 5′-GGA GAG ACT ATC AAG ATA GT-3′ (forward), 5′-ATG GTC AGT AGA CTT TTA CA-3′ (reverse); TNF-α, 5′- AGC ACA GAA AGC ATG ATC CG-3′ (forward), 5′-GTT TGC TAC GAC GTG GGC TA-3′ (reverse); IL-6, 5′-CGA TGA TGC ACT TGC AGA AA-3′ (forward), 5′-TGG AAA TTG GGG TAG GAA GG-3′ (reverse); IL-1β, 5′-GGA CAG AAT ATC AAC CAA CAA GTG ATA-3′ (forward), 5′-GTG TGC CGT CTT TCA TTA CAC AG-3′ (reverse).
2.8. Preparation of cytoplasmic and nuclear extracts and immuno-blotting detection
RAW 264.7 cells were pretreated with solvent fractions of A. yomena for 2 h and then stimulated with LPS (1 μg/mL) for 1 h. Cytoplasmic and nuclear extracts were prepared according to the kit manuals (NE-PER nuclear and cytoplasmic extraction reagents, Thermo Scientific, CA, USA) and BCA reagent (Gendepot, Houston, TX, USA) was used to determine the protein content of the cell lysates. For each sample, an equal amount of protein was resolved using sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred onto a polyvinylidene fluoride (PVDF) membrane, and incubated with the appropriate antibody. Immuno-detection was performed using an enhanced chemiluminescence (ECL) detection kit; the immunosignals were captured using a Chemi-doc image detector (Bio-Rad, Hercules, CA, USA).
2.9. Determination of cytokine levels
TNF-α, IL-6 and IL-1β levels in RAW 264.7 cells culture medium were quantified using ELISA kits according to the manufacturer's instructions (R&D Systems, Minneapolis, MN, USA).
2.10. Measurement of NF-κB luciferase activity
RAW 264.7 cells were transiently transfected with a pNF-κB-luciferase vector (Promega) using lipofectamine 2000 (Invitrogen) following the manufacturer's protocol. Briefly, 5 × 104 cells were placed in a 24-well plate and allowed to grow to 80–90% confluence for 24 h. The cells were then treated with DNA-Transfast reagent mixture (50 μL) and incubated for 16 h. The amount of DNA added was 0.25 μg/well. After 16 h of incubation, each well was overlaid with 1 mL of complete growth medium and transfection was carried out for 48 h. After transfection, cells were pretreated with fractions (25 μg/mL) for 2 h and then stimulated with LPS (1 μg/mL) for 1 h. Luciferase activity in the cells was measured using Dual-Luciferase Reporter Assay System (Promega) following the manufacturer's protocol. Briefly, growth medium was removed and the cells were washed with 1 mL ice-cold PBS. After complete removal of PBS, passive lysis buffer (100 μL) was added and the plate incubated at room temperature for 15 min with shaking. After incubation, the luciferase activity was measured by adding cell lysate (20 μL) to the luciferase assay reagent (100 μL). Relative luciferase activity was determined by measuring the firefly luciferase activity and normalizing it to the Renilla luciferase activity.
2.11. Statistical analysis
Statistical analyses were performed with SPSS v12.0 (SPSS, Chicago, IL, USA). Data are represented as the mean ± SEM from three independent experiments, unless stated otherwise. Statistical analyses were done using the Student's t-test, with p < 0.05 was considered significant.
3.1. Effect of A. yomena on RAW 264.7 cell viability
The inhibitory effect of A. yomena on RAW 264.7 cells viability was determined by measuring the intracellular ATP content (Fig. 1). Cells were treated with fractions of A. yomena at various concentrations (0, 5, 10, 25, and 50 μg/mL) and then co-incubated with LPS (1 μg/mL) for 24 h. The various fractions of A. yomena had no cytotoxic effects on RAW 264.7 cells at 25 μg/mL. In contrast, the viability of cells treated with 50 μg/mL was 69.1–110.9%. Therefore, all fractions of A. yomena at 25 μg/mL were selected for subsequent experiments.
3.2. Effect of A. yomena on inhibition of intracellular ROS production in LPS-stimulated RAW 264.7 cells
First, we examined the effects of A. yomena on LPS-induced ROS production in RAW 264.7 cells. The RAW 264.7 cells were treated with LPS at 1 μg/mL for 24 h, and flow cytometry with DCFH-DA staining was used to detect the ROS production in the cells. A marked increase was observed in the LPS stimulation group, while 24 h incubation with A. yomena fractions significantly inhibited the LPS-induced intracellular ROS. However, there were no significant differences among fractions (Fig. 2).
3.3. Effects of A. yomena on LPS-induced NO and PGE2 production
To analyze the potential anti-inflammatory properties of A. yomena, we used RAW 264.7 cells, which can produce NO and PGE2 upon stimulation with LPS. Cells were pre-incubated with A. yomena for 2 h and then stimulated with 1 μg/mL LPS for 24 h. The PBS group did not receive either LPS or sample treatments. After cell culture media were collected, nitrite and PGE2 levels were determined, and most of the A. yomena fractions were found to have reduced NO production (Fig. 3A). A. yomena fractions were also found to inhibit PGE2 production (Fig. 3B). In particular, NO and PGE2 secretion decreased by 43.3% and 18.1%, respectively, in cells exposed to the dichloromethane fraction of A. yomena.
3.4. Effects of A. yomena on LPS-induced iNOS and COX-2 protein and mRNA expressions
Western blot and RT-PCR analyses were performed to determine whether the inhibitory effects of A. yomena on pro-inflammatory mediators (NO and PGE2) were related to the modulation of the expressions of iNOS and COX-2. In un-stimulated RAW 264.7 cells, iNOS and COX-2 protein and mRNA were not detected, but LPS-treated cell were up-regulated their protein levels, and pre-treatment with A. yomena fractions inhibited this effect (Fig. 4A). In general, these results indicate that the inhibitory effects of A. yomena on LPS-induced NO and PGE2 production involved suppression of iNOS and COX-2 expression. Furthermore, RT-PCR analysis showed that mRNA expression levels of iNOS and COX-2 correlated with their protein levels (Fig. 4B).
3.5. Effects of A. yomena on LPS-induced cytokine production
Since A. yomena was found to potently inhibit the pro-inflammatory mediators, we further investigated its effects on LPS-induced TNF-α, IL-6, and IL-1β release using ELISA. The dichloromethane fraction of A. yomena reduced TNF-α, IL-6, and IL-1β production (Fig. 5A). To evaluate whether the decrease in A. yomena-induced cytokine release was due to the regulation of the mRNA gene in LPS-stimulated RAW 264.7 cells, we performed RT-PCR analysis. Similar to results from cytokine secretion, A. yomena down-regulated LPS-induced TNF-α, IL-6, and IL-1β mRNA expression (Fig. 5B). These data indicated that A. yomena suppressed cytokine release at the transcriptional level.
3.6. Effects of A. yomena on NF-κB activity and translocation of NF-κB subunits
Because the activation of NF-κB is critical for iNOS, COX-2, TNF-α, IL-6, and IL-1β activation by LPS,17–20 NF-κB luciferase assay was performed to determine whether A. yomena influenced NF-κB activation. As shown in Fig. 6A, treatment of A. yomena hexane and dichloromethane fractions (25 μg/mL) significantly decreased NF-κB activation in LPS-stimulated RAW 264.7 cells as measured with a firefly luciferase activity assay. Since p50 and p65 are the major components of NF-κB, which is activated by LPS in macrophages, we examined p50 and p65 translocation to the nucleus by immuno-blotting. RAW 264.7 cells were incubated with LPS in the presence or absence of A. yomena fractions for 1 h. Negligible levels of p50 or p65 protein were detected in control cell nuclei, but treatment with LPS for 1 h caused their nuclear translocation. A. yomena pre-treatment attenuated p50 and p65 levels in nuclear fractions as assessed by Western blot (Fig. 6B and C). A. yomena regulated NF-κB activation after LPS stimulation; A. yomena also regulated NF-κB in TNFα-induced cells. Unlike LPS stimulation, hexane fraction of A. yomena inhibited p50 and p65 translocation in cytosol in TNFα-stimulated RAW 264.7 cells (Supplementary Fig. 1). This is because LPS and TNF-α were stimulating different pathway.Further studies are planned to reveal NF-κB inhibition signaling by A. yomena in LPS-, TNFα-stimulated RAW 264.7 cells. These observations suggest that A. yomena inhibits NF-κB activation by preventing the LPS and TNFα-induced nuclear translocations of p50 and p65.
We investigated the anti-inflammatory activity of solvent fractions of A. yomena in mouse RAW 264.7 macrophages. In this study, we demonstrated that stimulation of RAW 264.7 cells by LPS enhanced their accumulation of intracellular ROS. NO and ROS are known to be crucial inflammatory mediators. ROS accumulation is an important factor in the pathogenesis of many inflammatory diseases, including cancer.21 ROS have also been implicated in the expression of inflammatory genes via the redox-based activation of the NF-κB signaling pathway.22 Thus, treatment of cells with A. yomena significantly reduced LPS-induced ROS production.
The pro-inflammatory cytokines, prostaglandins, and NO produced by activated macrophages play critical roles in inflammatory diseases such as sepsis and arthritis.23,24 Hence, the inhibition of pro-inflammatory cytokines or iNOS and COX-2 expressions in inflammatory cells offers a new potential therapeutic strategy for treatment of inflammation. In the present study, A. yomena inhibited COX-2 and iNOS expression in macrophage cells, most likely by acting at the transcriptional level. However, EtoAc and BuOH fractions did not inhibit COX-2 mRNA and protein levels (Fig. 4) but inhibited PGE2 production (Fig. 3). The EtoAc and BuOH fractions demonstrated a similar mechanism of non-steroidal anti-inflammatory drugs (NSAIDs) in inhibiting PGE2 production. In previous studies, NSAIDs were reported to upregulate COX-2 expression in mice.39 Similarly, ajoene, a natural compound present in garlic, was also reported to increase COX-2 gene and protein expression, but it inhibited NO, PGE2 release and iNOS expression.40 In murine macrophages, prostaglandins have been suggested to act as negative feedback regulators of COX-2 expression.41 Hence, inhibition of prostaglandin production may cause upregulation of COX-2 expression, as shown in our study. It is known that salicylic acid, a typical NSAID component, is contained in the essential oil of A. yomena.42 However, further study is needed to determine exactly which component of that essential oil acts on NSAIDs.
Additionally, there is a complex ‘cross-talk’ between the iNOS and COX-2 pathways, as has been evidenced by numerous studies. There are reports suggesting that NO seems to decrease COX-2 expression.43 In the case of BuOH fractions, increased NO affected the COX-2 expression. However, not all published reports are consistent. Inhibitors of nitric oxide synthase activity such as NGmonomethyl-l-arginine (L-NMMA) have been demonstrated to increase COX-2 protein expression, with decrease in NO production in rodent macrophages.44 Reduced NO levels may contribute to the observed increased COX-2 protein and mRNA expression with EtoAc fractions.
Cytokines such as TNF-α, IL-6 and IL-1β are classified as pro-inflammatory cytokines in vitro and in vivo.25 Moreover, the production of TNF-α is critical for the synergistic induction of NO synthesis in IFN-γ and/or LPS-stimulated macrophages.26 TNF-α elicits a number of physiologic effects, such as septic shock, inflammation, cachexia, and cytotoxicity.27 IL-6 is believed to be an endogenous mediator of LPS-induced fever. Kim et al.13 confirmed the inhibitory effect of IL-6 by isolation compounds from A. yomena extract. In the present study, we found that A. yomena significantly inhibited TNF-α and IL-6 release and their mRNA expressions. Our study also demonstrated the inhibitory effect of A. yomena extract and fractions on iNOS, COX-2, NO, and PGE2 production by stimulants. Although some differences in materials and mechanism between two studies, these two studies show very similar effects.
NF-κB is the main regulatory transcription factor involved in cellular responses to specific stimuli.28–31 In addition, NF-κB plays an important role in maintenance of cell survival and expression of diverse inflammatory mediators including nitrite, PGE2 and inflammatory cytokines.32,33 Investigation of the inhibitory effects of the solvent fractions of A. yomena on NF-κB activation showed that dichloromethane fraction blocked nuclear translocation of p65 and p50 under LPS stimulation in this study. These findings are consistent with those of other reports that NF-κB response elements are present on the promoter of iNOS, TNF-α and IL-6 genes.34,35
However, in the present study, protein levels of both p50 and p65 also decreased in the cytoplasmic fractions, as seen in Fig. 6. Because it has been demonstrated that NF-κB activity can be correlated with cell damage and apoptosis, some studies have chosen to examine another variable of cellular integrity and a marker of the endogenous pathway of apoptosis—translocation of cytochrome c from the mitochondrial intermembrane space to cytosol.45 In addition, Yun et al.46 reported that isoquercitrin isolated from AY extracts was increased in cytosolic cytochrome c levels, suggesting that treatment with AY extract contributed to the maintenance of cellular membrane integrity. Therefore, further investigation is required to explore how the fractions affect the NF-κB pathway and cell apoptosis, and whether they inhibit p65 and p50 degradation in cytosol. Recently, phenolic compounds including asteryomenin, esculetin, 4-O-b-d-glucopyranoside-3-hydroxy methyl benzoate, caffeic acid, isoquercitrin, isorhamnetin-3-O-glucoside, and apigenin from A. yomena were reported to have anti-inflammatory effects. In particular, caffeic acid and apigenin were found to potently inhibit IL-6 production in MG-63 cells.13 Caffeic acid and apigenin were also reported to possess various pharmacologic effects, such as antioxidative and anti-inflammatory activities.36 Caffeic acid and its conjugates such as chlorogenic and caftaric acids have been demonstrated to be powerful antioxidants.37 Apigenin showed a potent anti-inflammatory activity as well.38 These reports are consistent with our results, suggesting either that potent, unidentified anti-inflammatory compounds are present in the dichloromethane fraction of A. yomena or that caffeic acid, apigenin and other compounds exert a synergistic effect on the inhibition of inflammatory mediator production in LPS-stimulated RAW 264.7 cells. Subsequently, we have isolated various presumed active compounds including isoquercitrin, caffeic acid, apigenin and rutin from solvent fractions of A. yomena, and further studies are underway to find the mechanisms of anti-inflammation and provide a more in-depth analysis involving in vivo TPA (12-O-tetra decanoylphorbol-acetate)-induced ear edema mice models.
In conclusion, our findings suggest that A. yomena is a potent inhibitor of LPS-induced NO, PGE2, cytokine production in macrophage cells, and that it acts at the transcription level. Moreover, the inhibitory effects of A. yomena are associated with NF-κB inactivation. Since NF-κB is a transcription factor that regulates the transcription of many genes associated with inflammation, its inhibition by A. yomena offers a possible approach to the treatment of severe inflammatory diseases.
This study was supported by the Research Program for Agricultural Science & Technology Development (Project No. PJ01082601), the National Academy of Agricultural Science, and the Rural Development Administration, Korea.
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Appendix A. Supplementary data
The following is the supplementary data related to this article:
Supplementary data related to this article can be found at https://doi.org/10.1016/j.jcma.2017.06.017