- Research article
- Open Access
- Open Peer Review
Papaver nudicaule (Iceland poppy) alleviates lipopolysaccharide-induced inflammation through inactivating NF-κB and STAT3
- Jae-Hyeon Oh†1,
- Miyong Yun†2, 3,
- Dain Park3, 4,
- In Jin Ha4,
- Chang-Kug Kim1,
- Do-Wan Kim1,
- Eun-Ok Kim3Email author and
- Seok-Geun Lee3, 4, 5Email authorView ORCID ID profile
© The Author(s). 2019
- Received: 13 November 2018
- Accepted: 8 April 2019
- Published: 29 April 2019
Papaver nudicaule belongs to the Papaveraceae family, which is planted as an annual herbaceous species generally for ornamental purpose. Papaver rhoeas in the same family has been reported to have various pharmacological activities such as antioxidant and analgesic effects. In contrast, little is known about the pharmacological activity of Papaver nudicaule. In this study, the anti-inflammatory activity of Papaver nudicaule extracts and the action mechanisms were investigated in RAW264.7 macrophage cells.
To investigate the anti-inflammatory activity of five cultivars of Papaver nudicaule with different flower color, samples were collected from their aerial parts at two growth stages (60 and 90 days) and their ethanol extracts were evaluated in the lipopolysaccharide (LPS)-treated RAW264.7 cells by measuring nitric oxide (NO) and prostaglandin E2 (PGE2) levels. Interleukin 1-beta (IL-1β), Interleukin-6 (IL-6) and Tumor necrosis factor alpha (TNF-α) production were also analyzed by RT-PCR and multiplex assays. Nuclear Factor-kappa-light-chain-enhancer of activated B cells (NF-κB) and Signal transducer and activator of transcription 3 (STAT3) signaling pathways were examined using western blotting and luciferase reporter assays to reveal the action mechanism of Papaver nudicaule extracts in their anti-inflammatory activity.
All of the Papaver nudicaule extracts were effective in reducing the LPS-induced NO, which is an important inflammatory mediator, and the extract of Papaver nudicaule with white flower collected at 90 days (NW90) was selected for further experiments because of the best effect on reducing the LPS-induced NO as well as no toxicity. NW90 lowered the LPS-induced PGE2 level and decreased the LPS-induced Nitric oxide synthase 2 (NOS2) and Cyclooxygenase 2 (COX2). In addition, NW90 reduced the LPS-induced inflammatory cytokines, IL-1β and IL-6. Furthermore, NW90 inhibited the LPS-induced activation of NF-κB and STAT3.
These results indicate that NW90 may restrain inflammation by inhibiting NF-κB and STAT3, suggesting the potential therapeutic properties of Papaver nudicaule against inflammatory disease.
- Papaver nudicaule
Acute inflammation is one of the defensive reactions of biological tissues against external stimuli and is a protective response involving immune cells and molecular mediators. However, hyperinflammatory responses can cause severe sepsis, resulting in multiple organ failure as well as high mortality [1–3]. Therefore, anti-inflammation is an important issue in controlling various diseases. The macrophage plays an important role in the biological defense in the early stage of inflammation by producing inflammatory mediators, including cytokines. Several inflammatory mediators such as NO, PGE2, and cytokines (IL-6, IL-1β, and TNF-α) have been reported to play key roles in the inflammatory response . For this purpose, inhibiting macrophage function inclusive of inflammatory mediators has potential as a therapeutic agent in the treatment of various inflammatory diseases [5–7].
Papaver nudicaule (Iceland poppy, Family: Papaveraceae) is a short-lived perennial plant which has been cultivated mainly for ornamental purpose in many countries including Asia and Europe . As a matter of fact, five cultivars of Papaver nudicaule blooms have been identified (yellow, orange, pink, scarlet and white). The cultivar of white color is the dominant one, while the others are recessive . In Tibet, Europe, and North Asia, the flowers and seeds have been used as mild diaphoretic by folk medicines [10, 11] and the leaves have been used as a source of vitamin C . Despite the existence of these folk remedies, their pharmacological activity and action mechanism has not been revealed yet.
In this study, we investigated the inhibitory effects of ethanol extracts of the five cultivars of Papaver nudicaule on the lipopolysaccharide (LPS)-induced inflammation in RAW264.7 cells and its mechanism.
Preparation of Papaver nudicaule extracts
Five kinds of Papaver nudicaule with different flower color and the abbreviation
Cell culture and reagents
RAW264.7 murine macrophage cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS and 1% antibiotic/antimycotic in a humidified incubator with 5% CO2 at 37 °C. LPS (Escherichia coli serotype 0111: B4) and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). A Nitric oxide detection kit was purchased from iNtRON Biotechnology (Sungnam, Republic of Korea).
Cell viability assays
Cell viability was measured by MTT assays. Cells (1 × 104 cells/well) were seeded in 96-well plates and incubated overnight. Cells were treated with LPS and Papaver nudicaule extracts for 24 h , and then 20 μl of 2 mg/ml MTT solution was added to each well. The cells were incubated at 37 °C for 3 h. Formazan crystals were dissolved by DMSO and resulting absorbance value was measured using microplate reader (Molecular Devices, California, USA) at 570 nm. Data are presented as mean ± standard deviation (SD) from at least three independent experiments in triplicate.
Nitric oxide assays
The nitrite concentration in the culture supernatant was analyzed as an indicator of NO production using Griess reagent. RAW264.7 cells (5 × 105 cells/well) were seeded in 6-well plates and incubated overnight. Cells were pretreated with Papaver nudicaule extracts 1 h prior to LPS (100 ng/ml) treatment for 24 h . Then the culture supernatants were mixed with Griess reagent [equal volumes of 1% (w/v) sulfanilamide in 5% (v/v) phosphoric acid and 0.1% (w/v) naphtylethylenediamine-HCL], incubated at room temperature for 10 min, and the absorbance at 540 nm was measured using a microplate reader. Data are presented as mean ± SD from at least three independent experiments in triplicate.
Real-time quantitative PCR
Cells (5 × 105 cells/well) were seeded in 6-well plates and incubated overnight. Then the cells were pretreated with Papaver nudicaule extracts 1 h prior to LPS (100 ng/ml) treatment for 24 h. Total RNA was isolated using TRI Reagent solution (Ambion, Waltham, MA, USA) according to the manufacturer’s instructions. Total RNA (1 μg) isolated from cells was reverse transcribed to cDNA using PrimeScript first-strand cDNA synthesis kit (Takara Korea Biomedical Inc., Seoul, Republic of Korea) according to the manufacturer’s instructions. Amplification of each cDNA was monitored using Sensi FAST SYBR No-ROX kit (Bioline, Taunton, MA, USA) on a StepOnePlus instrument (Waltham, Massachusetts, USA). Specific primers used as following: IL-6 (forward: 5′-CCACGGCCTTCCCTACTTC-3′, and reverse: 5′-TTGGGAGTGGTATCCTCTGTGA-3′), TNF-α (forward: 5′-CACCGTCAGCCGATTTGC-3′, and reverse: 5′-TTGACGGCAGAGAGGAGGTT-3′), IL-1β (forward: 5′-AGTTGACGGACCCCAAAAGAT-3′, and reverse: 5′-GGACAGCCCAGGTCAAAGG-3′), GAPDH (forward: 5′-CAAGGCTGTGGGCAAGGT-3′, and reverse: 5′-GGAAGGCCATGCCAGTGA-3′). GAPDH was used as an internal control. Data are presented as mean ± SD from at least three independent experiments in triplicate.
Western blotting analysis
Whole cell lysates were prepared and western blotting was performed as described . Cells (3 × 106 cells) were seeded in 100 mm dishes and incubated overnight. The cells were pre-incubated with Papaver nudicaule extracts for 1 h and then incubated with LPS (100 ng/ml) and the extracts for 24 h. Primary antibodies against COX2, NF-κB p65, phospho-NF-κB p65, IκBα, phospho-IκBα, STAT3, and phospho-STAT3 were purchased from Cell Signaling Technology (Danvers, MA, USA), NOS2 from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and β-actin from Sigma-Aldrich. Secondary antibodies HRP-conjugated anti-mouse IgG and anti-rabbit (1:5000–1:10000; Jackson Immuno Research Laboratories, Inc., PA, USA) were used for western blotting. Densitometric analysis of each protein band was calculated using the ImageJ software (https://imagej.nih.gov/ij/). Data are presented as mean ± SD from at least three independent experiments.
Multiplex cytokine assays
Cells (5 × 105 cells/well) were pre-incubated with Papaver nudicaule extracts for 1 h in 6-well plates, incubated with LPS (100 ng/ml) and the extracts for 24 h. IL-1β, IL-6, and TNF-α were measured using a Magnetic Luminex Assay kit (R&D Systems, MN, USA) and Luminex 200 array system after collecting the culture medium. Standard curves for each cytokine were generated using the kit-supplied reference cytokines. Data are presented as mean ± SD from at least three independent experiments in triplicate.
Enzyme-linked immunosorbent assay (ELISA) kits were purchased from R&D system (Minneapolis, MN, USA) for IL-1β and Biochem Inc. (Farmingdale, NY, USA) for PGE2. To measure IL-1β and PGE2 secretion in the culture supernatants, cells (5 × 105 cells/well) were pre-incubated with Papaver nudicaule extracts for 1 h in 6 well plates and incubated with LPS (100 ng/ml) and extracts for 24 h. The culture supernatants were collected and the concentration of PGE2 and IL-1β was measured using a microplate reader. Data are presented as mean ± SD from at least three independent experiments in triplicate.
Transient transfection and luciferase reporter assays
NF-κB-Luc and pSTAT3-Luc vectors were previously described [16, 17]. RAW264.7 cells were transiently transfected with NF-κB-Luc or pSTAT3-Luc using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Transfection efficiency (> 24% for NF-κB-Luc and > 34% pSTAT3-Luc) was confirmed by measuring the bioluminescence intensity with flow cytometry (FACS Canto II, BD Biosciences, San Jose, CA) . One day after transfection, the cells were re-plated into 24 well plates (5 × 104 cells/well) and incubated overnight. Then the cells were pre-treated with Papaver nudicaule extracts 1 h prior to LPS (100 ng/ml) treatment. Following incubation for 3 or 24 h, luciferase assays were performed using a luciferase assay system (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Data are presented as mean ± SD from at least three independent experiments in triplicate.
Statistical analysis was performed with the SigmaPlot 10.0 software (Systat Software, Inc., San Jose, CA, USA). An unpaired Student’s t-test was performed between mock and LPS treatment or LPS alone and co-treatment of LPS with Papaver nudicaule extract to calculate the P-value . P < 0.05 was considered significant.
Extracts of Papaver nudicaule decrease the LPS-induced NO and PGE2
Extract of NW90 reduces the LPS-induced inflammatory cytokines
Extract of NW90 inhibits the LPS-induced NF-κB and STAT3 activation
In this study, the anti-inflammatory activities of Papaver nudicaule against LPS-induced inflammation in RAW264.7 cells were examined. NW90 inhibited the LPS-induced production of NO and PGE2 by regulating the expression of NOS2 and COX2 and also suppressed the LPS-induced production of inflammatory cytokines, IL-1β and IL-6. We further confirmed that NW90 inactivated the LPS-induced NF-κB and STAT3 activation. These results indicate that Papaver nudicaule is an herbal plant with anti-inflammatory activity through inactivating NF-κB and STAT3, suggesting a potential to be developed as an anti-inflammatory agent. NF-κB is a major downstream transcription factor regulating the expression of inflammation-related genes during the induction of inflammatory stimuli such as LPS [26, 27] and is also associated with many chronic inflammatory diseases such as inflammatory bowel disease, atopic dermatitis and rheumatoid arthritis . Therefore, the anti-inflammatory activity of Papaver nudicaule through NF-κB regulation is likely to be applied to chronic inflammatory disease. In addition, STAT3 is another important transcription factor involved in the immune response and inflammation and is known to collaborate with NF-κB to control inflammation . Many inflammatory cytokines induced by NF-κB or STAT3 can positively feedback to activate STAT3 and NF-κB . IL-6 induced by NF-κB activates STAT3, resulting in the increased expression of STAT3-target genes . Therefore, dual inhibition of NF-κB and STAT3 is an attractive therapeutic strategy for treating inflammatory diseases and we in the present study suggest Papaver nudicaule as a novel candidate for developing a new anti-inflammatory drug targeting both NF-κB and STAT3.
Studies on the development of anti-inflammatory drug and dietary food using herbal plants as complementary medicine and using phytochemicals have been going for a long time . Various phytochemicals such as flavonoids and alkaloids have been isolated from various plants and their biological activities have been confirmed [32, 33]. Chemical compounds and their pharmacological activities of other species in Papaver such as Papaver somniferum and Papaver rhoeas have been characterized well [14, 34]. In contrast, few studies about Papaver nudicaule have been done. Any pharmacological or biological activity of Papaver nudicaule has not been uncovered yet, and just a few alkaloids including chelidonine and allocryptopine have been known in Papaver nudicaule [18, 35, 36]. Although chelidonine is a minor alkaloid in Papaver nudicaule, recent studies indicated that chelidonine extracted from Chelidonium majus suppresses the TNF-α and LPS-induced inflammation by inhibiting NF-κB in HCT 116 and RAW264.7 cells, respectively [37, 38]. We, thus, analyzed the two known compounds chelidonine and allocryptopine in Papaver nudicaule by LC-MS analysis. Allocryptopine was detected in every extract of all five cultivars of Papaver nudicaule including NW90 (Additional file 2: Figure S1). However, chelidonine was not observed in all our samples (data not shown). It is considered that this type of difference is generally caused by various reasons such as usage of another part of the plant (leaf, flower, stem, root, etc.), environmental issues for cultivation, and the cultivation period. We used Papaver nudicaule cultivated in the Republic of Korea, but the other group collected it from Mongolia [18, 36].
In the present study we notice that Papaver nudicaule alleviates the LPS-induced inflammation by suppressing NOS2, COX2 and inflammatory cytokines IL-1β and IL-6 through inhibiting NF-κB and STAT3 pathways, indicating a potential pharmacological activity of Papaver nudicaule for the first time. In contrast to the other Papaver species, Papaver nudicaule has not received any attention in the field of pharmacology or herbal medicine. Beginning our first presentation, further studies about Papaver nudicaule in the context of other pharmacological and biological activities and pharmaco-chemical analysis will warranty that Papaver nudicaule could be a useful herbal plant for human beings, not just an ornamental poppy.
Our experimental results indicate that Papaver nudicaule extracts have the anti-inflammatory activity to decrease the inflammatory response induced by LPS through inhibiting NF-κB and STAT3 pathways. Our findings suggest that Papaver nudicaule could be a potentially useful herbal plant with an anti-inflammatory effect.
There are no special acknowledgments.
This study was performed with the support of the National Institute of Agricultural Science (Project no. PJ01184702), Rural Development Administration, Republic of Korea and also supported by the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education (NRF-2015R1A4A1042399, NRF- 2016R1D1A1B03932971 and NRF- 2018R1D1A1B07047610). The funding bodies played no role in the design of the study and data collection, analysis, and interpretation of data and in writing the manuscript.
Availability of data and materials
Data and materials are available from authors on reasonable request.
J-HO, MY, IJH, C-KK, E-OK, and S-GL conceived and designed the experiments. J-HO, MY, DP, E-OK, and S-GL conducted the experiments, analyzed the data, and generated the figures. J-HO, D-WK, and IJH contributed the plant collection and sample preparation. S-GL supervised the whole project, and MY, E-OK and S-GL wrote the manuscript. All authors read and approved the final manuscript.
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