Papaver Nudicaule

Oh et al. BMC Complementary and Alternative Medicine (2019) 19:90 https://doi.org/10.1186/s12906-019-2497-5 RESEARCH ARTICLE Open Access Papaver nudicaule (Iceland poppy) alleviates lipopolysaccharide-induced inflammation through inactivating NF-κB and STAT3 Jae-Hyeon Oh1†, Miyong Yun2,3†, Dain Park3,4, In Jin Ha4, Chang-Kug Kim1, Do-Wan Kim1, Eun-Ok Kim3* and Seok-Geun Lee3,4,5* Abstract Background: 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. Methods: 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. Results: 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. Conclusions: 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. Keywords: Papaver nudicaule, Inflammation, Macrophage, NF-κB, STAT3 * Correspondence: [email protected]; [email protected] †Jae-Hyeon Oh and Miyong Yun contributed equally to this work. 3Department of Science in Korean Medicine, Kyung Hee University, Seoul 02447, Republic of Korea Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Oh et al. BMC Complementary and Alternative Medicine (2019) 19:90 Page 2 of 9 Background Genomics Division of the National Institute of Agricul- Acute inflammation is one of the defensive reactions of tural Science [13]. Here we abbreviate the extract of biological tissues against external stimuli and is a protect- Papaver nudicaule with white flower harvested at 60 ive response involving immune cells and molecular medi- days after seeding to NW60 and the same way for the ators. However, hyperinflammatory responses can cause other extracts (Table 1). The specimens used in this severe sepsis, resulting in multiple organ failure as well as study were deposited in the Genomics Division of the high mortality [1–3]. Therefore, anti-inflammation is an National Institute of Agricultural Science (Republic of important issue in controlling various diseases. The Korea). Aerial parts of Papaver nudicaule were lyophi- macrophage plays an important role in the biological lized and then ground into a fine powder. The ethanol defense in the early stage of inflammation by producing extraction methods are previously described [14]. 2 g of inflammatory mediators, including cytokines. Several each sample was ultrasonicated for 30 mins with 5 ml of inflammatory mediators such as NO, PGE2, and cytokines ethanol, and then centrifugation was performed for 15 (IL-6, IL-1β, and TNF-α)havebeenreportedtoplaykey mins at 13,000 rpm at 4 °C. The supernatants were fil- roles in the inflammatory response [4]. For this purpose, tered through the 0.2 μm polytetrafluoroethylene syringe inhibiting macrophage function inclusive of inflammatory filter (Thermo Scientific, Waltham, MA). Each solvent mediators has potential as a therapeutic agent in the treat- was then evaporated using speed vacuum (Thermo/Sav- ment of various inflammatory diseases [5–7]. ant SPC VAC 2010, Waltham, MA). Each sample Papaver nudicaule (Iceland poppy, Family: Papavera- extracted was dissolved in DMSO at a concentration of ceae) is a short-lived perennial plant which has been 500 mg/ml based on the weight of the ground powder cultivated mainly for ornamental purpose in many coun- after initial lyophilization and stored at − 70 °C until tries including Asia and Europe [8]. As a matter of fact, used in the experiments. five cultivars of Papaver nudicaule blooms have been identified (yellow, orange, pink, scarlet and white). The Cell culture and reagents cultivar of white color is the dominant one, while the RAW264.7 murine macrophage cells were maintained in others are recessive [9]. In Tibet, Europe, and North Dulbecco’s modified Eagle’smedium(DMEM)containing Asia, the flowers and seeds have been used as mild dia- 10% FBS and 1% antibiotic/antimycotic in a humidified phoretic by folk medicines [10, 11] and the leaves have incubator with 5% CO2 at 37 °C. LPS (Escherichia coli been used as a source of vitamin C [12]. Despite the serotype 0111: B4) and 3-(4, 5-dimethylthiazol-2-yl)-2, existence of these folk remedies, their pharmacological 5-diphenyl-tetrazolium bromide (MTT) were purchased activity and action mechanism has not been revealed yet. from Sigma-Aldrich (St. Louis, MO, USA). A Nitric oxide In this study, we investigated the inhibitory effects of detection kit was purchased from iNtRON Biotechnology ethanol extracts of the five cultivars of Papaver nudi- (Sungnam, Republic of Korea). caule on the lipopolysaccharide (LPS)-induced inflammation in RAW264.7 cells and its mechanism. Cell viability assays Cell viability was measured by MTT assays. Cells (1 × Materials and methods 104 cells/well) were seeded in 96-well plates and incu- Preparation of Papaver nudicaule extracts bated overnight. Cells were treated with LPS and Papa- The aerial parts of Papaver nudicaule harvested at two ver nudicaule extracts for 24 h [15], and then 20 μlof2 different growth stages (60 and 90 days) were provided mg/ml MTT solution was added to each well. The cells by the National Institute of Agricultural Science, Rural were incubated at 37 °C for 3 h. Formazan crystals were Development Administration (Republic of Korea). All dissolved by DMSO and resulting absorbance value was five cultivars of Papaver nudicaule with different flower measured using microplate reader (Molecular Devices, colors were used as noticed in Table 1. Every voucher California, USA) at 570 nm. Data are presented as mean specimen was identified by Dr. Do-Wan Kim in the ± standard deviation (SD) from at least three independent experiments in triplicate. Table 1 Five kinds of Papaver nudicaule with different flower color and the abbreviation Nitric oxide assays Species Color Abbreviation The nitrite concentration in the culture supernatant was Papaver nudicaule White NW analyzed as an indicator of NO production using Griess 5 Papaver nudicaule Orange NO reagent. RAW264.7 cells (5 × 10 cells/well) were seeded Papaver nudicaule Yellow NY in 6-well plates and incubated overnight. Cells were pretreated with Papaver nudicaule extracts 1 h prior to Papaver nudicaule Scarlet NS LPS (100 ng/ml) treatment for 24 h [15]. Then the cul- Papaver nudicaule Pink NP ture supernatants were mixed with Griess reagent [equal Oh et al. BMC Complementary and Alternative Medicine (2019) 19:90 Page 3 of 9 volumes of 1% (w/v) sulfanilamide in 5% (v/v) phos- Multiplex cytokine assays phoric acid and 0.1% (w/v) naphtylethylenediamine- Cells (5 × 105 cells/well) were pre-incubated with HCL], incubated at room temperature for 10 min, and Papaver nudicaule extracts for 1 h in 6-well plates, the absorbance at 540 nm was measured using a micro- incubatedwithLPS(100ng/ml)andtheextractsfor plate reader. Data are presented as mean ± SD from at 24 h. IL-1β,IL-6,andTNF-α were measured using a least three independent experiments in triplicate. Magnetic Luminex Assay kit (R&D Systems, MN, USA) and Luminex 200 array system after collecting Real-time quantitative PCR the culture medium. Standard curves for each cyto- Cells (5 × 105 cells/well) were seeded in 6-well plates and kine were

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