Chlojaponilactone B from Chloranthus japonicus: Suppression of

Article pubs.acs.org/jnp

Chlojaponilactone B from Chloranthus japonicus: Suppression of Inflammatory Responses via Inhibition of the NF-κB Signaling Pathway Jing-Jun Zhao, Yan-Qiong Guo, De-Po Yang, Xue Xue, Qin Liu, Long-Ping Zhu, Sheng Yin,* and Zhi-Min Zhao* School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, Guangdong 510006, People’s Republic of China S Supporting Information *

ABSTRACT: Bioassay-guided fractionation of an ethanolic extract of Chloranthus japonicus led to the isolation of the known lindenanetype sesquiterpenoid chlojaponilactone B (1). This compound exhibited pronounced inhibition of nitric oxide (NO) production in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. Further anti-inflammatory assays showed that 1 suppressed the levels of some key inflammation mediators, such as iNOS, TNF-α, and IL-6, in a dose-dependent manner, and reduced the ear thickness and neutrophil infiltration in 12-O-tetradecanoylphorbol13-acetate (TPA)-stimulated mice. A mechanistic study revealed that compound 1 exerted its anti-inflammatory effects via the suppression of the NF-κB signaling pathway, which inhibited NF-κB-dependent transcriptional activity, IκBα phosphorylation, and p65 nuclear translocation. In contrast, chlojaponilactone B (1) was found to exert little influence on the MAPK signaling pathway.

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the treatment of inflammation-related disorders such as traumatic injury, rheumatic arthritis, swelling, blood stasis, and pain.12 Previous studies involving this species have led to the isolation of a number of sesquiterpenoids and sesquiterpenoid oligomers,13−15 of which some display biological activities, including anti-HIV16 and cytotoxic13 effects and the inhibition of cell adhesion molecule expression.17 However, little research has been conducted into the anti-inflammatory properties and associated mechanisms of the chemical constituents in C. japonicus. In a continuing search for anti-inflammatory agents from plants used in traditional Chinese medicine,18,19 chlojaponilactone B (1), a lindenane-type sesquiterpenoid lactone previously isolated from this plant by Yan et al.,15 was identified as an anti-inflammatory lead compound using an array of screening procedures. Herein, several in vitro and in vivo anti-inflammatory activities and the potential mechanism of action of 1 are described.

nflammation is a protective response initiated by the immune system to defend against noxious stimuli, as well as infections and tissue injury.1−3 Although inflammation has been investigated intensively over the last two decades, owing to its biological complexity and its close relationship with diverse devastating pathological conditions, in addition to the often serious side effects of existing anti-inflammatory drugs, the discovery and development of novel anti-inflammatory therapeutics remains a crucial challenge.3,4 The initial stages of inflammation involve the production of nitric oxide (NO) and prostaglandin generated by inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), respectively, associated with an up-regulation in the expression of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1 beta (IL-1β).5 In this process, nuclear transcription factor kappa B (NF-κB), a family of structurally related eukaryotic transcription factors, plays a vital role via the regulation of genes that encode proinflammatory cytokines (e.g., IL-1, IL-6, and TNF-α), chemokines (e.g., IL-8, MIP-1α, and MCP1), inducible enzymes (iNOS and COX-2), adhesion molecules (e.g., ICAM, VCAM, and E-selectin), and growth factors.6−9 Therefore, compounds that down-regulate the expression of these inflammatory mediators involved in NF-κB signaling pathway might be potential anti-inflammatory candidates. Natural products provide effective structurally diverse small molecules of use in the identification of lead compounds for the development of anti-inflammatory drugs.10,11 Chloranthus japonicus Sieb. (Chloranthaceae), a perennial herbaceous plant growing in shady places in southern mainland China, has had a long history of use in traditional Chinese medicine for © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Bioassay-Guided Isolation of Chlojaponilactone B (1) and Its Inhibitory Effects on Lipopolysaccharide (LPS)Induced NO Production in RAW 264.7 Cells. NO is an important inflammatory marker that exerts multiple immunemodulating effects and plays a key role in the regulation of inflammatory responses.20 LPS-induced NO production in RAW 264.7 cells is a classical and widely used model for preliminary screening of anti-inflammatory agents in vitro. In a Received: April 21, 2016

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Figure 1. Structures of 1−10 and their inhibitory effect on NO production in LPS-induced RAW 264.7 cells.

Figure 2. Effect of 1 on the production of (A) TNF-α and (B) IL-6 in LPS-induced RAW 264.7 macrophages. RAW 264.7 cells were treated with increasing concentrations of 1 (0.3, 1, 3, and 9 μM) for 30 min followed by LPS stimulation for another 24 h. The culture medium was subjected to an ELISA assay to quantify the levels of TNF-α and IL-6 secretion. Values are represented as means ± SD of three independent experiments (**p < 0.01 versus the group treated with LPS only).

Figure 3. Effect of 1 on LPS-induced iNOS and COX-2 protein expression in RAW 264.7 macrophages. Cells were pretreated with the indicated concentrations of 1 for 30 min before stimulation with LPS for 12 h. Cell lysates were subjected to Western blot analysis with iNOS, COX-2, and βtubulin (internal control) antibodies. (A) Results are presented as representative of three independent experiments and summarized in the bar graphs. (B) Data are presented as means ± SD of three independent experiments (**p < 0.01 vs the group treated with LPS only).

were identified as chlojaponilactone B (1),15 chloranthalactone A (2),21 5-hydroxychloranthalactone A (3),21 chloranthalactone B (4),21 8-epi-chlorajapolide F (5),22 chlorajapolide F (6),22 chloranthalactone E (7),23 shizukanolide (8),24 sarcandralactone A (9),25 and chlorajapolide C (10)13 by comparison of their physical and spectroscopic data ([α]D, 1H and 13C NMR, ESIMS) with values reported in the literature. Among them, compound 1 was identified as a potent NO inhibitor with an IC50 value of 1.4 ± 0.06 μM. The positive control N6-(1imino)-L-lysine (L-NIL) dihydrochloride exhibited an IC50 value

bioassay-guided isolation process, an ethanolic extract of C. japonicus was suspended in H2O and partitioned successively with petroleum ether, EtOAc, and n-BuOH. Each fraction was tested for inhibitory effects on NO generation in LPS-induced RAW 264.7 cells, and the EtOAc fraction with inhibitory activity (80.14% inhibition at 100 μg/mL) was selected for further chemical investigation. Subsequent purification of this fraction using various chromatographic methods led to the identification of 10 structurally related lindenane-type sesquiterpenoids (1−10, Figure 1). These 10 known compounds B

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of 7.5 ± 1.53 μM, while compounds 2−10 showed only weak activity (Table S20, Supporting Information). The number of related lindenane-type sesquiterpenoids obtained from other plant species is not enough to analyze accurately the structure− activity relationship. It was conjectured that the activity of compound 1 depends on the double bond between C-8 and C9, and the acetyl group at C-6, from the results of NO production evaluation. In addition, The inhibitory effects on NO production of sesquiterpene dimers seem to be more potent than those of the monomers according to the literature.26,27 Chlojaponilactone B (1) Decreased the Expression of Inflammatory Mediators TNF-α, IL-6, iNOS, and COX-2 in LPS-Stimulated RAW 264.7 Cells. The pro-inflammatory cytokines TNF-α and IL-6 and the inducible enzymes iNOS and COX-2 are important inflammatory mediators that contribute to acute inflammation by inducing vasodilation, increased vascular permeability, apoptosis, edema, and fever.28,29 These mediators become up-regulated during the inflammation process, and compounds that down-regulate them are considered to possess potential anti-inflammatory properties. To probe the possible anti-inflammatory properties of 1 further, its inhibitory effects on the LPS-induced expression of TNF-α, IL-6, iNOS, and COX-2 in RAW 264.7 cells were investigated. As shown in Figures 2 and 3, the enhanced levels of TNF-α, IL-6, and iNOS on the stimulation of LPS were dramatically decreased (p < 0.01) by pretreatment of 1 in a dose-dependent manner, while the level of COX-2 was also slightly down-regulated. These results suggested that 1 was able to reduce the inflammatory response in RAW 264.7 cells. Chlojaponilactone B (1) Ameliorated 12-O-Tetradecanoylphorbol-13-acetate (TPA)-Induced Ear Edema in Mice. Increased skin edema is a typical and first indication of skin irritation, which reflects a local inflammation process.30 To evaluate the in vivo anti-inflammatory effect of 1, a wellestablished animal model using TPA-induced ear edema31 on BALB/c mice was used. Hydrocortisone (1%) was used as a positive control, and topical applications of 1 at 10 mg/mL ameliorated TPA-induced ear edema (23.51% ± 2.8% reduction, p < 0.05), being less active than the positive control group (56.33% ± 7.4% reduction), while the therapeutic effect was decreased in a dose-dependent manner with the application of doses of 5 and 2.5 mg/mL (Figure S22, Supporting Information). The hematoxylin and eosin-stained ear sections from the experimental mice were then analyzed with the EVOS cell imaging system. As shown in Figure 4, application of 10 mg/ mL of 1 reduced TPA-induced epidermal hyperplasia and substantial inflammatory cell infiltration in the ear. Such an effect was comparable to the positive control group treated with 1% hydrocortisone. Accordingly, compound 1 was found to show a promising anti-inflammatory effect in vivo. NF-κB is representative of a class of inducible dimeric transcription factors that regulate the transcription of a large number of genes, particularly those involved in the immune and inflammatory responses. NF-κB has been established as one of the most significant transcription factors, which remains activated during the inflammatory process and participates in regulating the expression of multiple inflammatory mediators, such as TNF-α, IL-6, iNOS, and COX-2.6−9 For exploration of the underlying anti-inflammatory action mechanism of 1, its potential effect on the NF-κB signaling pathway was examined.

Figure 4. Histological sections of mouse ear skin biopsies. Animals were pretreated with topical applications of 1 on the right ear (2.5, 5, and 10 mg/mL). Hydrocortisone (1%) was used as a positive control for 30 min before TPA was applied for 4 h. The left ear received acetone only and was used as a solvent control. Ear samples were sectioned at a thickness of 4 μm, stained with hematoxylin and eosin, and observed under a magnification of 200 times; n = 8 mice per group. (A) Mouse ears treated with solvent only; (B) mouse ears treated with TPA and solvent; (C) mouse ears treated with TPA and 1 (10 mg/mL); and (D) mouse ears treated with TPA and 1% hydrocortisone.

Chlojaponilactone B (1) Suppressed the Transcription of the NF-κB-Dependent Reporter Gene in LPS-Induced RAW 264.7/NF-κB-luc Cells. A luciferase assay was performed in RAW 264.7/NF-κB-luc cells (i.e., the RAW 264.7 cell line stably transfected with an NF-κB-driven luciferase reporter plasmid) to evaluate the inhibition of 1 on the NF-κB-dependent reporter gene transcription. As shown in Figure 5, compound 1 significantly inhibited LPS-up-regulated

Figure 5. Compound 1 inhibits LPS-induced NF-κB activation. RAW 264.7/NF-κB-luc cells were treated for 30 min with the indicated concentrations of 1, solvent vehicle (0.5% DMSO), or 20 μM PDTC as a positive control prior to stimulation with 500 ng/mL LPS for 6 h. Data are represented as means ± SD of three independent experiments (**p < 0.01 vs the group treated with LPS and solvent).

NF-κB-dependent transcription in a dose-dependent manner and was more effective than a well-known NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC) (p < 0.01). Chlojaponilactone B (1) Prevented the Degradation of IκB and Restrained NF-κB Nuclear Translocation in LPS-Induced RAW 264.7 Cells. NF-κB is most often referred to the p65/p50 dimer, which is bound to an inhibitor of NF-κB (IκB) and is inactive in the cytoplasm. Upon stimulation, IκB is phosphorylated and dissociates from the IκB:NF-κB complex, C

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Figure 6. Effect of 1 on LPS-activated IκB-α phosphorylation in RAW 264.7 macrophages. RAW 264.7 cells were preincubated with 1 (2.5, 5, or 10 μM) or solvent vehicle (0.5% DMSO) for 30 min prior to stimulation with LPS for 1 h. Western blot analysis was performed for the detection of IκB-α and phospho-IκB-α (p-IκB-α). β-Tubulin was used as an internal loading control. Representative blots from three independent experiments are presented (A), and the results are summarized in bar graphs (B). Data are represented as means (*p < 0.05 and **p < 0.01 compared to the group treated with LPS only).

Figure 7. Effect of 1 on the translocation of the NF-κB p65 subunit into the nucleus of LPS-activated RAW 264.7 macrophages. Cells were pretreated with 10 μM 1 for 30 min followed by stimulation with LPS (500 ng/mL) for 3 h, then fixed and immunostained with mouse anti-p65 antibody followed by Alexa 594-labeled anti-rabbit IgG (red). DAPI was used to delineate the nucleus of each cell (blue). (A) Representative images of stained RAW 264.7 cells (20× magnification). (B) Quantitative analysis of NF-κB fluorescence in uninduced, induced, and 1-treated RAW 264.7 cells. Values represent the difference of fluorescence intensity in the nucleus and cytoplasm and as means ± SD of three independent experiments (**p < 0.01 compared to the group treated with LPS and solvent).

three vital MAPK kinases, (a) JNK, (b) p38, and (c) ERK1/2, was studied further. However, 1 exhibited no potent inhibitory effects on the LPS-induced phosphorylation pattern of any of the three kinases studied in RAW 264.7 cells (Figure S23, Supporting Information).

freeing NF-κB to translocate into the nucleus, where it can drive selectively the expression of a large number of inflammation-related genes.32 Thus, if the degradation of IκB is inhibited, such nuclear translocation may be prevented, and the subsequent downstream actions also are suppressed. Therefore, the influence of 1 on LPS-induced degradation of IκB-α in RAW 264.7 cells was investigated. As shown in Figure 6, stimulation of LPS resulted in a notable increase in IκB-α phosphorylation, while treatment of 1 exerted a dosedependent inhibitory effect on the degradation of IκB-α. Furthermore, immunofluorescent staining was performed to determine the translocation of p65 (a subunit of NF-κB and marker of its activation) in the nucleus. As shown in Figure 7, p65 was distributed primarily in the cytosol of the unstimulated macrophages, while the stimulation of LPS (500 ng/mL) markedly increased the level of p65 in the nucleus. Treatment of 1 restrained this translocation to the nucleus and provided support for its inhibitory effect on IκB degradation. In addition, mitogen-activated protein kinase (MAPK), a family of serine/threonine protein kinases that mediates the synthesis of inflammatory mediators at the level of transcription and translation, is also an important signaling pathway closely related to the inflammatory process.33−35 To examine whether 1 affects the MAPK pathway, the phosphorylation status of

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a PerkinElmer 341 polarimeter. NMR spectra were measured on a Bruker AM-400 spectrometer at 25 °C. ESIMS were measured on a Finnigan LCQ Deca instrument. Silica gel (300−400 mesh, Qingdao Haiyang Chemical Co., Ltd.), C18 reversed-phase silica gel (12 nm, S-50 μm, YMC Co., Ltd.), Sephadex LH-20 gel (Amersham Biosciences), and MCI gel (CHP20P, 75−150 μm, Mitsubishi Chemical Industries Ltd.) were used for column chromatography. All solvents used were of analytical grade (Guangzhou Chemical Reagents Company, Ltd.). Each pure compound was dissolved in DMSO at a concentration of 40 mM (final concentration of DMSO was limited to 0.5%) and diluted with Dulbecco’s modified Eagle’s medium (DMEM) to appropriate concentrations before use. Dimethyl sulfoxide, lipopolysaccharide, Geneticin (G418), 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 6,4′diamidino-2-phenylindole (DAPI), pyrrolidine dithiocarbamate (PDTC), and 12-O-tetradecanoylphorbol-13-acetate (TPA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Hydrocortisone D

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500 μg/mL G418. Both cell lines were placed at 37 °C in a humidified incubator containing 5% CO2. Cytotoxicity Assay. The cytotoxicity of compound 1 on RAW 264.7 cells was determined via an MTT assay, a commonly used method of measuring the number of metabolically active cells in in vitro studies.36,37 RAW 264.7 cells were placed in 96-well plates (5 × 103/well) overnight. The cells were then treated with different concentrations of 1 for 24 h. An equal concentration of the solvent vehicle (DMSO, 0.5%) was included as a control. Subsequently, 20 μL of MTT (5 mg/mL in sterile PBS) was added to each well for an additional 4 h incubation period. Finally, the medium was removed, 100 μL of DMSO was added to each well, and the absorbance (A) was detected at 490 nm using a microplate reader (Molecular Devices, Sunnyvale, CA, USA). Nitric Oxide Production. The inhibitory activity of compound 1 on LPS-induced NO production was determined by a colorimetric assay based on the Griess reaction, as previously described.38 RAW 264.7 cells were cultured in 96-well plates at a density of 5 × 104 cells per well for 24 h, then pretreated with 1 (test group) for 30 min before stimulation with 500 ng/mL LPS. Wells with no test compounds received only LPS and served as controls. Wells with neither a test chemical nor LPS treatment served as blank controls. An equal concentration of the solvent vehicle (DMSO, 0.5%) was included in each case. After 24 h stimulation, the medium in each well was collected and reacted with the Griess reagent. The absorbance was measured at 540 nm using a microplate reader. The known iNOs inhibitor L-NIL was used as a positive control. Determination of Pro-inflammatory Cytokine Levels. RAW 264.7 cells were pretreated with compound 1 (0.3, 1, 3, or 9 μM) for 30 min. The cells were stimulated with LPS (500 ng/mL) for 24 h, and the supernatant was harvested for testing in a pro-inflammatory cytokine assay. The concentrations of TNF-α and IL-6 in the culture medium were determined using commercial ELISA kits according to the instructions of each manufacturer. TPA-Induced Ear Edema Assay in Mice. The in vivo antiinflammatory activity of compound 1 was evaluated using a wellestablished animal model of TPA-induced ear edema in BALB/c mice. TPA was dissolved in acetone and used as an inducer of skin inflammation. A volume of 20 μL (2 μg of 1) was delivered to the right ear of the mouse (10 μL to both the inner and outer surfaces) to induce skin inflammation. A volume of 20 μL of 1 (2.5, 5, and 10 mg/ mL), acetone, or 1% hydrocortisone as a positive control was applied to the right ear 30 min before TPA treatment. The left ear remained untreated and only received the same volume of acetone as a solvent control. After 4 h of TPA exposure, the mice were sacrificed and a stainless steel hole puncher with a diameter of 7 mm was used to take the central sections of the right and left ears. The sections of both the right and left ears were weighed, and the weight difference was used to evaluate the level of ear edema. The following values were then calculated with the formula

was obtained from Aladdin (Los Angeles, CA, USA). N6-(1Iminoethyl)-L-lysine (L-NIL) was supplied by Cayman Chemical (Ann Arbor, MI, USA). Dulbecco’s modified Eagle’s medium, fetal bovine serum (FBS), and Alexa 594-labeled anti-rabbit IgG were obtained from Invitrogen (Grand Island, NY, USA). Luciferase assay system reagent was provided by Promega (Madison, WI, USA), and Griess reagent was purchased from Beyotime (Shanghai, People’s Republic of China). Mouse TNF-α and IL-6 ELISA kits were provided by 4A Biotech Co. (Beijing, People’s Republic of China). All primary antibodies were acquired from Cell Signaling Technology (Danvers, MA, USA). Plant Material. Whole plants of Chloranthus japonicus were collected in May 2013 at Anning City, Yunnan Province, People’s Republic of China, and authenticated by Prof. You-Kai Xu, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences. A voucher specimen (accession number: SKW201305) has been deposited at the School of Pharmaceutical Sciences, Sun Yat-sen University. Extraction and Isolation. The air-dried powder of whole C. japonicus plants (1.0 kg) was extracted using 95% EtOH (3 × 3 L) at room temperature to yield 100 g of crude extract. The extract was suspended in H2O (1 L) and partitioned successively with petroleum ether (3 × 1 L), EtOAc (3 × 1 L), and n-BuOH (3 × 1 L). The dried EtOAc extract (60 g) was subjected to MCI gel column chromatography (CC) with a MeOH/H2O gradient (3:7 → 10:0) to obtain five fractions (I−V). Fraction I (2.5 g) was chromatographed over C18 reversed-phase (RP-18) silica gel eluted with MeOH/H2O (5:5 → 10:0) to obtain three fractions (Ia−Ic). Fraction Ib (400 mg) was separated by silica gel CC (petroleum ether/CH2Cl2, 5:1 → 1:5), followed by Sephadex LH-20 CC, using EtOH, to produce compound 7 (200 mg). Fraction II (6.5 g) was subjected to silica gel CC (petroleum ether/acetone, 15:1 → 2:1) to provide five fractions (IIa− IIe). Fraction IIa (600 mg) was separated by RP-18 silica gel eluted with MeOH/H2O (5:5 → 10:0), followed by Sephadex LH-20 CC (CH2Cl2/MeOH, 1:1), to produce 6 (100 mg). Fraction IV (1.5 g) was separated by RP-18 CC, using a gradient of MeOH/H2O (6:4 → 10:0), to produce three fractions (IVa−IVc). Fraction IVb (630 mg) was successively subjected to silica gel CC (petroleum ether/EtOAc, 30:1 → 1:1), RP-18 silica gel CC (MeOH/H2O, 7:3 → 10:0), and Sephadex LH-20 eluted with MeOH, to yield 4 (32 mg), 1 (75 mg), and 9 (35 mg). Further purification of fraction IVc (275 mg) was conducted by silica gel CC (petroleum ether/acetone, 80:1 → 1:1) to produce 5 (18 mg) and 2 (46 mg). Fraction V was chromatographed over silica gel CC (petroleum ether/acetone, 30:1 → 2:1), followed by RP-18 silica gel CC (MeOH/H2O, 8:2 → 10:0) to yield 8 (17 mg), 3 (27 mg), and 10 (13 mg). The compounds were identified by comparison of their physical and spectroscopic data ([α]D, 1H and 13C NMR, ESIMS) with literature values. The purity of compounds 1−10 was estimated to be greater than 95%, as determined by their 1H NMR spectra (Figures S1−19, Supporting Information). Animals. Male and female BALB/c mice weighing 18−22 g were purchased from the Laboratory Animal Center of Sun Yat-sen University (Guangzhou, People’s Republic of China). The animals were housed in polypropylene cages (four mice per cage) and maintained on a standard laboratory diet and water ad libitum. They were held in an air-conditioned room for 2 to 3 days before the experiments for adaptation. The room temperature (22−24 °C), humidity (40−60%), and 12:12 h light and dark cycles were controlled automatically. All experimental procedures were performed under the approval of Sun Yat-sen University Animal Policy and Welfare Committee (protocol no. IACUC-DD-15-1204). Cell Culture. The RAW 264.7 mouse macrophage cell line was purchased from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology (Chinese Academy of Sciences, Shanghai, People’s Republic of China). The cells were cultured in DMEM supplemented with 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin. RAW 264.7/NF-κB-luc cells, provided by Professor Xiaojuan Li, Institute of Pharmacology, Southern Medical University (Guangzhou, People’s Republic of China), were cultured in DMEM supplemented with 10% FBS, 100 units/mL penicillin, 100 μg/mL streptomycin, and

edema rate (%) = [(weight right − weight left)/weight left] × 100% For histological analysis, ear samples were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned at a thickness of 4 μm, and then stained with hematoxylin and eosin. A representative area was selected for qualitative light microscopic analysis, and the magnification of 200× was used to evaluate the cell-mediated inflammatory response under the EVOS cell imaging system (Life Technologies, Waltham, MA, USA). NF-kB Reporter Gene Assay. To determine the effect of compound 1 on the NF-κB signaling pathway, a luciferase assay was performed to analyze the transcription of an NF-κB-dependent reporter gene as described previously.39 RAW 264.7/NF-κB-luc cells were seeded into 96-well plates at a density of 5 × 105 per well. After 24 h incubation, cells were pretreated with 1 (2.5, 5, 10, and 20 μM) for 30 min before LPS (500 ng/mL) stimulation. An equal concentration of the solvent vehicle (DMSO, 0.5%) was always included as a control. After 6 h stimulation, the medium was removed and the cells were lysed with the lysis buffer in the luciferase assay E

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Journal of Natural Products system. Afterward, the cell lysate was mixed with the luciferase assay reagent and the luminescence of firefly luciferase was immediately quantified using a microplate reader. PDTC was used as a positive control. p65 Subunit Translocation. RAW 264.7 cells (1 × 105/mL) were placed in 96-well plates overnight and then pretreated with 10 μM 1 for 30 min before stimulating with LPS (500 ng/mL). After 3 h stimulation, cells were first washed with 1% phosphate-buffered saline (PBS) and fixed with 4% formaldehyde for 20 min, followed by 1% Triton X-100 penetration for 10 min. Next, the cells were blocked with 5% bovine serum albumin for 1 h and incubated with NF-κB p65 antibody (diluted 1:100) at 4 °C overnight. Then, Alexa Fluor 594 labeled anti-rabbit IgG (diluted 1:1000 in blocking buffer) was added for an additional 1 h incubation at room temperature. The cells were then washed with PBS and stained with DAPI (100 ng/mL). The fluorescence signals were finally determined by a high-content screening system (Thermo Fisher, Waltham, MA, USA). Western Blot Analysis. RAW 264.7 cells (5 × 105 cells per well) were cultured in six-well plates for 24 h and incubated with serum-free DMEM for another 12 h. Then, the cells were pretreated with compound 1 for 30 min prior to stimulating with LPS (500 ng/mL) for 1 or 12 h (detection of iNOs and COX-2 protein). The cell lysate was prepared, and the total cell proteins in each sample were quantified using a BCA Protein Assay Kit (Pierce, Rockford, IL, USA). The proteins in each sample were separated by 10% SDS-PAGE and then transferred onto polyvinylidenedifluoride membranes (Bio-Rad, Hercules, CA, USA). The membranes were washed with Tris-buffered saline Tween-20 (TBST) and blocked in 5% skim milk for 1 h at room temperature. The membranes were incubated with the primary antibodies consisting of anti-IκB-α, antiphospho-IκB-α, anti-JNK, antiphospho-JNK, anti-p38, antiphospho-p38, anti-ERK1/2, antiphospho-ERK1/2, anti-iNOS, and anti-COX-2 antibodies (diluted 1:1000 in blocking buffer) at 4 °C overnight. The next day, the membranes were incubated with secondary antibodies (diluted 1:5000 in 5% skim milk) for 1 h at room temperature and then washed with TBST (containing 0.05% Tween-20). Blots were detected using an enhanced chemiluminescence detection system (Tanon, Shanghai, People’s Republic of China). β-Tubulin and GAPDH protein were used as the loading controls. Data Analysis. The data obtained are presented as the means ± SD (n = 3). Statistical analysis was performed by analysis of variance (ANOVA) using SPSS 12.0. Nonlinear regression (sigmoidal dose response) in GraphPad Prism 5 was used to calculate the IC50 values. A value of p < 0.05 was considered significant (*), and p < 0.01 as highly significant (**).

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ACKNOWLEDGMENTS

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REFERENCES

This work was supported by the Fundamental Research Funds for the Central Universities (No. 14yksh02) and the Science and Technology Program of Guangzhou, China (No. 20130000162).

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00355. 1 H and 13C NMR spectra of compounds 1, 2, and 4−10, 1 H NMR spectrum of compound 3, the optical rotation and ESIMS data of 1−10, and selected biological test results on compound 1 (PDF)

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*Tel/Fax (S. Yin): +86-020-39943090. E-mail: yinsh2@mail. sysu.edu.cn. *Tel/Fax (Z. M. Zhao): +86-020-39943043. E-mail: [email protected]. Notes

The authors declare no competing financial interest. F

DOI: 10.1021/acs.jnatprod.6b00355 J. Nat. Prod. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.jnatprod.6b00355 J. Nat. Prod. XXXX, XXX, XXX−XXX