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STATs activation
Author’s Accepted Manuscript Essential oil of Artemisia argyi suppresses inflammatory responses by inhibiting JAK/STATs activation Lin-Lin Chen, Hao-Jun Zhang, Jung Chao, JunFeng Liu www.elsevier.com/locate/jep
PII: DOI: Reference:
S0378-8741(16)31824-4 http://dx.doi.org/10.1016/j.jep.2017.04.017 JEP10827
To appear in: Journal of Ethnopharmacology Received date: 10 November 2016 Revised date: 16 April 2017 Accepted date: 20 April 2017 Cite this article as: Lin-Lin Chen, Hao-Jun Zhang, Jung Chao and Jun-Feng Liu, Essential oil of Artemisia argyi suppresses inflammatory responses by inhibiting JAK/STATs activation, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2017.04.017 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Essential oil of Artemisia argyi suppresses inflammatory responses by inhibiting JAK/STATs activation
Lin-Lin Chen1*, Hao-Jun Zhang2, Jung Chao3, Jun-Feng Liu1
1
Key Laboratory of Traditional Chinese Medicine Resource and Compound Prescription, Ministry
of Education, Hubei University of Chinese Medicine, Wuhan 430065, PR China 2
Department of Pharmacology, China-Japan Friendship Hospital, Beijing 100029, China
3
Department and Institute of Pharmacology, National Yang-Ming University, Taipei 112, Taiwan
*
Corresponding author at: Key Laboratory of Traditional Chinese Medicine Resource and
Compound Prescription, Ministry of Education, Hubei University of Chinese Medicine, 1 Huang-Jia-Hu
West
Road,
Wuhan
430065,
PR
China.
Tel.:
+86
027
68890247;
[email protected] (L.-L. Chen)
Abstract Ethnopharmacological relevance Artemisia argyi is a herbal medicine traditionally used in Asia for the treatment of bronchitis, dermatitis and arthritis. Recent studies revealed the anti-inflammatory effect of essential oil in this plant. However, the mechanisms underlying the
therapeutic potential have not been well elucidated. The present study is aimed to verify anti-inflammatory effect and investigate the probable mechanisms. Materials and methods The essential oil from Artemisia argyi (AAEO) was initially tested against LPS-induced production of inflammatory mediators and cytokines in RAW264.7 macrophages. Protein and mRNA expressions of iNOS and COX-2 were determined by Western blotting and RT-PCR analysis, respectively. The effects on the activation of MAPK/NF-κB/AP-1 and JAK/STATs pathway were also investigated by western blot. Meanwhile, in vivo anti-inflammatory effect was examined by histologic and immunohistochemical analysis in TPA-induced mouse ear edema model. Results The results of in vitro experiments showed that AAEO dose-dependently suppressed the release of pro-inflammatory mediators (NO, PGE2 and ROS) and cytokines (TNF-α, IL-6, IFN-β and MCP-1) in LPS-induced RAW264.7 macrophages. It down-regulated iNOS and COX-2 protein and mRNA expression but did not affect the activity of these two enzymes. AAEO significantly inhibited the phosphorylation of JAK2 and STAT1/3, but not the activation of MAPK and NF-κB cascades. In animal model, oral administration of AAEO significantly attenuated TPA-induced mouse ear edema and decreased the protein level of COX-2. Conclusion AAEO suppresses inflammatory responses via down-regulation of the JAK/STATs signaling and ROS scavenging, which could contribute, at least in part, to the
anti-inflammatory effect of AAEO.
Keywords: Artemisia argyi; Anti-inflammatory; JAK/STATs
Chemical compounds studied in this article: cineole (PubChem CID: 2758), camphor (PubChem CID: 2537), α-(−)-thujone (PubChem CID: 261491), borneol (PubChem CID: 64685), indomethacin (PubChem CID: 3715), 1400W (PubChem CID: 1433)
1. Introduction Inflammation is a host defense mechanism which is considered as a part of innate immune system. The bacterial or viral invasions trigger inflammatory responses by the activation of immune cells such as macrophages, neutrophils, and dendritic cells. Macrophages play a pivotal role in the regulation of innate immune response, which is vital for the recognition and elimination of invasive microbial pathogens (Roger et al., 2001). The activated cells produces various cytokines, chemokines, and inflammatory mediators [including nitric oxide (NO), prostaglandin (PG) E2, reactive oxygen species (ROS), tumor necrosis factor (TNF)-α, interferons (IFNs), monocyte chemoattractant protein (MCP)-1 and interleukins ] (Ramana et al., 2006; Choi et al., 2011), which leads to an increase in intracellular signaling and chemotactic responses of other inflammatory cells, thereby protects the body against infection. Although the pro-inflammatory cytokines and mediators play essential roles in the regulation of
immune response, dysregulation of their production can cause damage to the host and lead to a variety of acute and chronic disorders, including cancer, diabetes, septic shock, autoimmune diseases, and atherosclerosis (Tabas and Glass, 2013). The strategies for anti-inflammation are to negatively modulate the expression of genes or activity of enzymes involved in inflammatory responses, thus affects the release of pro-inflammatory cytokines. Therefore, intracellular signaling such as mitogen activated protein kinases (MAPK) and some related transcription factors, including nuclear factor-κB (NF-κB) and activator protein (AP)-1, have been regarded as target molecules in anti-inflammatory drug development. Traditional non-steroidal anti-inflammatory drugs (NSAIDs) are widely used to treat inflammatory disorders, but the gastrointestinal, renal and cardiovascular side effects limit their long term use (Harirforoosh et al., 2014). Actually the continued search for more effective and safe anti-inflammatory agents which work on different targets never stops. As plenty of plant species were found to contain various compounds exhibiting anti-inflammatory effects (Gautam and Jachak., 2009), it is becoming a new trend for botanicals used as an alternative to conventional chemotherapy in the treatment of inflammatory diseases. Artemisia argyi is a herbaceous plant distributed in most regions of East Asia. It has been widely used in traditional Chinese medicine for treating eczema, hemorrhage, and dysmenorrhea (Chinese Pharmacopoeia, 2015). Dried leaves of this herb are also used in acupuncture clinics in China to treat various diseases, especially chronic conditions such as osteoarthritis, asthma, gastrointestinal disorders, and insomnia (Ge
et al., 2016). A. argyi is reported to possess multiple pharmacological activities including
anti-inflammatory,
antimicrobial,
antiasthma,
analgesic,
antivirus,
antioxidant, antitumor and immunomodulatory effects (Jiang et al., 2005; Ge et al., 2016; Huang et al., 2012; Bao et al., 2013;), most of which are contributed by the essential oil existing in A. argyi leaves. Though the anti-inflammatory activity of Artemisia genus has been studied intensively and several anti-inflammatory compounds were also found in this plant (Moscatelli et al., 2006; Min et al., 2009; Choi et al., 2013; Habib and Waheed, 2013; Jeong et al., 2014; Park et al., 2015; Zeng et al., 2014), there is no much substantial evidence supporting the anti-inflammatory property of this herbal medicine and the molecular targets for the effects still remain unclear, which limited its application into clinical practice. Therefore this study was aimed to evaluate the anti-inflammatory potential of the essential oil of A. argyi (AAEO), and investigate the underlying mechanisms by lipopolysaccharide (LPS)-stimulated murine macrophages and acute in vivo inflammatory model in mice.
2. Materials and methods 2.1. Antibodies and reagents The mouse monoclonal antibodies against iNOS, COX-2, p65, c-Jun, JNK, phospho-JNK, ERK1/2, phospho-ERK1/2, p38, phospho-p38, STAT1, phospho -STAT1, STAT3, phospho-STAT3, JAK2, phospho-JAK2, β-actin, PARP and α-tublin were purchased from Cell Signaling Technology (Beverly, MA, USA). The DMEM medium and fetal bovine serum (FBS) was purchased from GIBCO (Grand Island,
NY). Unless otherwise indicated, all other reagents were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). The essential oil of Artemisiae Argyi folium (batch number 201406001) was provided by Hubei Lishizhen Medicine Group Co., Ltd. (Qichun, China), which was obtained from the leaves of A. argyi collected in Qichun County, Hubei Province. Voucher specimens (QC140531-2) are deposited in Hubei Lishizhen Medicine Group Co., Ltd.
2.2.
Gas chromatography-mass spectrometry (GC-MS) analysis of AAEO
The essential oils were obtained from partially crushed leaves of A. argyi by hydrodistillation with a Clevenger apparatus for 5 h as recommended by Chinese Pharmacopoeia. The oil obtained was subsequently dried over anhydrous sodium sulphate, sealed and placed in the dark under refrigeration until used. GC/MS analysis of the essential oil was carried out on a GC-MS (Thermo Trace GC Ultra-ISQ mass spectrometer) equipped with a TR-5MS capillary column (30 m × 0.2 mm, 0.25 μm, Thermo, USA) in the electron impact mode (Ionization energy: 70 eV), and data were recorded in the full-scan acquisition mode with the range of m/z 40–500. The carrier gas was helium at a flow rate of 1.0 mL min−1. Split injection (1 μl) was conducted with a split ratio of 1:20. The injector temperature was set at 260°C. The ion source temperature of mass spectrometer was 200°C. The temperature program was started at 60°C, remaining at this temperature for 2 min, and heated at 5°C/min to 170°C, then held for 2 min, followed by 20°C/min to 250°C and holding for 2 min. Volatile compounds were identified by comparing the obtained mass spectra of the analytes
with those of authentic standards from the NIST and Wiley libraries
2.3.
Cell culture
Mouse macrophage RAW 264.7 cells were cultured in DMEM supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin and 100 µg/mL streptomycin. Cells was cultured at 37°C, 5% CO2 in a humidified incubator. The essential oils were dissolved in dimethyl sulfoxide (DMSO) to make a 60 mg/ml stock solution and the working dosage was freshly prepared in the basal medium with a final DMSO concentration of less than 0.5%.
2.4.
Cell viability test
The cells were cultured in triplicate at a density of 2×104 cells/well in 96-well plates and incubated overnight, then treated respectively with AAEO (270, 90, 30, 10 μg/mL) at the present of 1 μg/mL LPS, and further incubated for 16 h. At 4 h prior to culture termination, 20 μL of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl -tetrazolium bromide (MTT) solution (5 mg/mL in a phosphate-buffered saline, pH 7.4) was added and the cells were continuously cultured until termination. The medium was aspirated, and the formazan crystals were dissolved in 150 μL of DMSO for 15min. The optical density at 490 nm was measured by microplate reader (Tecan Sunrise, Switzerland).
2.5.
NO and PGE2 assays
RAW264.7 cells in 10% FBS-DMEM without phenol red were plated in 96-well plates (5×104 cells/well) and incubated for 16 h. The cells were treated with different concentrations of samples for 30 min, followed by treatment with LPS (1 μg/ml) for an additional 16 h. Then the media were collected and analyzed for nitrite accumulation as an indicator of NO production by the Griess reaction. Briefly, 100 μl of Griess reagent (Promega, Madison, WI) was added to 50 μl of each cell culture medium in 96-well plates. After incubated at room temperature in dark for 10 min, the absorbance was measured at 540 nm and nitrite concentration was determined by a sodium nitrite standard curve. The collected medium from each well was also analyzed for PGE2 using an enzyme immunoassay kit according to the manufacturer’s instructions (Cayman Chemical Co., Ann Arbour, MI.).
2.6.
Determination of ROS Generation
The level of intracellular ROS was determined by the change in fluorescence resulting from the oxidation of the fluorescent probe H2DCFDA. Briefly, serum-starved macrophages (5×104 cells/well in a 96 well-plate) were exposed to AAEO for 30 min, then cells were incubated with LPS (1 μg/ml) as an inducer of ROS production at 37°C for 16 h. Subsequently, cells were incubated with 50 μM H2DCFDA for 1 h at 37°C in the dark. The fluorescence was determined on a SpectraMax M5 plate reader (Molecular Devices, Sunnyvale, Calif) at 535 nm with excitation at 485 nm.
2.7.
Cytokine assays
The concentrations of TNF-α, IL-6, IL-10, IFN-β and MCP-1 in the cell supernatants were measured by enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions.
2.8.
Western blot analysis
After LPS stimulation for indicated times, the cells were collected and washed twice with cold PBS. Cells were lysed in Laemmli sample buffer (containing 5% 2-mercaptoethanol and protease inhibitor cocktail) and heated at 95°C for 5 min. Cell lysates were centrifuged at 12,000 g for 10 min at 4°C. Cytoplasmic and nuclear proteins were prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents based on the manufacturer's instruction (Pierce, Rockford, IL). Protein concentration was determined through BCA method. The proteins were separated by SDS-PAGE on 8-12% gels and transferred to nitrocellulose membranes. The membranes were blocked with 5% nonfat milk in TBST buffer (150 mM NaCl, 20 mM Tris-HCl, and 0.02% Tween 20, pH7.4) for 1 h, and then incubated over night at 4°C with primary anti-bodies, washed with TBST for three times, and incubated with appropriate HRP-conjugated secondary antibodies at room temperature for 1 h. After washed three times with TBST, the membranes were developed using Western Lightning Plus-ECL (Perkin-Elmer, Waltham, MA, USA) for 1 min and was visualized using the FluorChem Q Imaging System (Protein Simple, Santa Clara, CA). β-actin was used as a loading control for whole-cell protein. PARP (a nuclear protein) and α-tubulin (a
cytosolic protein) were used as loading controls for nuclear and cytosolic proteins, respectively. Relative expression of proteins over loading controls was measured using ImageJ software (NIH, USA).
2.9.
Real-time PCR Analysis
The total RNA was isolated using a High Pure RNA Isolation Kit (Roche, Mannheim, Germany), and first-strand cDNA was synthesized from 1 μg of total RNA by SuperScriptTM II Reverse Transcriptase (Invitrogen). PCR was performed using selective primers for the mouse iNOS (5’-GGCAGCCTGTGAGACCTTTG-3’ and
5’-GCATTGGAAGTGAAGCGTTTC-3’), COX-2
(5’-CCAGAGCAGAGAGATGAAA-3’ and 5’-GGTACAGTTCCATGACATC-3’), and β-actin (5’-TGGAATCCTGTGGCATCCATGAAAC-3’ and 5’-TAAAACGCAGCTCAGTAACAGTCCG-3’) with a T100 Thermal Cycler (Bio-Rad, Hercules, CA, USA). Amplification conditions were as follows: 95°C initial denaturation for 5 min followed by 39 cycles of 95°C for 15 s and 60°C for 30 s. Data were processed using the ΔΔCT method. β-actin served as an internal reference gene.
2.10. Assay of iNOS and COX-2 enzyme activity The inhibitory activities of AAEO on iNOS and COX-2 were measured, respectively, using Nitric Oxide Synthase (NOS) Inhibitor Screening Kit (BioVision) and COX (ovine) Inhibitor Screening Assay kit (Cayman Chemicals) according to the
manufacturer’s instructions.
2.11. Experimental animals Male C57 BL/6 mice (eight weeks old, 25–30 g) were acclimatize at least 4 days prior to experiments under the conditions of 21±2°C, 55–60% relative humidity and 12 h light/dark cycle. They were fasted for 12 h before the experiment with free access to water. Studies were performed according to the guidelines of the Institutional Animal Care and Use Committee of Hubei University of Chinese Medicine.
2.12. TPA-induced ear edema in mice 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear edema was performed according to a previously reported method (Inoue et al. 1989). TPA (2.0 μg) dissolved in acetone (20 μL) was topically applied to the inner and outer surfaces of the right ear of mice. Vehicle, AAEO (750, 250, 83 mg/kg), or indomethacin (IMC, 20 mg/kg) was given orally administration 30 min before the TPA application. The mice were sacrificed by cervical dislocation after 4 h, and ear biopsies were collected with a punch (diameter of 4.5 mm) and weighed. Edema ratio = [(weight of right ear punch– weight of left ear punch)/ weight of left ear punch]
2.13. Histological and Immunohistochemical analysis Histopathologic analysis was performed according to standard protocols. Ear
biopsies from each group were collected and fixed in10% neutral-buffered formalin, embedded in paraffin, cut in to 5 μm sections, and stained with hematoxylin eosin (H&E). Stained tissue sections were analyzed by a blinded pathologist using light microscopy to assess the inflammatory response. The immunohistochemical study was performed as previously reported with minor modifications (Chiang et al., 2004). Tissue sections were deparaffinized in xylene and rehydrated in graded alcohols. Antigenic retrieval was performed by pressure cooking in 10 mM citric acid buffer (pH 6.0). Hydrogen peroxide (3%) was used to quench endogenous peroxidase activity for 10 min. For blocking buffer, 10% normal goat serum was used for 30 min. Sections were then incubated with primary antibody for 1 hour at room temperature. Biotinylated goat anti-rabbit IgG antibody and ABC solution (Vector Laboratories, Burlingame, CA, USA) were applied sequentially. Diaminobenzidine (DAB) was used to visualize a positive signal. Immunohistograms were taken with an Olympus DP-70 camera on an Olympus IX51 microscope.
2.14. Statistical analysis All results were expressed as mean±SD. The differences between LPS treated group with control were analyzed by Student’s t-test. Comparison of different concentrations of AAEO treatment with LPS treated groups were analyzed by one-way ANOVA followed by a Dunnett post-hoc test using Graphpad Prism 6 and considered significantly different at p < 0.05.
3. Results 3.1. GC-MS analysis of AAEO The chemical composition of the essential oil was analyzed by GC/MS as shown in Fig. 1. Seventeen components including monoterpenes, sesquiterpenes, alcohols, and aromatic compounds were identified (Tab. 1), which account for the 95.7% of the total content, and the major components were found to be cineole (33.4%), camphor (16.6%), α-(−)-thujone (12.9%) and borneol (12.8%).
Fig. 1. GC-MS analysis of the essential oil of A. argyi Table 1. Chemical composition of essential oil of A. argyi Rt(min)
Compound
mf
Peak area (%)
6.28 7.16 8.52 8.62 8.82 9.55 10.08 11.16 11.39 11.74 12.90 13.69
Camphene 1-Octen-3-ol o-Cymene (+)-Dipentene Cineole γ-Terpinene Cyclohexanol cis-4-(isopropyl)-1-methylcyclohex-2-en-1-ol α-(−)-Thujone (+)-Thujone Camphor (−)-Borneol
C10H16 C8H16O C10H14 C10H16 C10H18O C10H16 C6H12O C10H18O C10H16O C10H16O C10H16O C10H18O
1.29 1.02 1.26 2.03 33.42 1.39 3.00 1.51 12.92 1.04 16.59 12.77
13.89 14.44 15.26 17.29 21.59
Terpinen-4-ol α-Terpineol (E)-Carveol Bornyl acetate β-Caryophyllene
C10H18O C10H16 C10H16O C12H20O2 C15H24
2.82 1.45 0.53 0.72 1.93
3.2. Inhibitory activity of AAEO on NO, PGE2 and ROS production in RAW264.7 cells induced by LPS The effects of AAEO on NO and PGE2 production in LPS-induced RAW 264.7 cells were examined. After treating cells with LPS (1 μg/ml) for 16 h, NO and PGE2 concentrations in the medium were elevated considerably by over 40 and 120 fold, respectively. In the cells treated with different concentrations of the samples together with LPS, a significant inhibition on NO, PGE2 and ROS productions were detected in a dose-dependent manner (Fig. 2B and C). In the assay, an over 50% inhibition of NO production was shown at 270 μg/ml, and more remarkable inhibitions were observed in PGE2 production, which showed an over 70% inhibition at 10 μg/ml and complete inhibition at 90 μg/ml. AAEO also significantly inhibited the LPS induced intracellular production of ROS in a dose-dependent manner, with a maximum inhibitory ratio of 86.0% at 270 μg/ml, as shown in Fig. 2D. No significant effect on cell viability was observed at the each concentration of AAEO in MTT assay (Fig. 2A), indicating that the inhibition by AAEO was not mediated by cytotoxicity.
Fig. 2. Effects of AAEO on the viability (A) and production of nitrite (B), PGE2 (C) and ROS (D) in LPS-stimulated RAW 264.7 macrophages. Cells were treated with the indicated concentration of AAEO for 0.5 h followed by the addition of LPS (1μg/ml) for 16 h. Data are expressed as Mean ± SD from a triplicate experiments (###p < 0.001 vs. control, **p < 0.01 and ***p < 0.001 vs. LPS alone).
3.3. Inhibitory effect of AAEO on cytokines in RAW264.7 induced by LPS Results displayed that the levels of TNF-α, IL-6, IL-10, IFN-β and MCP-1 were dose-dependently inhibited by AAEO (Fig. 3A-E). LPS (1 μg/ml) promoted the generation of these cytokines tremendously (p < 0.01), and the treatment of AAEO could significantly inhibit them even at the lowest concentration (10 μg/ml) with the inhibitory ratios of 17.8%, 70.6%, 41.4%, 15.5% and 30.0%, respectively. The inductions of IL-6 and MCP-1 were almost completely suppressed at the highest concentration (270 μg/ml), however, the inhibition on TNF-α was relatively moderate, which could reach only up to 33.8%.
Fig. 3. Effect of AAEO on the production of TNF-α (A), IL-6 (B), IL-10 (C), INF-β (D) and MCP-1 (E) in LPS-stimulated RAW 264.7 macrophages. Cells were treated with the indicated concentration of AAEO for 0.5 h followed by the addition of LPS (1 μg/ml) for 16 h. Data are expressed as Mean ± SD from a triplicate experiments (###p < 0.001 vs. control, **p < 0.01 and ***p < 0.001 vs. LPS alone).
3.4. Inhibitory effect of AAEO on LPS-induced iNOS and COX-2 mRNA and protein expression in RAW264.7 cells As opposed to the inactivated cells, iNOS and COX-2 mRNA and protein expressions substantially increased in LPS-induced RAW 264.7 macrophages. AAEO dose-dependently suppressed the iNOS mRNA expression induced by LPS. However,
only the highest dose (270 μg/ml) can significantly decrease COX-2 mRNA expression (Fig. 4A and B). Furthermore, The COX-2 and iNOS protein expressions were determined by Western blotting. Fig. 4C and D showed that LPS-stimulated iNOS expression was effectively inhibited by AAEO treatment (24% and 97% inhibition) at 90 and 270 μg/ml, whereas the protein expression of COX-2 was only decreased obviously at the highest concentration (270 μg/ml), which was consistent with that in mRNA expression.
Fig. 4. Effect of AAEO on iNOS and COX-2 mRNA and protein expressions in LPS-stimulated RAW 264.7 macrophages. (A, B) Cells were treated with the indicated concentration of AAEO for 0.5 h followed by LPS stimulation (1μg/ml) for 6 h. Total RNA was extracted for RT-PCR analysis. (C, D) Cells were treated with the indicated concentration of AAEO for 0.5 h followed by LPS stimulation (1μg/ml) for 16 h. The protein levels were assessed by western blot analysis. β-actin was served as internal control for normalization of mRNA and protein expression. Data are expressed as Mean ± SD from three independent experiments (###p < 0.001 vs. control, **p < 0.01 and ***p < 0.001 vs. LPS alone).
3.5. Inhibitory activity of AAEO on iNOS and COX-2 enzyme activity In the enzymic inhibitor screening assay, AAEO were tested for its ability to inhibit the enzyme activities of iNOS and COX-2. 1400W and indomethacin were used as positive controls for the two enzymes, respectively. AAEO did not show significant inhibitory effects against both enzymes over the concentration range tested (10-270 μg/ml).
3.6. Effect of AAEO on MAPK/NF-κB/AP-1 pathway in RAW264.7 cells induced by LPS Several signaling pathways known to mediate inflammation including MAPKs and NF-κB were investigated to explore the possible molecular mechanisms underlying the anti-inflammatory effect of AAEO. JNK, p38 and ERK1/2, the three key proteins in MAPK signaling pathway were examined. As phosphorylation is required for the activation of MAPKs, phospho-specific MAPK antibodies were used in Western blotting analysis. After the stimulation of LPS for 30 min, the phospho-MAPK proteins were dramatically increased in RAW264.7 cells, however, no apparent inhibition on the phospho-MAPK proteins was observed in AAEO co-treated cells at all concentrations tested. NF-κB subunits (p65 and/or p50) are normally sequestered in the cytosol as an inactive complex by binding to repressor IκB-α in non-stimulated cells. Upon the stimulation of LPS, IκB-α is phosphorylated by IκB kinase (IKK) and inactivated by degradation, and then free NF-κB is translocated into the nucleus where it induces the expression of multiple inflammatory genes (Akira and Takeda,
2004).We further examined the distribution of the NF-κB p65 subunit in the cytosolic and nuclear extracts of RAW264.7 cells, and revealed that the amount of p65 in the nucleus increased after stimulation with LPS for 1 h, however, AAEO treatment did not reduce nuclear localization of the p65 protein. Detection of AP-1 subunit (c-Jun) was also performed using specific antibodies and no effect was found on AAEO treated cells. The detection of PARP and α-tubulin confirmed that little cross-contamination existed during the extraction of the two fractions (data not shown).
3.7. Effect of AAEO on phosphorylation of JAK2 and STAT1/3 in RAW264.7 cells induced by LPS JAK/STATs signaling participates in the regulation of genes involved in the acute primary response to infection and inflammation response (Stark and Darnell, 2012). Examining the effects of AAEO on this pathway may help in gaining further insight into the mechanism of AAEO in controlling inflammation. Multiple time points from 1 to 16 h were examined, and 8 to 16 h was thought as suitable time for AAEO to exert the efficacy (Fig. 5A). AAEO can dose-dependently inhibit the phosphorylation of STAT1 (Tyr701), STAT3 (Tyr705) and the upstream JAK2 in LPS-activated RAW264.7 cells, without affecting the total levels of JAK and STATs protein (Fig. 5B,C).
Fig. 5. Effect of AAEO on the LPS-induced activation of the JAK/STATs pathway in RAW 264.7 cells. (A) RAW264.7 cells were pretreated with AAEO for the indicated time periods and stimulated with LPS. (B, C) Cells were treated with AAEO (10, 30, 90, 270 μg/mL) for 16 h followed by the stimulation of LPS (1 μg/mL) for 1 h. Total protein was extracted and analyzed by Western blot using specific antibodies. β-actin was used as the internal control for normalization. Data are expressed as Mean ± SD from three independent experiments (**p < 0.01 and ***p < 0.001 vs. LPS alone)
3.8. Inhibitory effect of AAEO on TPA-induced ear edema in mice To evaluate the anti-inflammatory activity of AAEO in vivo, its effect against TPA-induced ear edema in mice was tested. Four hours’ exposure to TPA resulted in distinct increases in skin thickness (Fig. 6A). As expected, the dose-dependent inhibition of ear edema by AAEO was observed, with a marked effect even at the lowest dose (41% inhibition at 83 mg/kg). The positive control indomethacin also
produced an inhibition up to 84%, as compared with TPA-alone group. The histological analysis of the sections of mice ears were conducted by both hematoxylin and eosin staining and immunohistochemistry, as illustrated in Fig. 6B, TPA resulted in marked increases in skin thickness with clear evidence of edema, epidermal hyperplasia, substantial neutrophil infiltration, and connective tissue disruption. AAEO treatment reduced ear thickness and the associated pathological indicators to an extent comparable with indomethacin. The effects of AAEO on TPA-induced COX-2 expression in mouse skin were analyzed by both immunohistochemical and Western blot analysis. COX-2 expression increased dramatically in the epidermal layer upon TPA treatment for 4 h, and was significantly abolished by AAEO (Fig. 6C, D).
Fig. 6. Effect of orally administered AAEO on TPA-induced ear thickness (A), histological lesion (B) and COX-2 expression (C, D). Ears were treated with acetone vehicle, TPA plus either normal saline, AAEO (83, 250, 750 mg/kg), or IMC for 30 min and then treated with TPA for 4 h before harvest. Skin punch biopsies were harvested for thickness measurement. Protein was analyzed for COX-2 by western blotting. Paraffin-embedded tissues were subjected to hematoxylin and eosin staining for histological analysis or immunostained by specific COX-2 protein (representative sections of 250mg/kg group are shown in group C). Data are expressed as Mean ± SD (n=5, **p < 0.01 and ***p < 0.001 vs. TPA alone).
4. Discussion A. argyi was used as a traditional remedy for various diseases in Asia, modern studies have proved its modulatory effects on the inflammation process such as rhinitis, dermatitis and arthritis, furthermore, preparation of the essential oil has been clinically prescribed for chronic bronchitis in China for years (Miao et al., 2012, Jiang et al., 2016). GC-MS analysis in this study provided detailed chemical composition of AAEO, in which several predominant chemicals including cineole, borneol, camphor, and thujone were found to possess anti-inflammatory activities according to previous reports (Ehrnhöfer-Ressler, et al., 2013; Miguel, 2010). These reports imply that AAEO may be associated with anti-inflammatory properties due to the presence of the
above components. To assess this herbal medicine as a potential anti-inflammatory agents, the ability of AAEO to modulate inflammation was confirmed in vitro and in vivo, and the molecular mechanism of the activity were further investigated by using macrophage-mediated inflammatory responses and TPA-induced mouse ear edema model. The present results clearly demonstrated that AAEO could significantly suppress the production of NO and PGE2, and intensively inhibited IL-6, IL-10, IFN-β and MCP-1, but only have a relatively slight inhibition on TNF-α in LPS-stimulated RAW 264.7 macrophages. Consistent with the effects observed in vitro, AAEO could markedly ameliorate TPA-induced mouse ear edema in animal experiments, which implied it has anti-inflammatory activity in vivo. By interacting with Toll-like receptor 4 (TLR4), LPS activates intracellular signaling pathways, including MAPK and NF-κB pathway, to up-regulate the expressions of various inflammatory mediators, which include NO, PGEs, and pro-inflammatory cytokines (Du et al., 2009; Qi et al., 2013). Among these, two of the most prominent are NO and PGE2, which are the products of the inducible NO synthase (iNOS) and cyclooxygenase 2 (COX-2), respectively (Qi et al., 2013). The interference of AAEO with iNOS and COX-2 expression, as well as enzyme activities were first investigated. In accordance with the inhibition on NO production, AAEO of 30-270 μg/ml markedly inhibited the iNOS mRNA and protein expression in LPS-induced macrophages. However, it did not display inhibitory effects on both mRNA and protein expression of COX-2 until the dose was elevated to 270 μg/ml. We believe that AAEO contains some components with weak activities against
COX-2 gene expression, which effect becomes apparent at higher dosage. In addition, AAEO exhibited little effects on the enzyme activities of iNOS and COX-2. These findings indicated that AAEO inhibited LPS-induced NO and PGE2 production through the suppression of iNOS and COX-2 at transcriptional level in different degrees. The lack of effects on COX-2 regulation is somewhat surprising when compared to the inhibition on PGE2 production. The reasons for this discrepancy are unclear, but could be due to some terpenes such as cineole suppressing the release of arachidonic acid from the phospholipid stores of cells, which directly reduced the synthetic precursor of PGE2 (Juergens, 1999). The expressions of iNOS and COX-2 in macrophages are regulated by multiple signaling pathways associated with inflammation, mostly through the activation of MAPK and NF-κB cascades. The MAPK pathways, which are extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38 MAPK pathways in particular (Herlaar and Brown, 1999), also regulate the synthesis of inflammation mediators at the level of transcription and translation through activation of NF-κB and AP-1 transcription factors (Kaminska, 2005). However, Western blot assay revealed the increased phosphorylation of MAPKs including JNK, p38, and ERK1/2, which were not affected by AAEO at the tested concentrations. NF-κB and AP-1 are major transcription factors to regulate the expressions of pro-inflammatory enzymes and cytokines such as iNOS, COX-2, TNF-α, IL6, IFN-β and MCP-1 (Chiang et al., 2005; Choi et al., 2011). The appearance of the p65 subunit (Rel A) in the cytosolic and nuclear extracts of cells was monitored, and equal levels of p65
were observed in the nuclear extracts, indicating that the translocation of p65 to the nucleus was not responsive to AAEO. The same phenomenon was observed in c-Jun, the subunit of AP-1. We thus propose that the inhibition of AAEO on cytokine production was not due to the regulation of NF-κB and AP-1 translocation. The JAK/STATs signaling pathway has been implicated in cytokine-mediated gene expression in immune responses. Cytokines such as IL6 or IFN-β binding to their receptors initiate the cascade, which activates the phosphorylation of JAKs and further phosphorylates STATs. Phosphorylated STATs are released and translocate into the nucleus to regulate the transcription of target genes including iNOS and COX-2 (Yu et al., 2009; Kou et al., 2011). Some evidences have proved that the JAK/STATs pathway is crucial in LPS-induced iNOS expression (Okugawa et al., 2003), for NF-κB alone is insufficient for the optimal induction of the iNOS genes (Ganster et al., 2001). The activation of STAT1 by IFN-β production was required for the expression of iNOS in LPS-stimulated macrophages (Ohmori and Hamilton, 2001; Samardzic, 2001), while STAT3 is crucial for regulating the expression of pro-inflammatory cytokines and chemokines such as IL-1β, IL-6 and MCP-1. Meanwhile, the anti-inflammatory cytokine IL-10, which performs an irreplaceable role in negatively regulating inflammation, is also transcriptionally regulated by STAT3 (Benkhart et al., 2000). LPS-induced STAT1/3 activation leads to the productions of several cytokines including IFN-β, IL-6 and IL-10, which in turn augment the JAK/STATs signaling. The cytokine suppression supported our hypothesis that by blocking the production of IFN-β, IL6 and IL-10, AAEO treatment
negatively influenced the phosphorylated protein levels of STAT1/3, and the phosphorylation of JAK2, signaling pathway upstream of STATs in LPS-activated RAW264.7 cells as well. These results indicated that down-regulation of JAK/STAT signaling activation is a possible mechanism for AAEO to inhibit LPS-induced iNOS and COX-2 gene expression as well as the production of some pro-inflammatory cytokines and chemokines in macrophages. Moreover, since essential oil possesses antioxidant activity for scavenging ROS (Miguel, 2010, Huang et al., 2012), and the production of ROS is a key step involved in JAK/STATs activation in macrophages (Simon et al., 1998), it is plausible that AAEO inhibits JAK/STATs signaling by ROS scavenging. It is of interest to note that AAEO dose-dependently reduced LPS-induced ROS production in RAW264.7 macrophages, which implicates the inhibitory effect of AAEO on JAK/STATs might be attributed to its antioxidant activity toward ROS. AAEO has been reported to attenuate inflammation responses in different animal models (Ge et al., 2016; Miao et al., 2012; Jiang et al., 2016). TPA-induced ear edema model is an ideal model for screening drugs with anti-inflammatory potential. Topical application of TPA induced an acute inflammatory response in mouse ear with massive
edema
formation,
and
AAEO
markedly
attenuated
TPA-induced
inflammation in a dose-dependent manner, with percentage of inhibition up to 84%, which was comparable with the positive control indomethacin. Consistent with the effect in vitro, AAEO suppressed COX-2 expression in the ears from the mice exposed to TPA (Fig.6C, D), indicating that it may be beneficial for COX-2-related
cutaneous inflammation. In conclusion, our results demonstrated that AAEO exert anti-inflammatory activity by inhibiting the production of inflammatory mediators, including NO, PGE2, TNF-α, IL-6, etc. The anti-inflammatory mechanism of AAEO may be related to negative regulation of JAK/STATs and ROS scavenging, diminished the protein and mRNA expression levels of iNOS and COX-2, rather than MAPK and NF-κB pathway (Fig. 7). These findings provide a new molecular insight into the mechanism of AAEO on inflammation and suggest it can be developed into a novel therapeutic agent for inflammatory-related diseases.
Fig. 7. Schematic diagram of the mechanisms underlying the suppression of AAEO on LPS-mediated inflammatory response.
Acknowledgement This work was supported by New products of TCM Senile Diseases Co-Innovation
Center of Hubei.
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PII: DOI: Reference:
S0378-8741(16)31824-4 http://dx.doi.org/10.1016/j.jep.2017.04.017 JEP10827
To appear in: Journal of Ethnopharmacology Received date: 10 November 2016 Revised date: 16 April 2017 Accepted date: 20 April 2017 Cite this article as: Lin-Lin Chen, Hao-Jun Zhang, Jung Chao and Jun-Feng Liu, Essential oil of Artemisia argyi suppresses inflammatory responses by inhibiting JAK/STATs activation, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2017.04.017 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Essential oil of Artemisia argyi suppresses inflammatory responses by inhibiting JAK/STATs activation
Lin-Lin Chen1*, Hao-Jun Zhang2, Jung Chao3, Jun-Feng Liu1
1
Key Laboratory of Traditional Chinese Medicine Resource and Compound Prescription, Ministry
of Education, Hubei University of Chinese Medicine, Wuhan 430065, PR China 2
Department of Pharmacology, China-Japan Friendship Hospital, Beijing 100029, China
3
Department and Institute of Pharmacology, National Yang-Ming University, Taipei 112, Taiwan
*
Corresponding author at: Key Laboratory of Traditional Chinese Medicine Resource and
Compound Prescription, Ministry of Education, Hubei University of Chinese Medicine, 1 Huang-Jia-Hu
West
Road,
Wuhan
430065,
PR
China.
Tel.:
+86
027
68890247;
[email protected] (L.-L. Chen)
Abstract Ethnopharmacological relevance Artemisia argyi is a herbal medicine traditionally used in Asia for the treatment of bronchitis, dermatitis and arthritis. Recent studies revealed the anti-inflammatory effect of essential oil in this plant. However, the mechanisms underlying the
therapeutic potential have not been well elucidated. The present study is aimed to verify anti-inflammatory effect and investigate the probable mechanisms. Materials and methods The essential oil from Artemisia argyi (AAEO) was initially tested against LPS-induced production of inflammatory mediators and cytokines in RAW264.7 macrophages. Protein and mRNA expressions of iNOS and COX-2 were determined by Western blotting and RT-PCR analysis, respectively. The effects on the activation of MAPK/NF-κB/AP-1 and JAK/STATs pathway were also investigated by western blot. Meanwhile, in vivo anti-inflammatory effect was examined by histologic and immunohistochemical analysis in TPA-induced mouse ear edema model. Results The results of in vitro experiments showed that AAEO dose-dependently suppressed the release of pro-inflammatory mediators (NO, PGE2 and ROS) and cytokines (TNF-α, IL-6, IFN-β and MCP-1) in LPS-induced RAW264.7 macrophages. It down-regulated iNOS and COX-2 protein and mRNA expression but did not affect the activity of these two enzymes. AAEO significantly inhibited the phosphorylation of JAK2 and STAT1/3, but not the activation of MAPK and NF-κB cascades. In animal model, oral administration of AAEO significantly attenuated TPA-induced mouse ear edema and decreased the protein level of COX-2. Conclusion AAEO suppresses inflammatory responses via down-regulation of the JAK/STATs signaling and ROS scavenging, which could contribute, at least in part, to the
anti-inflammatory effect of AAEO.
Keywords: Artemisia argyi; Anti-inflammatory; JAK/STATs
Chemical compounds studied in this article: cineole (PubChem CID: 2758), camphor (PubChem CID: 2537), α-(−)-thujone (PubChem CID: 261491), borneol (PubChem CID: 64685), indomethacin (PubChem CID: 3715), 1400W (PubChem CID: 1433)
1. Introduction Inflammation is a host defense mechanism which is considered as a part of innate immune system. The bacterial or viral invasions trigger inflammatory responses by the activation of immune cells such as macrophages, neutrophils, and dendritic cells. Macrophages play a pivotal role in the regulation of innate immune response, which is vital for the recognition and elimination of invasive microbial pathogens (Roger et al., 2001). The activated cells produces various cytokines, chemokines, and inflammatory mediators [including nitric oxide (NO), prostaglandin (PG) E2, reactive oxygen species (ROS), tumor necrosis factor (TNF)-α, interferons (IFNs), monocyte chemoattractant protein (MCP)-1 and interleukins ] (Ramana et al., 2006; Choi et al., 2011), which leads to an increase in intracellular signaling and chemotactic responses of other inflammatory cells, thereby protects the body against infection. Although the pro-inflammatory cytokines and mediators play essential roles in the regulation of
immune response, dysregulation of their production can cause damage to the host and lead to a variety of acute and chronic disorders, including cancer, diabetes, septic shock, autoimmune diseases, and atherosclerosis (Tabas and Glass, 2013). The strategies for anti-inflammation are to negatively modulate the expression of genes or activity of enzymes involved in inflammatory responses, thus affects the release of pro-inflammatory cytokines. Therefore, intracellular signaling such as mitogen activated protein kinases (MAPK) and some related transcription factors, including nuclear factor-κB (NF-κB) and activator protein (AP)-1, have been regarded as target molecules in anti-inflammatory drug development. Traditional non-steroidal anti-inflammatory drugs (NSAIDs) are widely used to treat inflammatory disorders, but the gastrointestinal, renal and cardiovascular side effects limit their long term use (Harirforoosh et al., 2014). Actually the continued search for more effective and safe anti-inflammatory agents which work on different targets never stops. As plenty of plant species were found to contain various compounds exhibiting anti-inflammatory effects (Gautam and Jachak., 2009), it is becoming a new trend for botanicals used as an alternative to conventional chemotherapy in the treatment of inflammatory diseases. Artemisia argyi is a herbaceous plant distributed in most regions of East Asia. It has been widely used in traditional Chinese medicine for treating eczema, hemorrhage, and dysmenorrhea (Chinese Pharmacopoeia, 2015). Dried leaves of this herb are also used in acupuncture clinics in China to treat various diseases, especially chronic conditions such as osteoarthritis, asthma, gastrointestinal disorders, and insomnia (Ge
et al., 2016). A. argyi is reported to possess multiple pharmacological activities including
anti-inflammatory,
antimicrobial,
antiasthma,
analgesic,
antivirus,
antioxidant, antitumor and immunomodulatory effects (Jiang et al., 2005; Ge et al., 2016; Huang et al., 2012; Bao et al., 2013;), most of which are contributed by the essential oil existing in A. argyi leaves. Though the anti-inflammatory activity of Artemisia genus has been studied intensively and several anti-inflammatory compounds were also found in this plant (Moscatelli et al., 2006; Min et al., 2009; Choi et al., 2013; Habib and Waheed, 2013; Jeong et al., 2014; Park et al., 2015; Zeng et al., 2014), there is no much substantial evidence supporting the anti-inflammatory property of this herbal medicine and the molecular targets for the effects still remain unclear, which limited its application into clinical practice. Therefore this study was aimed to evaluate the anti-inflammatory potential of the essential oil of A. argyi (AAEO), and investigate the underlying mechanisms by lipopolysaccharide (LPS)-stimulated murine macrophages and acute in vivo inflammatory model in mice.
2. Materials and methods 2.1. Antibodies and reagents The mouse monoclonal antibodies against iNOS, COX-2, p65, c-Jun, JNK, phospho-JNK, ERK1/2, phospho-ERK1/2, p38, phospho-p38, STAT1, phospho -STAT1, STAT3, phospho-STAT3, JAK2, phospho-JAK2, β-actin, PARP and α-tublin were purchased from Cell Signaling Technology (Beverly, MA, USA). The DMEM medium and fetal bovine serum (FBS) was purchased from GIBCO (Grand Island,
NY). Unless otherwise indicated, all other reagents were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). The essential oil of Artemisiae Argyi folium (batch number 201406001) was provided by Hubei Lishizhen Medicine Group Co., Ltd. (Qichun, China), which was obtained from the leaves of A. argyi collected in Qichun County, Hubei Province. Voucher specimens (QC140531-2) are deposited in Hubei Lishizhen Medicine Group Co., Ltd.
2.2.
Gas chromatography-mass spectrometry (GC-MS) analysis of AAEO
The essential oils were obtained from partially crushed leaves of A. argyi by hydrodistillation with a Clevenger apparatus for 5 h as recommended by Chinese Pharmacopoeia. The oil obtained was subsequently dried over anhydrous sodium sulphate, sealed and placed in the dark under refrigeration until used. GC/MS analysis of the essential oil was carried out on a GC-MS (Thermo Trace GC Ultra-ISQ mass spectrometer) equipped with a TR-5MS capillary column (30 m × 0.2 mm, 0.25 μm, Thermo, USA) in the electron impact mode (Ionization energy: 70 eV), and data were recorded in the full-scan acquisition mode with the range of m/z 40–500. The carrier gas was helium at a flow rate of 1.0 mL min−1. Split injection (1 μl) was conducted with a split ratio of 1:20. The injector temperature was set at 260°C. The ion source temperature of mass spectrometer was 200°C. The temperature program was started at 60°C, remaining at this temperature for 2 min, and heated at 5°C/min to 170°C, then held for 2 min, followed by 20°C/min to 250°C and holding for 2 min. Volatile compounds were identified by comparing the obtained mass spectra of the analytes
with those of authentic standards from the NIST and Wiley libraries
2.3.
Cell culture
Mouse macrophage RAW 264.7 cells were cultured in DMEM supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin and 100 µg/mL streptomycin. Cells was cultured at 37°C, 5% CO2 in a humidified incubator. The essential oils were dissolved in dimethyl sulfoxide (DMSO) to make a 60 mg/ml stock solution and the working dosage was freshly prepared in the basal medium with a final DMSO concentration of less than 0.5%.
2.4.
Cell viability test
The cells were cultured in triplicate at a density of 2×104 cells/well in 96-well plates and incubated overnight, then treated respectively with AAEO (270, 90, 30, 10 μg/mL) at the present of 1 μg/mL LPS, and further incubated for 16 h. At 4 h prior to culture termination, 20 μL of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl -tetrazolium bromide (MTT) solution (5 mg/mL in a phosphate-buffered saline, pH 7.4) was added and the cells were continuously cultured until termination. The medium was aspirated, and the formazan crystals were dissolved in 150 μL of DMSO for 15min. The optical density at 490 nm was measured by microplate reader (Tecan Sunrise, Switzerland).
2.5.
NO and PGE2 assays
RAW264.7 cells in 10% FBS-DMEM without phenol red were plated in 96-well plates (5×104 cells/well) and incubated for 16 h. The cells were treated with different concentrations of samples for 30 min, followed by treatment with LPS (1 μg/ml) for an additional 16 h. Then the media were collected and analyzed for nitrite accumulation as an indicator of NO production by the Griess reaction. Briefly, 100 μl of Griess reagent (Promega, Madison, WI) was added to 50 μl of each cell culture medium in 96-well plates. After incubated at room temperature in dark for 10 min, the absorbance was measured at 540 nm and nitrite concentration was determined by a sodium nitrite standard curve. The collected medium from each well was also analyzed for PGE2 using an enzyme immunoassay kit according to the manufacturer’s instructions (Cayman Chemical Co., Ann Arbour, MI.).
2.6.
Determination of ROS Generation
The level of intracellular ROS was determined by the change in fluorescence resulting from the oxidation of the fluorescent probe H2DCFDA. Briefly, serum-starved macrophages (5×104 cells/well in a 96 well-plate) were exposed to AAEO for 30 min, then cells were incubated with LPS (1 μg/ml) as an inducer of ROS production at 37°C for 16 h. Subsequently, cells were incubated with 50 μM H2DCFDA for 1 h at 37°C in the dark. The fluorescence was determined on a SpectraMax M5 plate reader (Molecular Devices, Sunnyvale, Calif) at 535 nm with excitation at 485 nm.
2.7.
Cytokine assays
The concentrations of TNF-α, IL-6, IL-10, IFN-β and MCP-1 in the cell supernatants were measured by enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions.
2.8.
Western blot analysis
After LPS stimulation for indicated times, the cells were collected and washed twice with cold PBS. Cells were lysed in Laemmli sample buffer (containing 5% 2-mercaptoethanol and protease inhibitor cocktail) and heated at 95°C for 5 min. Cell lysates were centrifuged at 12,000 g for 10 min at 4°C. Cytoplasmic and nuclear proteins were prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents based on the manufacturer's instruction (Pierce, Rockford, IL). Protein concentration was determined through BCA method. The proteins were separated by SDS-PAGE on 8-12% gels and transferred to nitrocellulose membranes. The membranes were blocked with 5% nonfat milk in TBST buffer (150 mM NaCl, 20 mM Tris-HCl, and 0.02% Tween 20, pH7.4) for 1 h, and then incubated over night at 4°C with primary anti-bodies, washed with TBST for three times, and incubated with appropriate HRP-conjugated secondary antibodies at room temperature for 1 h. After washed three times with TBST, the membranes were developed using Western Lightning Plus-ECL (Perkin-Elmer, Waltham, MA, USA) for 1 min and was visualized using the FluorChem Q Imaging System (Protein Simple, Santa Clara, CA). β-actin was used as a loading control for whole-cell protein. PARP (a nuclear protein) and α-tubulin (a
cytosolic protein) were used as loading controls for nuclear and cytosolic proteins, respectively. Relative expression of proteins over loading controls was measured using ImageJ software (NIH, USA).
2.9.
Real-time PCR Analysis
The total RNA was isolated using a High Pure RNA Isolation Kit (Roche, Mannheim, Germany), and first-strand cDNA was synthesized from 1 μg of total RNA by SuperScriptTM II Reverse Transcriptase (Invitrogen). PCR was performed using selective primers for the mouse iNOS (5’-GGCAGCCTGTGAGACCTTTG-3’ and
5’-GCATTGGAAGTGAAGCGTTTC-3’), COX-2
(5’-CCAGAGCAGAGAGATGAAA-3’ and 5’-GGTACAGTTCCATGACATC-3’), and β-actin (5’-TGGAATCCTGTGGCATCCATGAAAC-3’ and 5’-TAAAACGCAGCTCAGTAACAGTCCG-3’) with a T100 Thermal Cycler (Bio-Rad, Hercules, CA, USA). Amplification conditions were as follows: 95°C initial denaturation for 5 min followed by 39 cycles of 95°C for 15 s and 60°C for 30 s. Data were processed using the ΔΔCT method. β-actin served as an internal reference gene.
2.10. Assay of iNOS and COX-2 enzyme activity The inhibitory activities of AAEO on iNOS and COX-2 were measured, respectively, using Nitric Oxide Synthase (NOS) Inhibitor Screening Kit (BioVision) and COX (ovine) Inhibitor Screening Assay kit (Cayman Chemicals) according to the
manufacturer’s instructions.
2.11. Experimental animals Male C57 BL/6 mice (eight weeks old, 25–30 g) were acclimatize at least 4 days prior to experiments under the conditions of 21±2°C, 55–60% relative humidity and 12 h light/dark cycle. They were fasted for 12 h before the experiment with free access to water. Studies were performed according to the guidelines of the Institutional Animal Care and Use Committee of Hubei University of Chinese Medicine.
2.12. TPA-induced ear edema in mice 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear edema was performed according to a previously reported method (Inoue et al. 1989). TPA (2.0 μg) dissolved in acetone (20 μL) was topically applied to the inner and outer surfaces of the right ear of mice. Vehicle, AAEO (750, 250, 83 mg/kg), or indomethacin (IMC, 20 mg/kg) was given orally administration 30 min before the TPA application. The mice were sacrificed by cervical dislocation after 4 h, and ear biopsies were collected with a punch (diameter of 4.5 mm) and weighed. Edema ratio = [(weight of right ear punch– weight of left ear punch)/ weight of left ear punch]
2.13. Histological and Immunohistochemical analysis Histopathologic analysis was performed according to standard protocols. Ear
biopsies from each group were collected and fixed in10% neutral-buffered formalin, embedded in paraffin, cut in to 5 μm sections, and stained with hematoxylin eosin (H&E). Stained tissue sections were analyzed by a blinded pathologist using light microscopy to assess the inflammatory response. The immunohistochemical study was performed as previously reported with minor modifications (Chiang et al., 2004). Tissue sections were deparaffinized in xylene and rehydrated in graded alcohols. Antigenic retrieval was performed by pressure cooking in 10 mM citric acid buffer (pH 6.0). Hydrogen peroxide (3%) was used to quench endogenous peroxidase activity for 10 min. For blocking buffer, 10% normal goat serum was used for 30 min. Sections were then incubated with primary antibody for 1 hour at room temperature. Biotinylated goat anti-rabbit IgG antibody and ABC solution (Vector Laboratories, Burlingame, CA, USA) were applied sequentially. Diaminobenzidine (DAB) was used to visualize a positive signal. Immunohistograms were taken with an Olympus DP-70 camera on an Olympus IX51 microscope.
2.14. Statistical analysis All results were expressed as mean±SD. The differences between LPS treated group with control were analyzed by Student’s t-test. Comparison of different concentrations of AAEO treatment with LPS treated groups were analyzed by one-way ANOVA followed by a Dunnett post-hoc test using Graphpad Prism 6 and considered significantly different at p < 0.05.
3. Results 3.1. GC-MS analysis of AAEO The chemical composition of the essential oil was analyzed by GC/MS as shown in Fig. 1. Seventeen components including monoterpenes, sesquiterpenes, alcohols, and aromatic compounds were identified (Tab. 1), which account for the 95.7% of the total content, and the major components were found to be cineole (33.4%), camphor (16.6%), α-(−)-thujone (12.9%) and borneol (12.8%).
Fig. 1. GC-MS analysis of the essential oil of A. argyi Table 1. Chemical composition of essential oil of A. argyi Rt(min)
Compound
mf
Peak area (%)
6.28 7.16 8.52 8.62 8.82 9.55 10.08 11.16 11.39 11.74 12.90 13.69
Camphene 1-Octen-3-ol o-Cymene (+)-Dipentene Cineole γ-Terpinene Cyclohexanol cis-4-(isopropyl)-1-methylcyclohex-2-en-1-ol α-(−)-Thujone (+)-Thujone Camphor (−)-Borneol
C10H16 C8H16O C10H14 C10H16 C10H18O C10H16 C6H12O C10H18O C10H16O C10H16O C10H16O C10H18O
1.29 1.02 1.26 2.03 33.42 1.39 3.00 1.51 12.92 1.04 16.59 12.77
13.89 14.44 15.26 17.29 21.59
Terpinen-4-ol α-Terpineol (E)-Carveol Bornyl acetate β-Caryophyllene
C10H18O C10H16 C10H16O C12H20O2 C15H24
2.82 1.45 0.53 0.72 1.93
3.2. Inhibitory activity of AAEO on NO, PGE2 and ROS production in RAW264.7 cells induced by LPS The effects of AAEO on NO and PGE2 production in LPS-induced RAW 264.7 cells were examined. After treating cells with LPS (1 μg/ml) for 16 h, NO and PGE2 concentrations in the medium were elevated considerably by over 40 and 120 fold, respectively. In the cells treated with different concentrations of the samples together with LPS, a significant inhibition on NO, PGE2 and ROS productions were detected in a dose-dependent manner (Fig. 2B and C). In the assay, an over 50% inhibition of NO production was shown at 270 μg/ml, and more remarkable inhibitions were observed in PGE2 production, which showed an over 70% inhibition at 10 μg/ml and complete inhibition at 90 μg/ml. AAEO also significantly inhibited the LPS induced intracellular production of ROS in a dose-dependent manner, with a maximum inhibitory ratio of 86.0% at 270 μg/ml, as shown in Fig. 2D. No significant effect on cell viability was observed at the each concentration of AAEO in MTT assay (Fig. 2A), indicating that the inhibition by AAEO was not mediated by cytotoxicity.
Fig. 2. Effects of AAEO on the viability (A) and production of nitrite (B), PGE2 (C) and ROS (D) in LPS-stimulated RAW 264.7 macrophages. Cells were treated with the indicated concentration of AAEO for 0.5 h followed by the addition of LPS (1μg/ml) for 16 h. Data are expressed as Mean ± SD from a triplicate experiments (###p < 0.001 vs. control, **p < 0.01 and ***p < 0.001 vs. LPS alone).
3.3. Inhibitory effect of AAEO on cytokines in RAW264.7 induced by LPS Results displayed that the levels of TNF-α, IL-6, IL-10, IFN-β and MCP-1 were dose-dependently inhibited by AAEO (Fig. 3A-E). LPS (1 μg/ml) promoted the generation of these cytokines tremendously (p < 0.01), and the treatment of AAEO could significantly inhibit them even at the lowest concentration (10 μg/ml) with the inhibitory ratios of 17.8%, 70.6%, 41.4%, 15.5% and 30.0%, respectively. The inductions of IL-6 and MCP-1 were almost completely suppressed at the highest concentration (270 μg/ml), however, the inhibition on TNF-α was relatively moderate, which could reach only up to 33.8%.
Fig. 3. Effect of AAEO on the production of TNF-α (A), IL-6 (B), IL-10 (C), INF-β (D) and MCP-1 (E) in LPS-stimulated RAW 264.7 macrophages. Cells were treated with the indicated concentration of AAEO for 0.5 h followed by the addition of LPS (1 μg/ml) for 16 h. Data are expressed as Mean ± SD from a triplicate experiments (###p < 0.001 vs. control, **p < 0.01 and ***p < 0.001 vs. LPS alone).
3.4. Inhibitory effect of AAEO on LPS-induced iNOS and COX-2 mRNA and protein expression in RAW264.7 cells As opposed to the inactivated cells, iNOS and COX-2 mRNA and protein expressions substantially increased in LPS-induced RAW 264.7 macrophages. AAEO dose-dependently suppressed the iNOS mRNA expression induced by LPS. However,
only the highest dose (270 μg/ml) can significantly decrease COX-2 mRNA expression (Fig. 4A and B). Furthermore, The COX-2 and iNOS protein expressions were determined by Western blotting. Fig. 4C and D showed that LPS-stimulated iNOS expression was effectively inhibited by AAEO treatment (24% and 97% inhibition) at 90 and 270 μg/ml, whereas the protein expression of COX-2 was only decreased obviously at the highest concentration (270 μg/ml), which was consistent with that in mRNA expression.
Fig. 4. Effect of AAEO on iNOS and COX-2 mRNA and protein expressions in LPS-stimulated RAW 264.7 macrophages. (A, B) Cells were treated with the indicated concentration of AAEO for 0.5 h followed by LPS stimulation (1μg/ml) for 6 h. Total RNA was extracted for RT-PCR analysis. (C, D) Cells were treated with the indicated concentration of AAEO for 0.5 h followed by LPS stimulation (1μg/ml) for 16 h. The protein levels were assessed by western blot analysis. β-actin was served as internal control for normalization of mRNA and protein expression. Data are expressed as Mean ± SD from three independent experiments (###p < 0.001 vs. control, **p < 0.01 and ***p < 0.001 vs. LPS alone).
3.5. Inhibitory activity of AAEO on iNOS and COX-2 enzyme activity In the enzymic inhibitor screening assay, AAEO were tested for its ability to inhibit the enzyme activities of iNOS and COX-2. 1400W and indomethacin were used as positive controls for the two enzymes, respectively. AAEO did not show significant inhibitory effects against both enzymes over the concentration range tested (10-270 μg/ml).
3.6. Effect of AAEO on MAPK/NF-κB/AP-1 pathway in RAW264.7 cells induced by LPS Several signaling pathways known to mediate inflammation including MAPKs and NF-κB were investigated to explore the possible molecular mechanisms underlying the anti-inflammatory effect of AAEO. JNK, p38 and ERK1/2, the three key proteins in MAPK signaling pathway were examined. As phosphorylation is required for the activation of MAPKs, phospho-specific MAPK antibodies were used in Western blotting analysis. After the stimulation of LPS for 30 min, the phospho-MAPK proteins were dramatically increased in RAW264.7 cells, however, no apparent inhibition on the phospho-MAPK proteins was observed in AAEO co-treated cells at all concentrations tested. NF-κB subunits (p65 and/or p50) are normally sequestered in the cytosol as an inactive complex by binding to repressor IκB-α in non-stimulated cells. Upon the stimulation of LPS, IκB-α is phosphorylated by IκB kinase (IKK) and inactivated by degradation, and then free NF-κB is translocated into the nucleus where it induces the expression of multiple inflammatory genes (Akira and Takeda,
2004).We further examined the distribution of the NF-κB p65 subunit in the cytosolic and nuclear extracts of RAW264.7 cells, and revealed that the amount of p65 in the nucleus increased after stimulation with LPS for 1 h, however, AAEO treatment did not reduce nuclear localization of the p65 protein. Detection of AP-1 subunit (c-Jun) was also performed using specific antibodies and no effect was found on AAEO treated cells. The detection of PARP and α-tubulin confirmed that little cross-contamination existed during the extraction of the two fractions (data not shown).
3.7. Effect of AAEO on phosphorylation of JAK2 and STAT1/3 in RAW264.7 cells induced by LPS JAK/STATs signaling participates in the regulation of genes involved in the acute primary response to infection and inflammation response (Stark and Darnell, 2012). Examining the effects of AAEO on this pathway may help in gaining further insight into the mechanism of AAEO in controlling inflammation. Multiple time points from 1 to 16 h were examined, and 8 to 16 h was thought as suitable time for AAEO to exert the efficacy (Fig. 5A). AAEO can dose-dependently inhibit the phosphorylation of STAT1 (Tyr701), STAT3 (Tyr705) and the upstream JAK2 in LPS-activated RAW264.7 cells, without affecting the total levels of JAK and STATs protein (Fig. 5B,C).
Fig. 5. Effect of AAEO on the LPS-induced activation of the JAK/STATs pathway in RAW 264.7 cells. (A) RAW264.7 cells were pretreated with AAEO for the indicated time periods and stimulated with LPS. (B, C) Cells were treated with AAEO (10, 30, 90, 270 μg/mL) for 16 h followed by the stimulation of LPS (1 μg/mL) for 1 h. Total protein was extracted and analyzed by Western blot using specific antibodies. β-actin was used as the internal control for normalization. Data are expressed as Mean ± SD from three independent experiments (**p < 0.01 and ***p < 0.001 vs. LPS alone)
3.8. Inhibitory effect of AAEO on TPA-induced ear edema in mice To evaluate the anti-inflammatory activity of AAEO in vivo, its effect against TPA-induced ear edema in mice was tested. Four hours’ exposure to TPA resulted in distinct increases in skin thickness (Fig. 6A). As expected, the dose-dependent inhibition of ear edema by AAEO was observed, with a marked effect even at the lowest dose (41% inhibition at 83 mg/kg). The positive control indomethacin also
produced an inhibition up to 84%, as compared with TPA-alone group. The histological analysis of the sections of mice ears were conducted by both hematoxylin and eosin staining and immunohistochemistry, as illustrated in Fig. 6B, TPA resulted in marked increases in skin thickness with clear evidence of edema, epidermal hyperplasia, substantial neutrophil infiltration, and connective tissue disruption. AAEO treatment reduced ear thickness and the associated pathological indicators to an extent comparable with indomethacin. The effects of AAEO on TPA-induced COX-2 expression in mouse skin were analyzed by both immunohistochemical and Western blot analysis. COX-2 expression increased dramatically in the epidermal layer upon TPA treatment for 4 h, and was significantly abolished by AAEO (Fig. 6C, D).
Fig. 6. Effect of orally administered AAEO on TPA-induced ear thickness (A), histological lesion (B) and COX-2 expression (C, D). Ears were treated with acetone vehicle, TPA plus either normal saline, AAEO (83, 250, 750 mg/kg), or IMC for 30 min and then treated with TPA for 4 h before harvest. Skin punch biopsies were harvested for thickness measurement. Protein was analyzed for COX-2 by western blotting. Paraffin-embedded tissues were subjected to hematoxylin and eosin staining for histological analysis or immunostained by specific COX-2 protein (representative sections of 250mg/kg group are shown in group C). Data are expressed as Mean ± SD (n=5, **p < 0.01 and ***p < 0.001 vs. TPA alone).
4. Discussion A. argyi was used as a traditional remedy for various diseases in Asia, modern studies have proved its modulatory effects on the inflammation process such as rhinitis, dermatitis and arthritis, furthermore, preparation of the essential oil has been clinically prescribed for chronic bronchitis in China for years (Miao et al., 2012, Jiang et al., 2016). GC-MS analysis in this study provided detailed chemical composition of AAEO, in which several predominant chemicals including cineole, borneol, camphor, and thujone were found to possess anti-inflammatory activities according to previous reports (Ehrnhöfer-Ressler, et al., 2013; Miguel, 2010). These reports imply that AAEO may be associated with anti-inflammatory properties due to the presence of the
above components. To assess this herbal medicine as a potential anti-inflammatory agents, the ability of AAEO to modulate inflammation was confirmed in vitro and in vivo, and the molecular mechanism of the activity were further investigated by using macrophage-mediated inflammatory responses and TPA-induced mouse ear edema model. The present results clearly demonstrated that AAEO could significantly suppress the production of NO and PGE2, and intensively inhibited IL-6, IL-10, IFN-β and MCP-1, but only have a relatively slight inhibition on TNF-α in LPS-stimulated RAW 264.7 macrophages. Consistent with the effects observed in vitro, AAEO could markedly ameliorate TPA-induced mouse ear edema in animal experiments, which implied it has anti-inflammatory activity in vivo. By interacting with Toll-like receptor 4 (TLR4), LPS activates intracellular signaling pathways, including MAPK and NF-κB pathway, to up-regulate the expressions of various inflammatory mediators, which include NO, PGEs, and pro-inflammatory cytokines (Du et al., 2009; Qi et al., 2013). Among these, two of the most prominent are NO and PGE2, which are the products of the inducible NO synthase (iNOS) and cyclooxygenase 2 (COX-2), respectively (Qi et al., 2013). The interference of AAEO with iNOS and COX-2 expression, as well as enzyme activities were first investigated. In accordance with the inhibition on NO production, AAEO of 30-270 μg/ml markedly inhibited the iNOS mRNA and protein expression in LPS-induced macrophages. However, it did not display inhibitory effects on both mRNA and protein expression of COX-2 until the dose was elevated to 270 μg/ml. We believe that AAEO contains some components with weak activities against
COX-2 gene expression, which effect becomes apparent at higher dosage. In addition, AAEO exhibited little effects on the enzyme activities of iNOS and COX-2. These findings indicated that AAEO inhibited LPS-induced NO and PGE2 production through the suppression of iNOS and COX-2 at transcriptional level in different degrees. The lack of effects on COX-2 regulation is somewhat surprising when compared to the inhibition on PGE2 production. The reasons for this discrepancy are unclear, but could be due to some terpenes such as cineole suppressing the release of arachidonic acid from the phospholipid stores of cells, which directly reduced the synthetic precursor of PGE2 (Juergens, 1999). The expressions of iNOS and COX-2 in macrophages are regulated by multiple signaling pathways associated with inflammation, mostly through the activation of MAPK and NF-κB cascades. The MAPK pathways, which are extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38 MAPK pathways in particular (Herlaar and Brown, 1999), also regulate the synthesis of inflammation mediators at the level of transcription and translation through activation of NF-κB and AP-1 transcription factors (Kaminska, 2005). However, Western blot assay revealed the increased phosphorylation of MAPKs including JNK, p38, and ERK1/2, which were not affected by AAEO at the tested concentrations. NF-κB and AP-1 are major transcription factors to regulate the expressions of pro-inflammatory enzymes and cytokines such as iNOS, COX-2, TNF-α, IL6, IFN-β and MCP-1 (Chiang et al., 2005; Choi et al., 2011). The appearance of the p65 subunit (Rel A) in the cytosolic and nuclear extracts of cells was monitored, and equal levels of p65
were observed in the nuclear extracts, indicating that the translocation of p65 to the nucleus was not responsive to AAEO. The same phenomenon was observed in c-Jun, the subunit of AP-1. We thus propose that the inhibition of AAEO on cytokine production was not due to the regulation of NF-κB and AP-1 translocation. The JAK/STATs signaling pathway has been implicated in cytokine-mediated gene expression in immune responses. Cytokines such as IL6 or IFN-β binding to their receptors initiate the cascade, which activates the phosphorylation of JAKs and further phosphorylates STATs. Phosphorylated STATs are released and translocate into the nucleus to regulate the transcription of target genes including iNOS and COX-2 (Yu et al., 2009; Kou et al., 2011). Some evidences have proved that the JAK/STATs pathway is crucial in LPS-induced iNOS expression (Okugawa et al., 2003), for NF-κB alone is insufficient for the optimal induction of the iNOS genes (Ganster et al., 2001). The activation of STAT1 by IFN-β production was required for the expression of iNOS in LPS-stimulated macrophages (Ohmori and Hamilton, 2001; Samardzic, 2001), while STAT3 is crucial for regulating the expression of pro-inflammatory cytokines and chemokines such as IL-1β, IL-6 and MCP-1. Meanwhile, the anti-inflammatory cytokine IL-10, which performs an irreplaceable role in negatively regulating inflammation, is also transcriptionally regulated by STAT3 (Benkhart et al., 2000). LPS-induced STAT1/3 activation leads to the productions of several cytokines including IFN-β, IL-6 and IL-10, which in turn augment the JAK/STATs signaling. The cytokine suppression supported our hypothesis that by blocking the production of IFN-β, IL6 and IL-10, AAEO treatment
negatively influenced the phosphorylated protein levels of STAT1/3, and the phosphorylation of JAK2, signaling pathway upstream of STATs in LPS-activated RAW264.7 cells as well. These results indicated that down-regulation of JAK/STAT signaling activation is a possible mechanism for AAEO to inhibit LPS-induced iNOS and COX-2 gene expression as well as the production of some pro-inflammatory cytokines and chemokines in macrophages. Moreover, since essential oil possesses antioxidant activity for scavenging ROS (Miguel, 2010, Huang et al., 2012), and the production of ROS is a key step involved in JAK/STATs activation in macrophages (Simon et al., 1998), it is plausible that AAEO inhibits JAK/STATs signaling by ROS scavenging. It is of interest to note that AAEO dose-dependently reduced LPS-induced ROS production in RAW264.7 macrophages, which implicates the inhibitory effect of AAEO on JAK/STATs might be attributed to its antioxidant activity toward ROS. AAEO has been reported to attenuate inflammation responses in different animal models (Ge et al., 2016; Miao et al., 2012; Jiang et al., 2016). TPA-induced ear edema model is an ideal model for screening drugs with anti-inflammatory potential. Topical application of TPA induced an acute inflammatory response in mouse ear with massive
edema
formation,
and
AAEO
markedly
attenuated
TPA-induced
inflammation in a dose-dependent manner, with percentage of inhibition up to 84%, which was comparable with the positive control indomethacin. Consistent with the effect in vitro, AAEO suppressed COX-2 expression in the ears from the mice exposed to TPA (Fig.6C, D), indicating that it may be beneficial for COX-2-related
cutaneous inflammation. In conclusion, our results demonstrated that AAEO exert anti-inflammatory activity by inhibiting the production of inflammatory mediators, including NO, PGE2, TNF-α, IL-6, etc. The anti-inflammatory mechanism of AAEO may be related to negative regulation of JAK/STATs and ROS scavenging, diminished the protein and mRNA expression levels of iNOS and COX-2, rather than MAPK and NF-κB pathway (Fig. 7). These findings provide a new molecular insight into the mechanism of AAEO on inflammation and suggest it can be developed into a novel therapeutic agent for inflammatory-related diseases.
Fig. 7. Schematic diagram of the mechanisms underlying the suppression of AAEO on LPS-mediated inflammatory response.
Acknowledgement This work was supported by New products of TCM Senile Diseases Co-Innovation
Center of Hubei.
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