Contributed equally
There are three isoforms of nitric oxide synthase (NOS): Neuronal NOS, endothelial NOS and inducible NOS (iNOS). Stimulation of macrophages with LPS induces increased expression of iNOS, which leads to the increased production of NO during an inflammatory response (
To the best of our knowledge, the present study is the first to demonstrate the anti-inflammatory effect of tribulusamide D, a compound isolated from the ethanolic extract of
The fruits of
The air-dried and crushed fruits of
Among them, the fraction G46-51-7 (2.71 g) exhibited promising inhibitory activity against NO production and it was selected for further investigation. G46-51-7 (2.71 g) was passed through a column containing Sephadex® LH-20 gel using CHCl3:MeOH (1:1) as eluent, to produce sub-fractions (G46-52-1-21). Of the above 21 fractions, tribulusamide D (17.3 mg) was isolated from fraction G46-52-14, which was precipitated with MeOH. 1H- and 13C-nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Ascend™ 700 MHz NMR spectrometer (Bruker Corporation, Billerica, MA, USA) using dimethylsulfoxide (DMSO) as a solvent. Electrospray ionization mass (ESI-MS) was measured on a Thermo Finnigan TSQ Quantum mass spectrometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA).
Tribulusamide D is a white, amorphous powder; 1H-NMR (DMSO-d6, 700 MHz) δ: 10.4 (1H, s, OH), 9.39 (1H, s, OH), 9.16 (1H, s, OH), 8.29 (1H, t, J=5.6 Hz, NH), 7.90 (2H, d, J=8.4 Hz, H-2′ and H-6′), 7.27 (1H, d, J=16.1 Hz, H-7), 6.99 (1H, d, J=2.1 Hz, H-2), 6.88 (2H, d, J=9.1 Hz, H-3′ and H-5′), 6.87 (1H, dd, J=8.4, 2.1 Hz, H-6), 6.75 (1H, d, J=7.7 Hz, H-5), 6.52 (1H, d, J=15.4 Hz, H-8), 4.65 (2H, s, H-8′); 13C-NMR (DMSO-d6, 175 MHz) δ 193.9 (C-7′), 166.2 (C-9), 162.8 (C-4′), 147.9 (C-4), 146.0 (C-3), 140.0 (C-7), 130.9 (C-2′), 130.9 (C-6′), 127.0 (C-1′), 126.8 (C-1), 121.0 (C-6), 118.6 (C-8), 116.2 (C-5), 115.8 (C-3′), 115.8 (C-5′), 114.3 (C-2), 45.9 (C-8′); ESI-MS m/z 312 [M-H]-. The structure of tribulusamide D is presented in
DMSO (cat. no. D1370) and MTT (cat. no. M1415) were purchased from Duchefa Biochemie B.V (Haarlem, The Netherlands). LPS from
Image-iT™ FX Signal Enhancer, Alexa Fluor® 488-conjugated secondary antibody (goat anti-mouse, Thermo Fisher Scientific, Waltham, MA, USA, cat. no. A32723), ProLong® Gold Antifade reagent with DAPI (Thermo Fisher Scientific, Inc., cat. no. P36931). Anti-β-actin (cat. no. 5125), anti-phospho-JNK (T183/Y185) (cat. no. 4668), anti-JNK (cat. no. 9252), anti-phospho-extracellular signal-regulated kinase (ERK; T202/Y204) (cat. no. 9101), anti-ERK (cat. no. 9102), anti-phospho-p38 (T180/Y182) (cat. no. 9211) and anti-p38 (cat. no. 9212) primary antibodies were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA) and anti-iNOS antibody was purchased from Abcam (Cambridge, UK) (cat. no. ab3523). Anti-COX2 (cat. no. sc-376861), anti-NF-κB p65 (cat. no. sc-7151) and anti-lamin B (cat. no. sc-6216) antibodies, and goat (cat. no. sc-2354) and rabbit (cat. no. sc-2004) IgG-horseradish peroxidase conjugated secondary antibodies were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).
RAW 264.7 mouse macrophage cells (TIB-71) were purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 0.1 mg/ml streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.) in a humidified atmosphere of 95% air with 5% CO2 at 37°C.
RAW 264.7 cells were seeded in 96-well plates (4×104 cells/well) and were treated with tribulusamide D (25–100 µM) for 1 h prior to LPS (0.5 µg/ml) stimulation for 24 h. The negative control was treated with serum-free media (SFM; Gibco; Thermo Fisher Scientific, Inc.).
The amount of nitrite, a stable metabolite of NO, was measured by using Griess reagent (1% sulfanilamide and 0.1% naphthylethylenediamine dihydrochloride in 2.5% phosphoric acid; EMD Millipore, Billerica, MA, USA). Absorbance was subsequently measured at 540 nm, using a SpectraMax 190PC microplate reader (Molecular Devices, LLC, Sunnyvale, CA, USA). The quantity of nitrite was determined from a standard curve for sodium nitrite (Sigma-Aldrich; Merck KGaA).
RAW264.7 cells were plated at a density of 4×104 cells/well in 96-well plates. Cells were treated with tribulusamide D (25–100 µM) for 1 h prior to LPS (0.5 µg/ml) stimulation for 24 h. MTT (5 mg/ml in PBS) was added to each well and incubated for 90 min. The medium was removed from the wells by aspiration, 0.1 ml buffered DMSO was added to each well and the plate was shaken. The absorbance of each well was measured at a wavelength of 540 nm using a SpectraMax 190PC microplate reader. Data are presented as the mean ± standard deviation of three replicates.
ELISA was performed to determine the concentration of cytokines in RAW 264.7 cells
Levels of IL-6, IL-10 and TNF-α in culture medium were quantified using platinum IL-6 (cat. no. BMS603/2), IL-10 (cat. no. BMS614/2), TNF-α (cat. no. BMS607/3) ELISA kits (eBioscience, Inc., San Diego, CA, USA), and the PGE2 (cat. no. KGE004B) concentration in culture medium was quantified using a competitive enzyme ELISA kit (R&D Systems, Inc., Minneapolis, MN, USA), according to the manufacturer's instructions.
RAW264.7 cells were plated at a density of 1×106 cells/well in 6-well plates and treated with tribulusamide D (25–100 µM) for 1 h prior to LPS (0.5 µg/ml) stimulation for 24 h. The total RNA was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The integrity of the RNA was evaluated using 1% agarose gel electrophoresis and ethidium bromide staining. Briefly, 1 µg RNA was used as a template for each RT-PCR, using the SuperScript® III First-Strand Synthesis System and Taq DNA polymerase (Invitrogen; Thermo Fisher Scientific, Inc.). Invitrogen Platinum PCR SuperMix (Thermo Fisher Scientific, Inc.) was used which contains DNA polymerase. RT-PCR amplification was performed in My Gene Series Peltier Thermal Cycler (Long Gene Scientific Instruments, Hangzhou, China), using and AccuPower Pfu PCR premix (Bioneer Corporation, Daejeon, Korea). PCR conditions were as follows for 25 cycles: 30 sec denaturation at 95°C, 40 sec annealing between at 55 and 60°C, 60 sec extension at 72°C, and for final extension of 10 min. The final PCR products were electrophoresed in 1% agarose gel and stained with ethidium bromide. Images of the bands were captured using a Chemidoc XRS system (Bio-Rad Laboratories, Hercules, CA, USA) and observed using the Quantity One software version 4.6.3. The results compared with the housekeeping gene GAPDH. They were performed at least in triplicate. The sequences of the primers used for RT-PCR are shown in
RAW264.7 cells were plated at a density of 1×106 cells/well in 6-well plates and treated with tribulusamide D (25–100 µM) for 1 h prior to LPS (0.5 µg/ml) stimulation for different time points, as indicated. Cells were harvested and washed with PBS, and collected by centrifugation at 16,000 × g for 1 min at 4°C. To obtain the cellular lysate, cells were lysed on ice for 30 min in a RIPA buffer [50 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 1% NP-40, 0.1% sodium SDS, 1 mM dithiothreitol (DTT) and 1 mM phenylmethylsulfonyl fluoride (PMSF)], which contained protease inhibitors (Sigma-Aldrich; Merck KGaA). Total protein was quantified with the Quick Start Bradford 1x Dye reagent (Bio-Rad Laboratories) using bovine serum albumin (BSA, Sigma-Aldrich; Merck KGaA) for the standard. Protein extracts representing 40 µg total protein were separated on 10% SDS-PAGE gel using the BioRad Mini Protean 3 System (Bio-Rad Laboratories) and electroblotted onto Protran nitrocellulose membranes (Whatman; GE Healthcare Life Sciences, Chalfont, UK). Membranes were blocked in 5% BSA in PBS/0.025% Tween-20 (Sigma-Aldrich; Merck KGaA) for 1 h at room temperature. The primary antibodies used were specific for β-actin, p-JNK, JNK, p-ERK, ERK, p-p38, p38 (Cell Signaling Technology), iNOS (Abcam), COX-2, NF-κB p65 and Lamin B (Santa Cruz Biotechnology, Inc.). The primary antibodies were diluted (1:1,000) in 5% BSA in PBST and incubated with the membrane overnight at 4°C. The secondary antibody was applied at a 1:2,000 dilution in 5% BSA in PBST and incubated for 1 h at room temperature then processed for detection with the Supersignal West Pico Chemiluminescent substrate (Thermo Fisher Scientific, Inc.), using a Chemidoc XRS system (Bio-Rad Laboratories) and observed using the Quantity One software version 4.6.3. They were performed at least in triplicate.
RAW264.7 cells were plated at a density of 1×106 cells/well in 6-well plates and treated with tribulusamide D (25–100 µM) for 1 h prior to LPS (0.5 µg/ml) stimulation for 30 min. Cells were washed twice with ice-cold PBS prior to trypsinization and centrifugation at 90 × g and 4°C for 5 min. Cells were then centrifuged at 20,000 × g and 4°C for 5 min and resuspended in 200 µl buffer (10 mM HEPES at pH 7.9, 10 mM KCl, 1 mM DTT, 0.5 mM PMSF and 0.1 mM EDTA). After incubation on ice for 10 min, cells were lysed by the addition of 12.5 µl of 10% NP-40. Cells were then centrifuged at 20,000 × g for 2 min at 4°C, and the supernatants were collected as cytosolic extract. Pellets were resuspended in 50 µl of extraction buffer (20 mM HEPES at pH 7.9, 0.4 M NaCl, 1 mM DTT, 1 mM PMSF, 1 mM EDTA and 1% NP-40) and incubated on ice for 10 min. Nuclear extract was collected by centrifugation at 15,000 × g for 15 min at 4°C. Western blot analysis was performed in the same manner as aforementioned.
RAW264.7 cells were cultured on glass cover-slips in 6-well plates with 10% FBS at a density of 4×105 cells/well. Cells were treated with tribulusamide D (25–100 µM) for 1 h prior to LPS (0.5 µg/ml) stimulation for 24 h. The cells were washed with PBS for 5 min, fixed with 4% formaldehyde for 5 min on ice, permeabilized with 0.1% Triton X-100 for 2 min on ice, washed with PBS for 5 min, and blocked with PBS containing 5% BSA for 1 h at room temperature. Primary antibody (COX-2) was incubated for overnight (5% BSA in PBS; 1:200), washed with PBS, and followed by secondary antibody (Alexa Fluor 488-conjugated, goat anti-mouse) incubation for 1 h in darkroom (5% BSA in PBS, 1:300). After washing with PBS, the stained cells were visualized and photographed using a confocal laser scanning microscope (Zeiss AG, Oberkochen, Germany).
Statistical analysis was performed using a Student's t-test (Microsoft Excel 2007) and was based on at least three different experiments. P<0.05 was considered to indicate a statistically significant difference.
To determine the cytotoxic effect of tribulusamide D, cells were treated with various concentrations of tribulusamide D (25, 50 or 100 µM), which was followed by LPS stimulation (0.5 µg/ml). As demonstrated in
The effect of tribulusamide D on the expression of the pro-inflammatory cytokines IL-6, IL-10 and TNF-α in LPS-induced RAW 264.7 cells was investigated. As presented in
To further investigate the molecular mechanisms by which tribulusamide D regulates the LPS-induced inflammatory response, the present study investigated whether tribulusamide D regulates the translocation of NF-κB into the nucleus and the changes in activation of mitogenic signaling pathways, including ERK, JNK and p38 MAPK, which have pivotal roles in inflammatory responses (
Previous studies have explored the development of novel medicines using natural products.
The present study investigated the effect of tribulusamide D on LPS-induced stimulation of the inflammatory response and the expression of pro-inflammatory cytokines. NO is a pleiotropic inflammatory mediator, and it is generated by iNOS. iNOS is considered an important inducible enzyme that regulates inflammatory responses (
It is established that IL-6, IL-10 and TNF-α have key roles in the induction and inhibition of the immune reactions of other inflammatory mediators. TNF-α is an element of the innate immune response against stimuli, and IL-6 and IL-10 are the most important pro-inflammatory cytokines. The present study demonstrated that the mRNA and protein concentration of IL-6, IL-10 and TNF-α were decreased in LPS-stimulated cells treated with 100 µM of tribulusamide D. NF-κB and MAPKs signaling pathway has a critical role in the regulation of inflammatory gene expression (
In conclusion, the results of the present study demonstrated that tribulusamide D isolated from
The present study was conducted by the research fund of Dankook University in 2015.
cyclooxygenase-2
inducible nitric oxide synthase
interleukin
lipopolysaccharide
nitric oxide
prostaglandin
phenylmethylsulfonyl fluoride
tumor necrosis factor-α
nuclear factor-κB
mitogen-activated protein kinases
Regulatory effects of tribulusamide D on NO production and iNOS expression in LPS-stimulated RAW 264.7 cells. Cells were treated with tribulusamide D (25–100 µM) for 1 h prior to LPS (0.5 µg/ml) stimulation for 24 h. (A) Chemical structure of tribulusamide D. (B) Cytotoxicity was determined by MTT assay and (C) NO assay was performed using Griess reagent. L-NMMA (100 µM) was used as a positive control. (D) Expression of iNOS mRNA was determined by reverse transcription-polymerase chain reaction analysis. (E) Expression of iNOS protein was determined by western blot analysis. Data are presented as the mean ± standard deviation of three independent experiments. **P<0.01 vs. LPS-treated cells. NO, nitric oxide; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; L-NMMA, NG-methyl-L-arginine acetate salt; Dexa, dexamethasone.
Regulatory effect of tribulusamide D on the production of IL-6, IL-10 and TNF-α in LPS-stimulated RAW 264.7 cells. Cells were treated with tribulusamide D (25–100 µM) for 1 h prior to LPS (0.5 µg/ml) stimulation for 24 h. (A) Expression of IL-6, IL-10 and TNF-α mRNA was determined by reverse transcription-polymerase chain reaction analysis. ELISA was performed on cell culture supernatants to examine protein levels of (B) IL-6, (C) IL-10 and (D) TNF-α cytokines. Data are presented as the mean ± standard deviation of three independent experiments. *P<0.05; **P<0.01 vs. LPS-treated cells. IL, interleukin; TNF-α, tumor necrosis factor-α; LPS, lipopolysaccharide; Dexa, dexamethasone.
Regulatory effect of tribulusamide D on COX-2 expression in LPS-stimulated RAW 264.7 cells. Cells were treated with tribulusamide D (25–100 µM) for 1 h prior to LPS (0.5 µg/ml) stimulation for 24 h. (A) Expression of COX-2 mRNA was determined by reverse transcription-polymerase chain reaction. (B) Expression of COX-2 protein was determined by western blotting. (C) Cellular expression of COX-2 was microscopically detected by immunofluorescence. Magnification, ×200. DNA was stained with DAPI. COX-2, cyclooxygenase-2; LPS, lipopolysaccharide.
Regulatory effect of tribulusamide D on PGE2 production in LPS-stimulated RAW 264.7 cells. Cells were treated with tribulusamide D (25–100 µM) for 1 h prior to LPS (0.5 µg/ml) stimulation for 24 h. Quantitative measurement of PGE2 level was performed by using cell culture supernatants. Data are presented as the mean ± standard deviation of three independent experiments. *P<0.05 vs. LPS-treated cells. PGE2, prostaglandin E2; LPS, lipopolysaccharide.
Regulatory effect of tribulusamide D on NF-κB nuclear localization and phosphorylation of p38 mitogen-activated protein kinase in LPS-stimulated RAW 264.7 cells. Cells were treated with tribulusamide D (25–100 µM) for 1 h prior to LPS (0.5 µg/ml) stimulation for 30 min. (A) Cytosolic and nuclear extracts were analyzed via western blot analysis with anti-NF-κB p65, , anti-β-actin or anti-lamin B antibodies. (B) Cell lysates were subjected to western blot analysis with anti-p-ERK, anti-ERK, anti-p-JNK, anti-JNK, anti-p-p38 and anti-p38 antibodies. Results are representative of at least three independent replicates. NF-κB, nuclear factor-κB; LPS, lipopolysaccharide; p-, phospho-; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase.
Primer sequences used for reverse transcription-polymerase chain reaction.
Primer sequences | |||
---|---|---|---|
Target | Sense | Antisense | Accession number |
GAPDH | 5′-GTATGACTCCACTCACGGCAAA-3′ | 5′-GGTCTCGCTCCTGGAGAGATG-3′ | NM_008084 |
IL-6 | 5′-CACTTCACAAGTCGGAGGCTT-3′ | 5′-GCAAGTGCATCATCGTTGTTC-3′ | NM_031168 |
IL-10 | 5′-CCTGGTAGAAGTGATGCCCCAGGCA-3′ | 5′-CTATGCAGTTGATGAAGATGTCAAA-3′ | NM_010548 |
COX-2 | 5′-GGAGAGACTATCAAGATAGTGATC-3′ | 5′-ATGGTCAGTAGACTTTTACAGCTC-3′ | NM_011198 |
TNF-α | 5′-AGCCTGTAGCCCACGTCGTA-3′ | 5′-TCTTTGAGATCCATGCCGTTG-3′ | NM_013693 |
iNOS | 5′-ATCTGGATCAGGAACCTGAA-3′ | 5′-CCTTTTTTGCCCCATAGGAA-3′ | NM_0109273 |
IL, interleukin; COX-2, cyclooxygenase-2; TNF-α, tumor necrosis factor-α; iNOS, inducible nitric oxide synthase.