The effects of Diallyl sulfide Porphyromonas
gingivalis
lipopolysaccharide
stimulated
pro-inflammatory cytokine expressions and NF-κB
activation in human gingival fibroblasts
Earl Fu, DDS, DScD a, *, Man-Chin Tsai, MS a, *, Yu-Tang Chin, PhD a, b, Hsiao-Pei Tu, PhD a, c, Martin M. Fu, BDS, MS, DMSc a, d, Cheng-Yang Chiang, DDS, MScD a, and Hsien-Chung Chiu, DDS, PhD a
a: Research Institute of Dental Sciences and Department of Periodontology, School of Dentistry, National Defense Medical Center and Tri-Service General Hospital, Taipei, Taiwan, ROC
b: Program for Graduate Institute of Cancer Biology and Drug Discovery, College of Medical Science and technology, Taipei Medical University, Taipei 11031, Taiwan, ROC
c: Department of Dental Hygiene, China Medical University, Taichung, Taiwan, ROC d: Department of Oral Medicine, Infection and Immunity, Harvard School of Dental
Medicine, Boston, MA, USA
*: The authors contributed equally to this work.
Correspondence to:
Dr. Hsien-Chung Chiu, DDS, PhD School of Dentistry
National Defense Medical Center
PO Box 90048-507, Taipei, Taiwan, ROC Tel: +886-2-87927150; Fax: +886-2-87927145 Email: [email protected]
Abstract
Background and Objective: Diallyl sulfide (DAS), a flavor compound from garlic, has varied potential therapeutic activities. Periodontitis is a disease that develops as a result of host-mediated inflammation to periodontal pathogens. In this study, the effects of DAS on the common pro-inflammatory cytokines and nuclear factor-kappa B (NF-κB) in the human gingival fibroblasts (HGFs) being stimulated with
lipopolysaccharide (LPS) from Porphyromonas gingivalis (P. gingivalis), a potent periodontal pathogen, were evaluated.
Material and Methods: Cytotoxicities of DAS and LPS on HGFs were measured with MTS assay. The mRNA and protein expressions of pro-inflammatory cytokines, including interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α, from the HGFs treated with LPS with and without DAS were examined with reverse transcription– polymerase chain reaction and immunocytochemistry, respectively; and the activation and nuclear translocation of NF-κB with and without DAS were compared.
Results: DAS and LPS treatments within 3 mM and 10 g/ml, respectively, did not affect the survival rate of HGFs. LPS (1 g/ml) significantly increased the mRNA expressions of IL-1β, IL-6 and TNF-α; however, DAS (1 mM) inhibited these expressions. The protein expressions of TNF-α, IL-1β, as well as the NF-κB nuclear translocation were increased after LPS treatment, but decreased when there was a DAS pretreatment.
Conclusion: DAS diminished P. gingivalis LPS-stimulated cytokine expression and NF-κB activation in HGFs; we therefore suggest DAS may be beneficial on periodontal inflammation.
Introduction
Periodontitis is an inflammation-associated infectious disease that causes the destruction of tooth-supporting tissue structures and may eventually result in loss of the tooth. Periodontitis develops as a result of a host-mediated inflammatory response to bacterial plaque residing in gingival pockets . A few specific gram-negative bacteria have been acknowledged as the main etiologic pathogens such as
Porphyromonas gingivalis (P. gingivalis) and Aggregatibacter
actinomycetemcomitans . Lipopolysaccharide (LPS) is a major component of the
outer membrane of those bacteria and can elicit strong immune responses in gingiva. LPS has the ability to penetrate gingival connective tissue and induce a local inflammatory response that may lead to periodontal bone resorption in the end . LPS has been therefore broadly used to induce the experimental inflammation and to enhance the pro-inflammatory cytokine expression in the periodontium in many in vivo and in vitro studies . Tremendous effort has also been put into investigating suitable pharmacological agents against LPS, bacteria, and host immune responses as an adjunct treatment tool of periodontal therapy .
Garlic has been known to have several beneficial bio-activities, including anti-thrombosis , anti-oxidant and anti-carcinogen . Therefore, the garlic extract has been suggested to have protective effects on certain diseases . Diallyl sulfide (DAS), a flavor compound derived from garlic, has been reported to have varied potential therapeutic activities, including oxidant, infection, immunomodulation, anti-apoptosis, decreased loss of bone density, reduced cholesterol and blood pressure . DAS has been shown to in vivo decrease LPS-induced lung inflammation and in vitro inhibit LPS-induced inducible nitric oxide synthase (iNOS), prostaglandin E2 (PGE2) and cyclooxygenase-2 (COX-2) expressions in macrophages . However, the effects of
DAS on periodontitis and its inflammatory associated mediators are absent in the literature. The aim of this study was therefore to evaluate in vitro evaluate the inhibitory effects of DAS on the expressions of pro-inflammatory cytokines, including interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α, and on the activation of nuclear factor-kappa B (NF-κB) in human gingival fibroblasts (HGFs) treated with P.
Materials and Methods
Cell Culture
Primary HGFs were purchased from Coriell Cell Repository§ (AG09319 and AG09429) (Camden, NJ, USA). The cells were cultured in Dulbecco’s minimal essential medium (DMEM)/F12 medium (Invitrogen Corporation, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (FBS), 2 mM L-gluthamine, and 100 U/ml of penicillin/streptomycin in plastic culture flasks and maintained in an atmosphere of 5% CO2 at 37°C. Upon 80 % confluence, HGFs were detached by incubation with 0.05 % trypsin in EDTA solution for 5 min at 37°C. The cells were then plated in 10 cm culture dish or 6-well tissue culture plates (Nunc AS, Roskilde, Denmark). Confluent HGFs were starved in serum-free medium overnight before being used in the experiments. All of the HGFs used for the experiments were within five passages.
P. gingivalis LPS and Diallyl Sulfide Treatments
To study the inhibitory effects of DAS on the LPS-stimulated pro-inflammatory cytokines and NF-κB, HGFs were cultured with 10% FBS in DMEM/F12 medium to 70% confluence in six-well plates and eight-well chamber slides (Merck Millipore, Billerica, MA, USA). The medium was changed to serum-free DMEM/F-12 to starve the cells overnight. HGFs were first pretreated with DAS (Sigma-Aldrich Inc., St. Louis, MO, USA)at a concentration of 0, 0.5, and 1 mM in alcohol for 24 hours. The cells were then stimulated with P. gingivalis LPS (InvivoGen, San Diego, CA, USA) at a concentration of 1 μg/ml in endotoxin-free H2O for 3, 12, and 24 hours. Cells
were collected and used in the following experiments.
MTS Cell Viability Assay
HGFs were placed in the 96-well multi-plates containing DMEM/F-12 medium and 10% FBS, and cultured until confluent. After washed with phosphate-buffered saline (PBS), the medium was replaced with DMEM/F-12 medium containing 1% FBS. The cell viability of HGFs were evaluated 24 hours after culturing by the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulf-ophenyl)-2H-tetrazolium, inner salt] assay (CellTiter 96_AQueous One Solution; Promega, Madison, WI, USA)according to the manufacturer’s protocol. The survival rates of HGFs after the treatments of LPS, DAS, and LPS plus DAS were measured and compared, which were modified from a previous study .
Reverse Transcription–Polymerase Chain Reaction (RT-PCR)
To examine the effects of DAS pretreatment on the mRNA expressions of TNF-α, IL-1β and IL-6 in HGFs treated with P. gingivalis LPS, total RNA was extracted using TRIsure reagent (Bioline Ltd., London, UK). To avoid the contamination of genomic DNA, total RNA was treated with DNase I (Life Technologies Corporation, Carlsbad, California, U.S.A) according to the manufacturer's instructions, prior to first strand cDNA synthesis. As described previously, 1 μg of total RNA was reversed transcribed with Tetro RT enzyme into cDNA, and was used as the template for PCR reactions and analysis . The PCR reactions included an initial denaturation at 94C for 2 minutes and 30 seconds, followed by 21 cycles (glyceraldehyde-3-phosphate dehydrogenase, GAPDH) or 31 cycles (TNF-α, IL-1β and IL-6) at 94C for 30 seconds, annealing at 62C for 30 seconds, and then elongation at 72C for 55
seconds. The PCR primer sequences were human TNF-α, forward
5’-CAGAGGGAAGAGTTCCCCAG-3’ and reverse
5’-CCTTGGTCTGGTAGGAGACG-3’ (325 bp, Accession No. NM_000594); IL-1β, forward 5’- GACACATGGGATAACGAGGC-3’ and reverse 5’-ACGCAGGACAGGTACAGATT-3’ (248 bp, Accession No. NM_000576); IL-6, forward 5’- ATGAACTCCTTCTCCACAAGCGC-3’ and reverse 5’-GAAGAGCCCTCAGGCTGGACTG-3’ (628 bp, Accession No. NM_000600); GAPDH, Forward AGCCGCATCTTCTTTTGCGTC-3’ and reverse 5’-TCATATTTGGCAGGTTTTTCT-3’ (816 bp, Accession No. NM_002046). The amplicons were analyzed on 1% agarose gels and visualized with SYBR® Safe staining (Invitrogen Corporation) and a camera system (ChemiDoc XRS+ system) (Bio-Rad Laboratories, Hercules, CA, USA). The gel images were scanned and analyzed with an imaging software (Image Lab, Bio-Rad Laboratories, Waltham, MA, USA).
Immunocytochemistry and Confocal Laser Scanning Microscopy
HGFs were cultured in eight-well chamber slides for immunocytochemistry (ICC). The cells were pretreated with 0, 0.5, and 1 mM of DAS, then treated with P.
gingivalis LPS, fixed with methanol, blocked with 5% bovine serum albumin (BSA)
in PBS for 20 minutes, and then exposed to the primary antibody of anti-NF-κB (1:250 in 5% BSA) (Epitomics Inc., Burlingame, CA, USA), anti-IL-1β, anti-IL-6 or anti-TNF-α (1:200 in 5% BSA) (Abcam, Cambridge, UK) overnight at 4 C. The cells were then washed in PBS, exposed to the secondary antibody (FITC-conjugated goat anti-rabbit IgG, 1:200 in 5% BSA) for 30 minutes and counterstained with DAPI. The activation and nuclear translocation of NF-κB and expressions of IL-1β, anti-IL-6 and
TNF-α in HGFs were observed and compared by confocal laser scanning microscopy (LSM780) (Carl Zeiss MicroImaging, Inc., New York, NY, USA).
Western Blot
Western blot analysis was performed to quantify the protein expression levels of IκB-α in the total cell lysates of HGFs after treatment with LPS (1 μg/ml), DAS (0.5 and 1.0 mM) or combined. Protein samples were resolved with 1X RIPA buffer with protease inhibitor (Sigma-Aldrich Inc., St. Louis, MO, USA). Supernatants were removed after centrifuging for 10 min at 14000 x g. A 20-μg quantity of protein was loaded in each well with 4x sample buffer, and the protein samples were resolved by electrophoresis at 100 V for 2 hours. The resolved proteins were transferred from the polyacrylamide gel to Millipore Immobilon-PSQ Transfer PVDF membranes (Millipore, Billerica, MA, USA) with the Hoefer Semi-dry Transfer system (Hoefer, Inc., Holliston, MA, USA). The membranes were blocked with a solution of 2% bovine serum albumin (Sigma-Aldrich Inc., St. Louis, MO, USA) and 1% Tween 20 in Tris-buffered saline. The membranes were incubated with primary antibodies to IκB-α (Abcam, Cambridge, UK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (GeneTex Inc., San Antonio, Texas, USA) at 4°C overnight and washed, and the proteins were detected with HRP-conjugated secondary antibodies and ImmobilonTM Western HRP Substrate Luminol Reagent (Millipore). Images of the Western blots were visualized, recorded and analyzed by ChemiDoc™ XRS+ imaging system with Image Lab™ image acquisition and analysis software (Bio-Rad Laboratories, Hercules, CA, USA).
One-way ANOVA with Duncan’s post hoc test was used to determine the survival rates of HGFs, the dose and time effects of LPS on pro-inflammatory cytokines, and the effect of DAS on the LPS-stimulated cytokines.
Results
Cytotoxicity of LPS and DAS on HGFs
The survival rates of the HGFs receiving 0.1 to 10.0 µg/ml of LPS treatments were similar to the group without LPS (Figure 1A). For the cytotoxicity of DAS on HGFs, the survival rates maintained in similar when the concentration was not more than 3 mM. However, the survival rates were significantly decreased when the concentrations were 5 and 7 mM (Figures 1B and 1C). Similar trend of survival rate was observed in the treatments of 1.0 µg/ml LPS combined with different dosages of DAS (Figure 1C). In the rest of the experiments, LPS with the concentration of 1.0 g/ml and DAS with the concentrations of 0.5 and 1.0 mM were used unless otherwise specified.
IL-1β, IL-6 and TNF-α mRNA Expressions of HGFs after LPS Treatment
LPS treatments significantly increased the mRNA expressions of IL-1β, IL-6 and TNF-α (Figure 2A). The dose dependent effect of LPS was clearly observed in those increases of IL-1β and IL-6. For these two cytokines, the peak mRNA expressions were noted at 12 hours for IL-1β and at 24 hours for IL-6 after the LPS treatment, whereas TNF-α expression was highest 3 hours after the LPS treatment (Figure 2B).
Effects of DAS on IL-1β, IL-6 and TNF-α mRNA and Protein Expressions of HGFs after LPS Treatment
The mRNA expression of IL-1β was significantly increased in the HGFs received LPS; however, the expression was reduced to the original level without LPS if the cells were pretreated with 1.0 mM DAS (Figure 3A). Similar findings were also observed in the mRNA expressions of IL-6 and TNF-α (Figures 3B and 3C).
cytoplasm of HGFs; however, the increased expression was reduced when there was a treatment of 0.5 or 1.0 mM DAS (Figure 3D). Similar findings were also observed for the protein expressions of IL-6 and TNF-α in cytoplasm, especially in 1.0 mM DAS.
Effect of DAS on NF-κB Activation and Translocation in HGF after LPS Stimulation
Activation and nuclear translocation of NF-κB could be observed in HGFs after the treatment of P. gingivalis LPS (Figure 4D) as the intensity of staining (green in color) of NF-κB in cytoplasm and nuclei was increased. The amounts of these NF-κB activation and nuclear translocation were reduced when DAS was added (Figures 4E and 4F); however, their staining intensities were still more pronounced than that observed for the control. In addition, the dose-related effect of DAS on the intensity reductions seemed not to be observed, if DAS concentrations were less than 1.0 mM. The protein expressions of IκB-α in the HGFs after DAS, LPS, or the combined treatment of LPS and DAS were evaluated (Figure 4G). The reduced IκB-α was observed in that after LPS treatment only.
Discussion
In the present study, the anti-inflammatory effects of DAS were in vitro examined in the primary HGFs having the treatment of P. gingivalis LPS. HGFs are the most abundant connective tissue cells that are located in gingiva under gingival epithelium. In this study, the LPS from P. gingivalis significantly increased the mRNA expressions of IL-1β, IL-6 and TNF-α (Figure 2). This finding was consistent with the previous reports in the literature . Our results further revealed that the treatment of P. gingivalis LPS significantly increased the cellular and nuclear intensity of NF-κB (Figure 4D) indicating the activation and nuclear translocation of NF-κB after the LPS stimulation. NF-κB is a transcription factor playing a key role in the regulations of cell survival, proliferation, differentiation, as well as inflammation. Studies have shown that the pro-inflammatory cytokines are commonly involved with the activation of NF-κB . In the classical NF-kB signaling pathway, upon the stimulation by TNF-α or IL-1β, the IKK signalosome composed of IKKα, IKKβ, and IKKγ would be activated, and lead to the phosphorylation of IkB on two conserved N-terminal serine residues. Then phosphorylated IkB would be ubiquitinated and subsequently degraded by the S26 proteasome. The activated NF-kB in the cytoplasm would then translocate into nucleus and bind to a decameric consensus motif in target genes, resulting in their transcription . In the present study, the finding that DAS reversed the LPS-reduced IκB-α indicated the NF-kB signaling pathway was involved (Figure 4G). Because of the enhanced expression of pro-inflammatory cytokines and activation of NF-κB, the gingival fibroblasts are considered to be not only the structural framework but also the immune responder for gingival tissue . In the present study, the LPS of gram-negative bacterium P. gingivalis was selected and used because it is one of the main periodontopathic bacteria in periodontitis.
Therapeutic interventions targeting the inflammatory cytokines have become a focal point for controlling various inflammatory diseases. In the progression of periodontitis, inflammation plays a critical role. The present study showed that DAS not only significantly reduced P. gingivalis LPS-enhanced expression of pro-inflammatory cytokines but also declined the activation and nuclear translocation of NF-κB in HGFs (Figures 3D and 4E). Consistent with the results in the current study, a recent study has showed that DAS prevents induced COX-2 upregulation through a mechanism involving NF-kB in synovial cells and chondrocytes . DAS was selected and examined in the present study because DAS is one of the anti-inflammatory components from garlic. In addition, garlic also contains many other biologically and pharmacologically important compounds, such as allicin, and diallyldisulfide (DADS), which are beneficial to human health against several other diseases . A study has recently reported that a pretreatment of DADS prior to the treatment of LPS in microglial cells could significantly attenuate the production of a few pro-inflammatory cytokines and chemokines, including IL-1β, TNF-α, and monocyte chemoattractant protein-1 . DADs DADS reduced dose-dependently the number of lymphocyte subsets and monocytes in rats. The diallyl trisulfide protects the ischemic myocardium by preservation of endogenous hydrogen sulfide and increasing nitric oxide bioavailability. Dially trisulfide inhibits phorbol ester-induced tumor promotion, activation of AP-1, and expression of COX-2 in mouse skin by blocking JNK and Akt singaling. Diallyl tetrasulfane activates both the ROS-elF2alpha and Nrf2/HO-1 pathways in HCT116 cells . The bactericidal effects of DAS has also been documented in laboratory rodents . In LPS-stimulated macrophages, DAS inhibited the corresponding expressions of inducible NO synthase, COX-2, as well as PGE2 production .
Allicin, another flavor compound derived from garlic oil, has been shown to inhibit the spontaneous and TNF-induced secretion of pro-inflammatory cytokines, including IL-1β, but had not effect on cellular viability of epithelial cells. Because allicin suppressed the degradation of Iκ-B, it was suggested that the inhibition of NF-κB pathway of allicin would probably diminish the amplification of NF-NF-κB-mediated cytokine secretion . However, allicin is generally considered asan unstable compound .
In the present study, three common pro-inflammatory cytokines (i.e. IL-1β, IL-6, and TNF-α) were examined to validate the potential anti-inflammatory efficacy of DAS in the clinical therapy of periodontitis. Dose-dependent increases of mRNA for IL-1β and IL-6 were observed in HGFs after the P. gingivalis LPS treatments; however, this dose effect was not observed in TNF-α (Figure 2). The exact reasons are uncertain; however, our mRNA data in the time course illustrated that the highest expression of TNF-α was noted at the third hour after the LPS stimulation and the dose effect of TNF-α after the highest expression peak at the third hour (Figure 2B). A delayed measurement in the experiment may partly explain the reduced incensement at high dose (10.0 μg/ml) (Figure 2A: TNF-α). In addition, the cytotoxicity of LPS and DAS on HGFs were evaluated in this study (Figure 1). For DAS, the survival rates of HGFs were significantly decreased when the concentration was 5 or 7 mM, regardless with and without the presence of LPS (Figures 1B and 1C), which might indicate a cytotoxic effect of DAS, if the concentration greater than 3 mM, on HGFs. For LPS treatments, however, the survival rates were similar if the concentrations keeping in the range from 0.1 µg/ml to 10.0 µg/ml (Figure 1A). Although the detailed experiment are still needed, one of the reason is that the tested concentrations might be too low to detect the cytotoxicity.
In this study, DAS decreased the LPS-induced pro-inflammatory cytokine production and NF-kB activation, but the exact mechanism and the detailed signal molecules affected are still uncertain. Because the P. gingivalis LPS is a TLR2 ligand, whether the mechanisms observed in any other TLR2-ligand, such as Pam3CSK4 (35, 36), could be recovered in the DAS reduced activation in HGFs are also unknown. In human colon cancer colo 205 cells, nevertheless, DAS down-regulated PI3K, Ras, MEKK3, MKK7, ERK1/2, JNK1/2, and p38 expressions was observed down-regulated a certain signaling pathways, including ERK 1/2 and JNK 1/2, and then leaded to the inhibition of the matrix metalloproteinase (MMP)-2, -7, and -9 has been reported recently (37). Further detailed investigation on the mechanisms related to DAS-decreased the LPS-induced pro-inflammatory cytokine production and NF-kB activation is indicated. On Within the limitations of the experiments, we suggested that DAS, a flavor compound from garlic, diminished the P. gingivalis LPS-stimulated pro-inflammatory cytokine expression and the NF-κB activation in HGFs. The properties observed might be utilized for exploring its therapeutic potential in treatment of P. gingivalis-associated oral and periodontal inflammation.
Acknowledgements
This study was part supported by the grants from the Department of National Defense (103-M047) and the C.Y. Foundation for Advancement of Education, Sciences and Medicine, Taipei, Taiwan, ROC.
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Figure legends
Figure 1: Cell viability of HGFs after the treatments of LPS and/or DAS. The
survival rates of HGFs received 0, 0.1, 1.0 and 10.0 g/ml of LPS (A), 0 to 7 mM of DAS (B) and the combined treatment of LPS plus DAS (C). (Data present by mean ± SD, *: significantly different vs that of no treatment at p < .05)
Figure 2. Effect of the LPS treatment on the mRNA expression of IL-1β, IL-6 and TNF-α in HGFs. A) presents the effect of LPS dosages on the mRNA expression
of IL-β, IL-6 and TNF-α in HGFs in 5 hours (n = 3). B) shows the comparison among the observation periods up to 24 hours after the treatment of 1 μg/ml LPS (n =3). (a-d: the subsets after post hoc analysis after the significant differences were obtained, by one-way ANOVA)
Figure 3: Effects of DAS on the mRNA and protein expressions of IL-1β, IL-6 and TNF-α after of LPS stimulation in HGFs. A-C) present the effects of DAS
treatments (0.5 and 1.0 mM) on the relative mRNA expressions of IL-1ß, IL-6 and TNF-α in HGFs after the treatment of 1 μg/ml LPS (3, 12, 24 h for TNF- ɑ, IL-1ß and IL-6, respectively) (means ± standard deviations, all of the experiments were repeated three times and, a-c: the subsets after post hoc analysis, one-way ANOVA).
D) demonstrated the inhibitory effects of DAS on the protein expressions of IL-1β,
IL-6 and TNF-α in HGFs received LPS stimulation for 24 hours, observed by confocal LASER laser scanning microscopy.
Figure 4: Effect of DAS on the NF-κB nuclear translocation in HGFs after the treatment of LPS. A-F) Under the confocal LASER scanning microscope, the left
column presents the fluorescent intensity of NF-κB in cells, the middle column indicates the DAPI staining of the cell nuclei, while the right column presents the NF-κB nuclear translocation by merging the above two images. The DAS dosages used were either 0.5 or 1.0 mM. The LPS concentration was 1.0 μg/ml, whereas the control only has the solvent of DAS. G) demonstrated the effect of DAS on the protein expressions of IkB-α in HGFs received LPS stimulation, observed by Western blotting. The bar length is 50 µm.
