Introduction
Inflammation is the attempt of the body to remove harmful stimuli and initiate the healing process. However, chronic inflammatory processes are involved in the pathogenesis of common inflammation-associated diseases (1,2). Various infectious agents, such as bacteria and viruses, cause inflammation and macrophages play a critical role in initiating and propagating inflammatory responses by releasing pro-inflammatory mediators, such as nitric oxide (NO), prostaglandin E2 (PGE2) and cytokines to promote the inflammatory response (3,4).
Lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, activates a number of major inflammatory cellular effects by inducing several interconnecting mechanisms through specific surface molecules known as pathogen-associated molecular patterns, which bind to Toll-like receptors (TLRs), particularly TLR4, leading to the release of a wide variety of inflammatory inducers (5,6). The LPS-initiated signaling cascade occurs by binding of LPS to its receptor complex, and this leads to activation of the nuclear factor-κB (NF-κB) pathway (7,8). In normal cells, NF-κB is constitutively localized in the cytosol as a homodimer or heterodimer, which is associated with the NF-κB inhibitor, inhibitory-κBα (IκB). Upon stimulation with LPS, IκB proteins are phosphorylated, ubiquitinated and rapidly degraded by the ubiquitin-proteasome pathway, and the resulting free NF-κB migrates into the nucleus and activates transcription of NF-κB-dependent inflammatory enzymes and cytokines (5,9,10). These observations indicate that antagonistic agents of TLR4 can inactivate pro-inflammatory downstream signaling pathways by suppressing the differential target gene expression and cellular responses.
Several epidemiological studies have shown that consuming allium vegetables is associated with a decreased risk of various diseases (11,12). Among them, garlic (Allium sativam L.) has long been used for culinary and medicinal purposes in numerous cultures. Accumulating studies have demonstrated that garlic has a wide range of biological activities against a number of chronic diseases, including cardiovascular problems, diabetes, infections and even cancer (13–15). Garlic is a particularly rich source of organosulfur compounds (OSCs), such as diallyl sulfide (DAS), diallyl disulfide (DADS) and diallyl trisulfide (DATS), which are believed to be responsible for its flavor and aroma, as well as its potential health benefits (16–18). In particular, studies have shown that the antioxidant potential is in the order DATS > DADS > DAS (19–21). Na et al (22) found the same order for the anticancer potential, suggesting that the number of sulfur atoms plays a vital role in the biological activities of OSCs. Although DATS has been suggested recently as the strongest inhibitor of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) among OSCs (21,23), the molecular mechanisms underlying the anti-inflammatory effects of DATS have not been extensively explored.
Therefore, the present study was carried out to further investigate the potential anti-inflammatory effect and underlying molecular mechanism of DATS using a murine RAW 264.7 macrophage model, which can be stimulated with LPS to mimic a status of inflammation. The results suggest that DATS effectively suppresses LPS-induced inflammatory signaling by inactivating the NF-κB pathway and inhibiting TLR4.
Materials and methods
Cell culture
The murine RAW 264.7 macrophage cell line was obtained from the American Type Culture Collection (Manassas, VA, USA) and was cultured in Dulbecco’s modified Eagle’s medium (Gibco-BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco-BRL), 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine, at 37°C in a 5% CO2 humidified air environment.
DATS treatment and MTT assay
DATS was obtained from LKT Laboratories (St. Paul, MN, USA) and was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA) as a 100 mM stock solution and was stored in aliquots at −20°C. RAW 264.7 cells were incubated with different concentrations of DATS or 100 ng/ml LPS (Sigma-Aldrich) alone, or pretreated with DATS for 1 h before LPS for the cell viability assay. After 24 h, the medium was removed and the cells were incubated with 0.5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide (MTT; Sigma-Aldrich) solution for 2 h. The supernatant was discarded and the formazan blue, which was formed in the cells, was dissolved in DMSO. Optical density was measured at 540 nm with a microplate reader (Dynatech Laboratories, Chantilly, VA, USA) and growth inhibition was assessed as the percent viability, in which vehicle-treated cells were considered as 100% viable.
Measurement of NO production
Concentrations of NO in the culture supernatants were determined by measuring nitrite, a stable oxidation product of nitric oxide, using the Griess reagent (Sigma-Aldrich). Briefly, cells (5×105 cells/ml) were stimulated in 24-well plates with DATS and/or LPS for 24 h. Subsequently, 100 μl of each culture supernatant was mixed with an equal volume of Griess reagent. After 10 min incubation at room temperature, absorbance was measured at 540 nm and nitrite production was determined with an NaNO2 standard curve (24).
Determination of PGE<sub>2</sub>, tumor necrosis factor-α (TNF-α) and interleukin (IL)-1β production
The cells were incubated with DATS in either the presence or absence of LPS (100 ng/ml) for 24 h to measure the quantity of PGE2. A 100 μl aliquot of culture medium supernatant was collected and the concentration (pg/ml) of PGE2 in the cell culture medium was calculated based on the concentrations of the standard solution using a PGE2 enzyme-linked immunosorbent assay (ELISA) kit following the manufacturer’s instructions (Cayman Chemical Co., Ann Arbor, MI, USA). The levels of TNF-α and IL-1β were also measured with ELISA kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions (25).
RNA isolation and reverse transcriptase-polymerase chain reaction (RT-PCR)
Total RNA was isolated using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions, and reverse-transcribed using M-MLV reverse transcriptase (Promega, Madison, WI, USA) to produce cDNAs. RT-generated cDNAs encoding iNOS, COX-2, TNF-α and IL-1β genes were amplified by PCR using selective primers, which were purchased from Bioneer (Seoul, Korea). The PCR primers were as follows: mouse iNOS (5′-ATG TCC GAA GCA AAC ATC AC-3′ and 5′-TAA TGT CCA GGA AGT AGG TG-3′), COX-2 (5′-CAG CAA ATC CTT GCT GTT CC-3′ and 5′-TGG GCA AAG AAT GCA AAC ATC-3′), TNF-α (5′-ATG AGC ACA GAA AGC ATG ATC-3′ and 5′-TAC AGG CTT GTC ACT CGA ATT-3′) and IL-1β (5′-ATG GCA ACT GTT CCT GAA CTC AAC T-3′ and 5′-TTT CCT TTC TTA GAT ATG GAC AGG AC-3′). Following amplification, the PCR reactants were electrophoresed in 1% agarose gels and visualized by ethidium bromide (Sigma-Aldrich) staining. In a parallel experiment, glyceraldehyde-3-phosphate dehydrogenase was used as an internal control.
Protein extraction and western blotting
The cells were harvested and lysed with lysis buffer [20 mM sucrose, 1 mM EDTA, 20 μM Tris-Cl (pH 7.2), 1 mM dithiothreitol, 10 mM KCl, 1.5 mM MgCl2 and 5 μg/ml aprotinin] for 1 h. In a parallel experiment, nuclear and cytosolic proteins were prepared using nuclear extraction reagents (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. Protein concentration was measured using a Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s instructions. For western blot analysis, equivalent amounts of proteins were separated by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels and transferred to nitrocellulose membranes (Schleicher and Schuell, Keene, NH, USA). After blocking with 5% skimmed milk, the membranes were incubated with protein-specific antibodies for 1 h, subsequently incubated with appropriate enzyme-linked secondary antibodies [mouse IgG, HRP-linked whole antibody (NA931) and rabbit IgG, HRP-linked whole antibody (NA934)] (Amersham Corp., Arlington Heights, IL, USA) and visualized by enhanced chemiluminescence (Amersham Corp.) according to the manufacturer’s instructions. Anti-iNOS (#610330) and anti-COX-2 (#160106) antibodies were purchased from BD Biosciences (San Jose, CA, USA) and Cayman Chemical Co., respectively. Anti-IL-1β (SC7884), anti-TLR4 (SC10741), anti-MyD88 (SC11356), anti-NF-κB p65 (SC109), anti-actin (SC1616) and anti-GAPDH (SC32233) antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Anti-TNF-α (#3737) and anti-IκB (#4812) antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Anti-nucleolin (AB22758) antibody was obtained from Abcam (Cambridge, MA, USA).
Electrophoretic mobility assay (EMSA)
EMSA was performed with the nuclear extract. Synthetic complementary NF-κB (5′-AGT TGA GGG GAC TTT CCC AGG C-3′) binding oligonucleotides (Santa Cruz Biotechnology, Inc.) were 3′-biotinylated using the biotin 3′-end DNA labeling kit (Pierce) according to the manufacturer’s instructions, and annealed for 30 min at room temperature. Assays were loaded onto native 4% polyacrylamide gels pre-electrophoresed for 60 min in 0.5X Tris borate/EDTA before being transferred onto a positively charged nylon membrane (Hybond™-N+) in 0.5X Tris borate/EDTA at 100 V for 30 min. The transferred DNAs were cross-linked to the membrane at 120 mJ/cm2. Horseradish peroxidase-conjugated streptavidin was used according to the manufacturer’s instructions to detect the transferred DNA.
Immunofluorescence staining
The RAW 264.7 cells were cultured on glass coverslips in 6-well plates for 24 h, stimulated with LPS in the presence or absence of DATS, fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min at room temperature, and permeabilized with 100% MeOH for 10 min at 20°C. The anti-NF-κB p65 antibody was applied for 1 h followed by a 1 h incubation with fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). Subsequent to washing with PBS, nuclei were stained with 4,6-diamidino-2-phenyllindile (Sigma-Aldrich), and fluorescence was visualized using a fluorescence microscope (Carl Zeiss, Oberkochen, Germany). RAW 264.7 cells were stimulated with Alexa Fluor 488-conjugated LPS (100 ng/ml, AF-LPS; Invitrogen) for 30 min in the presence or absence of DATS for the LPS/TLR4 complex formation assay. The cells were fixed, stained with anti-TLR4 antibody for 90 min at 4°C and then incubated with secondary antibodies conjugated with Alexa Fluor 594 (1:200; Invitrogen) for 1 h. The stained cells were observed under a fluorescence microscope.
Measurement of TLR4 expression on cell surface
RAW 264.7 cells were incubated with AF-LPS in the presence or absence of DATS for 1 h. The cells were washed twice with PBS, harvested with 0.005% EDTA and analyzed by flow cytometry. Alexa 488 was excited using a 488 argon-ion laser line and detected on a channel FL1 using a 530 nm emission filter. The fluorescence emission of samples was recorded by a flow cytometer (26).
Statistical analysis
Data are presented as mean ± standard deviation. Statistical significance was determined using an analysis of variance followed by Student’s t-test. P<0.05 was considered to indicate a statistically significant difference.
Results
Effects of DATS and LPS on RAW 264.7 macrophage viability
To examine whether DATS is cytotoxic to RAW 264.7 macrophages, the cells were exposed to DATS for 24 h in the presence or absence of LPS, and cell viability was measured by the MTT assay. The results showed no effect of DATS on cell viability at concentrations of 10, 20 or 30 μM (Fig. 1A); however, at higher concentrations the MTT assay suggested an adverse influence of DATS on cell viability. Furthermore, no significant cytotoxic effects were observed at ≤30 μM DATS in the presence of LPS (Fig. 1B).
DATS reduces LPS-induced production of pro-inflammatory mediators and cytokines
Pro-inflammatory mediators including NO and PGE2, and pro-inflammatory cytokines, such as IL-1β and TNF-α, that were released into the culture medium were measured using the Griess reagent and ELISA to examine the inhibitory effect of DATS on LPS-induced inflammatory responses. According to the NO detection assay, LPS alone markedly induced NO production compared to that generated by the control. However, pretreatment with DATS significantly repressed the levels of NO production in LPS-stimulated RAW 264.7 cells in a concentration-dependent manner ≤30 μM (Fig. 2A). The levels of PGE2 and tested cytokines also increased significantly in the culture media of LPS-stimulated RAW 264.7 cells, but the increases were significantly attenuated in a concentration-dependent manner by DATS pretreatment (Fig. 2B–D).
DATS inhibits LPS-induced expression of pro-inflammatory enzymes and cytokines
The effects of DATS on the levels of iNOS, COX-2, TNF-α and IL-1β mRNA and protein expression was measured by RT-PCR and western blot analyses to elucidate the mechanism involved in the inhibition of pro-inflammatory mediators and cytokines generated by DATS in LPS-stimulated RAW 264.7 cells. mRNA and protein expression in unstimulated RAW 264.7 cells was undetectable or extremely low. However, expression increased markedly in response to LPS, which was significantly inhibited by pretreatment with DATS (Fig. 3). These results indicate that the reduced expression of pro-inflammatory enzymes and cytokines at the transcriptional levels contributed to the inhibitory effect of DATS on LPS-induced NO, PGE2, IL-1β and TNF-α production.
DATS blocks LPS-induced nuclear translocation of NF-κB p65 and degradation of IκBα
As previous studies suggested that NF-κB is an important transcription factor regulating the expression of pro-inflammatory enzymes and cytokines (5,9,10), whether DATS blocked the NF-κB signaling pathway was explored. The western blot analysis results showed that the amount of NF-κB p65 in the nucleus was markedly increased within 30 min of exposure to LPS alone, concomitant with degradation of IκBα in the cytosol. However, LPS-induced NF-κB p65 levels in the nuclear fractions decreased markedly by DATS pretreatment, and LPS-induced IκBα degradation was clearly blocked in a concentration-dependent manner by pretreatment with DATS (Fig. 4A). EMSA also showed that treatment with LPS causes an increase in NF-κB DNA-binding activity at 30 min, while pretreatment of the cells with DATS for 1 h resulted in a significant reduction in the DNA-binding activity of NF-κB (Fig. 4B). In parallel with immunoblotting data, the immunofluorescence images revealed that the nuclear accumulation of NF-κB p65 was not induced in the cells following treatment with DATS alone in the absence of LPS stimulation; however, it was strongly induced following LPS stimulation, and the shift of NF-κB to the nucleus was completely abolished subsequent to pretreating the cells with DATS (Fig. 4C). Taken together, these results suggest that DATS treatment inhibits LPS-induced NF-κB translocation by attenuating the IκBα degradation.
DATS inhibits LPS-induced TLR4 and myeloid differentiation factor 88 (MyD88) expression, and the interaction between LPS and TLR4
The effects of DATS was assessed on the expression of LPS-induced TLR4 and MyD88 (a TLR4-associated molecule) expression to determine the involvement of the TLR4 signaling pathway in the DATS-mediated anti-inflammatory potential. The increased levels of TLR4 and MyD88 proteins in LPS-treated RAW 264.7 cells were completely blocked in a concentration-dependent fashion by pretreatment with DATS (Fig. 5A). Furthermore, when cells were treated with AF-LPS alone, fluorescence of LPS and TLR4 were observed outside the cell membrane by the immunofluorescence assay (Fig. 5B). However, the fluorescence intensity of TLR4 was markedly attenuated in the presence of DATS, suggesting that DATS may inhibit the interaction between LPS and TLR4 on the RAW 264.7 cell surface. In addition, treatment with LPS in the presence of DATS significantly prevented the binding of LPS to the RAW 264.7 cell surface (Fig. 5C), indicating that DATS may interfere with the clustering of TLR4 with MyD88.
CLI-095, a TLR4 inhibitor, increases the anti-inflammatory potential of DATS
The effects of CLI-095, which blocks TLR4-mediated signaling, were evaluated to further confirm the involvement of the TLR4 signaling pathway in the DATS-mediated anti-inflammatory potential. CLI-095 reduced the LPS-induced production of pro-inflammatory mediators and cytokines by suppressing the transcriptional levels of their corresponding genes (Fig. 6). Furthermore, DATS and CLI-095 co-treatment synergistically inhibited LPS-induced synthesis of NO and PGE2, as well as expression of iNOS and COX-2. DATS and CLI-095 co-treatment synergistically inhibited LPS-induced release and expression of IL-1β and TNF-α (Fig. 7). These results verify that the inhibitory effects of DATS on the LPS-induced inflammatory responses may result from suppression of the TLR4 signaling pathway.
Discussion
In the present study, DATS was shown to be suitable for reducing LPS-induced NO and PGE2 synthesis in RAW 264.7 macrophages. This suppression was possibly due to inhibiting the upregulation of iNOS and COX-2 at the transcriptional level during macrophage activation by LPS. Parallel inhibitory effects on synthesis and expression of TNF-α and IL-1β were also observed in LPS-stimulated RAW 264.7 cells.
During the inflammatory process, LPS induces the overproduction of pro-inflammatory mediators, such as NO and PGE2, which are generated by the inducible enzymes iNOS and COX-2, respectively (3,4). NO is controlled by NO synthase and iNOS is markedly upregulated in inflammatory disorders. Another enzyme that plays a pivotal role in mediating inflammation is COX-2, which catalyzes the rate-limiting step in the synthesis of PGE2 from arachidonic acid. In addition to NO and PGE2, numerous inflammatory cytokines have been recently demonstrated to be initiators and mediators of the inflammatory response. Among these, TNF-α and IL-1β are the major pro-inflammatory cytokines produced by activated macrophages, and their excessive production has been linked to the development of chronic inflammatory diseases (27,28). Additionally, TNF-α and IL-1β production is critical for the synergistic induction of NO and PGE2 production in LPS-stimulated macrophage cells (27,29). Therefore, overproduction of these inflammatory factors is a histopathological characteristic of various inflammation-mediated diseases, and the selective inhibition of their production and function may be effectively therapeutic in the control of inflammatory disorders.
Consistent with the findings of the present study (Figs. 2 and 3), DATS inhibited LPS-induced NO and iNOS expression, as well as intracellular reactive oxygen species (ROS) generation, and the inhibitory effect of DATS on LPS-induced iNOS expression is possibly attributed to its antioxidant potential to inhibit NF-κB activation in RAW 264.7 macrophages (21). DATS has been reported to reduce the levels of LPS-induced PGE2 synthesis and several cytokines, including TNF-α, by inactivating mitogen-activated protein kinases and NF-κB signaling pathways in the same cell line (23). This study also revealed that DATS reduces LPS-induced ROS and activates transcription factor NF-E2 p45-related factor 2-mediated expression of heme oxygenase-1 and NAD(P)H quinine oxidoreductase 1, demonstrating that cross-talk between antioxidant and anti-inflammation in the inhibition of LPS-induced inflammation by DATS. In addition, DATS inhibits TNF-α production by inhibiting its transcriptional activity and downregulating NF-κB activation in inflamed mucosa of ulcerative colitis (30). Additionally, DATS ameliorates dextran sulfate sodium-induced mouse colitis presumably by blocking inflammatory signaling mediated by NF-κB and signal transducer and activator of transcription 3 transcription factors, as well as expression of their target proteins, including COX-2 and iNOS (31). A previous study has also suggested that DATS suppresses high glucose-induced cardiomyocyte apoptosis by inhibiting NADPH oxidase-related ROS and downstream c-Jun N-terminal kinase/NF-κB signaling (32). A previous study showed that DATS has a anti-cancer effect by inhibiting the levels of certain critical genes involved in cancer cell growth and metastasis by suppressing NF-κB activation (33). In agreement with these previous observations, the present data also revealed that the anti-inflammatory effects of DATS appeared to involve inhibiting NF-κB activation by blocking LPS-stimulated IκBα degradation and nuclear translocation of the NF-κB p65 protein (Fig. 4). Based on the present study findings and previous studies, DATS may exert its anti-inflammatory effect by suppressing the production of these pro-inflammatory mediators and cytokines by modulating the NF-κB signaling pathway.
By contrast, increasing evidence suggests that signaling pathways downstream of TLR4, a pattern recognition receptor that generates innate immune responses to pathogens such as LPS, play an important role in the pathogenesis of inflammation (6). TLR4 has a common TIR motif in the cytoplasmic domain and shares different adaptor proteins, including MyD88 for NF-kB activation (34). During infection, LPS directly binds with cellular TLR4 and induces a cascade of inflammatory events by triggering overproduction of inflammatory inducers through receptor dimerization and the recruitment of adapter molecules (35,36). Several studies have reported that certain anti-inflammatory agents compete with LPS binding to TLR4, resulting in the suppression of downstream signaling pathways (37,38). In addition, TLR4 and MyD88 are upregulated in various inflammatory processes induced by LPS (39,40). Consequently, the pharmacological reduction of LPS-inducible inflammatory regulators through inhibiting the association between LPS and TLR4 is regarded as an attractive therapeutic strategy for numerous acute and chronic inflammatory diseases. In the present study, increased expression of TLR4 and MyD88 proteins was concentration-dependently reduced in the presence of DATS (Fig. 5A). The data also indicate that DATS has inhibitory effects on the binding of LPS with TLR4 (Fig. 5B and C), suggesting a possible antagonistic effect of DATS against TLR4. In addition, combined pretreatment of DATS with CLI-095, a specific TLR4 signaling inhibitor (41,42), synergistically attenuated the release of NO and PGE2, as well as expression of iNOS and COX-2 mRNA and protein (Fig. 6). In accordance with these results, co-treatment also reduced the production and expression of TNF-α and IL-1β compared to those in the DATS or CLI-095 alone treated groups in LPS-treated RAW 264.7 macrophages (Fig. 7). These observations suggest that DATS may inhibit the initiation of intracellular inflammatory signaling cascades by attenuating the binding of LPS to TLR4 on macrophages, which is a pivotal upstream signal for NF-κB activation. Therefore, the antagonistic function of DATS against TLR4 may be responsible for the anti-inflammatory effects of DATS in LPS-stimulated RAW 264.7 macrophages.
In conclusion, the present data reveal that DATS inhibited LPS-induced iNOS, COX-2, TNF-α and IL-1β expression, which was accompanied by reducing the corresponding transcriptional gene expression through interfering with the clustering of LPS with TLR4. This resulted in blocking the activation of NF-κB signaling pathway in RAW 264.7 macrophages. These findings provide a further partial molecular description of the mechanism that underlies the anti-inflammatory properties of DATS.