MSM (cat. no. PHR1346) and LPS (cat. no. L2630) were obtained from MilliporeSigma. The following primary antibodies were purchased from Santa Cruz Biotechnology, Inc.: Ferrochelatase (FECH; cat. no. sc-377377), phosphorylated (p)-IκBα (cat. no. sc-8404), TLR2 (cat. no. sc-21759), TLR4 (cat. no. sc-293072), COX-1 (cat. no. sc-19998), COX-2 (cat. no. sc-19999, IKKα/β (cat. no. sc-7607), and β-actin (cat. no. sc-47778); from Cell Signaling Technology, Inc.: IL-1β (cat. no. 12703), p-ERK (cat. no. 9101), ERK (cat. no. 9102), IκBα (cat. no. 9242), p-IKKα/β (cat. no. 2697), NF-κB (cat. no. 8242), p-p38 (cat. no. 4511), p38 (cat. no. 8690), ATR (cat. no. 2790), ATM (cat. no. 2873), Chk2 (cat. no. 2662), BRCA1 (cat. no. 9010), p53 (cat. no. 9282), p-MDM2 (cat. no. 3521), MDM2 (cat. no. 86934) and the DNA Damage Antibody Sampler Kit (cat. no. 9947; containing p-ATM, p-ATR, p-Chk2, p-BRCA1 and p-p53 antibodies); from Abcam: Divalent metal transporter (DMT)1 (cat. no. ab55735), 5′-aminolevulinate synthase (ALAS)1 (cat. no. ab84962), STEAP3 (cat. no. ab151566), HO-1 (cat. no. ab137749), transferrin receptor (TfR; cat. no. ab84036), PKCα (cat. no. ab179523), p-PKCα (cat. no. ab59411), TNF-α (cat. no. ab183218), IL-6 (cat. no. ab6672) and TATA-binding protein (TBP; cat. no. ab818); from LifeSpan BioSciences, Inc.: ABCB10 (cat. no. LS-C381841) and FLVCR1 (cat. no. LS-C750126); from LS Bio; Vector Laboratories, Inc.: FPN (cat. no. NBP1-21502) and iNOS (cat. no. NB300-605); from Novus Biologicals; Bio-Techne: Mitoferrin (MFRN; cat. no. MBS6013473); and from Boster Biological Technology: ABCB6 (cat. no. PA1723). Horseradish peroxidase (HRP)-conjugated anti-rabbit (cat. no. 7074) and anti-mouse (cat. no. 7076) secondary antibodies were purchased from Cell Signaling Technology, Inc. NTS was provided by the NARA Bioetch.

Human THP-1 cells were purchased from the Korean Cell Line Bank; Korean Cell Line Research Foundation and cultured in RPMI-1640 medium (cat. no. L0498; Biowest) containing 10% fetal bovine serum (cat. no. A5670801; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin at 37°C in 5% CO₂. The cells (1×10⁶ cells/ml) were cultured for 72 h with 10 ng/ml LPS, with or without NTS, MSM or TLR4-C34 (cat. no. S0822; Selleck Chemicals).

The cells were stained with 5 µM FerroFarRed solution (cat. no. GC903-01; Goryo Chemical, Inc.) and incubated in a CO₂ incubator at 37°C for 30–40 min. After staining, the cells were washed with 1 ml pre-warmed serum-free RPMI-1640 medium and used for flow cytometric analysis (FACSCalibur; BD Biosciences). FlowJo v10 software (FlowJo; BD Biosciences) was used for analysis.

Whole-cell lysates were prepared by incubating untreated or LPS-treated THP-1 cells on ice with radioimmunoprecipitation lysis buffer (cat. no. 20-188; MilliporeSigma) containing protease and phosphatase inhibitors. Protein concentrations were measured using the Bradford method (Thermo Fisher Scientific, Inc.). The same amounts of protein (30 µg/well) were then separated by SDS-PAGE on 6–15% gels and the separated proteins were transferred onto nitrocellulose membranes. The blots were blocked for 1 h at room temperature with 5% skim milk (BD Biosciences) in TBS-Tween-20 (TBS-T) buffer [20 mM Tris-HCl (MilliporeSigma), pH 7.6; 137 mM NaCl (Formedium Limited); 0.1X (0.1%) Tween-20 (Scientific Sales, Inc.)]. The membranes were then incubated with primary antibodies diluted in 5% skim milk (1:1,000 dilution) overnight at 4°C with agitation. Subsequently, the membranes were washed with TBS-T and incubated for 1 h at room temperature with HRP-conjugated secondary antibodies (1:5,000). Detection was performed using a West-Q Pico ECL Solution (cat. no. W3652-020; GenDEPOT, LLC) and a LAS-4000 imaging device (FUJIFILM Wako Pure Chemical Corporation).

Total cellular RNA was extracted using an RNeasy Mini Kit (Qiagen GmbH) according to the manufacturer's protocol. The isolated RNA was quantified spectrophotometrically at 260 nm, and cDNA was synthesized at 42°C for 1 h and 95°C for 5 min with oligo d(T) primers and a first-strand cDNA synthesis kit (cat. no. K-2041; Bioneer Corporation). qPCR was then conducted using a thermal cycler (C1000 Thermal Cycler; Bio-Rad Laboratories, Inc.) as follows: 2 µl diluted cDNA was added to diluted forward and reverse primers (1 µl each, 100 pM) and 10 µl TB Green Advantage Premix (Takara Bio, Inc.). The thermocycling conditions were as follows: Initial denaturation at 95°C for 5 min; followed by 40 cycles of denaturation at 95°C for 40 sec, annealing at 58°C for 40 sec and extension at 72°C for 40 sec; and a final extension step at 72°C for 5 min. All measurements were performed in triplicate. Relative target gene expression was normalized to GAPDH. The calculations were performed using the 2^−ΔΔCq values obtained (31). The primer sequences are provided in Table I.

After cultured cells were washed with pre-chilled PBS, cell pellets were incubated with 10% BSA (cat. no. A3311; MilliporeSigma) on ice for 20 min. PE-conjugated anti-human CD284 (TLR4) antibody (1:200; cat. no. 312806; Biolegend, Inc.) and APC-conjugated anti-human CD282 (TLR2) antibody (1:200; cat. no. 309720; Biolegend, Inc.) was used to stain the cells on ice for 30 min. Stained cells were then washed with pre-chilled PBS. Cells were also stained with CM-H2DCFDA (5 µM; cat. no. C6827; Invitrogen; Thermo Fisher Scientific, Inc.) for cellular ROS and placed in a CO₂ incubator at 37°C for 30 min. The stained cells were washed with 1 ml prewarmed staining buffer (cat. no. 554656; BD Bioscience). Flow cytometric analysis was performed using a FACSCalibur flow cytometer. FlowJo v10 software was used for analysis.

The comet assay kit (cat. no. ab238544; Abcam) was used according to the manufacturer's protocol for measuring cellular DNA damage. A base layer of comet agarose was used to coat the slide, and a layer of cells (70% confluence) treated with 10 ng/ml LPS with the indicated concentrations of NTS or MSM was added, followed by another layer of 100 cells and agarose, followed by lysis. Electrophoresis was performed under neutral conditions, and cells were stained with DNA dye. Cell morphology was observed by fluorescence microscopy (IX71/DP72; Olympus Corporation).

A ChIP assay was performed using an Imprint Chromatin Immunoprecipitation Kit (cat. no. 17-295; MilliporeSigma) according to the manufacturer's protocol. THP-1 cells were fixed with 1% formaldehyde for 10 min at 37°C and quenched with 1.25 M glycine at room temperature for 30 min at 37°C. After washing with PBS, cells were suspended in a nucleus preparation buffer and sonicated in a shearing buffer under optimized conditions at 4°C for 10 times (20 kHz, 10 sec on/30 sec off). This sheared DNA was diluted with a dilution buffer (1:1 ratio), and 5 µl diluted sample was removed as an internal control. The diluted supernatant was incubated for 5 min at room temperature in wells pre-coated with antibodies (1:50 dilution) specific for IL-1β (cat. no. sc-12742; Santa Cruz Biotechnology, Inc.), TNF-α (cat. no. 6945; Cell Signaling Technology, Inc.) or IL-6 (cat. no. sc-57315; Santa Cruz Biotechnology, Inc.) at room temperature for 90 min. Normal mouse IgG (1:10 dilution; cat. no. M8695; MilliporeSigma) and anti-RNA polymerase II (1:10 dilution; cat. no. R1530; MilliporeSigma) were used as negative and positive controls, respectively. The unbound DNA was washed off with wash buffer, and the bound DNA was collected by cross-link reversal using a DNA release buffer containing proteinase K. The released DNA and the DNA from the internal controls were purified with GenElute Binding Column G. DNA was then quantified and RT-qPCR was performed using the aforementioned reagents; the thermocycling conditions were as follows: Initial denaturation at 95°C for 5 min; followed by 50 cycles of denaturation at 95°C for 20 sec, annealing at 58°C for 20 sec and extension at 72°C for 20 sec. All measurements were performed in triplicate. Relative target gene expression was normalized to GAPDH. The calculations were performed using the 2^−ΔΔCq values obtained.

Nuclear protein extracts were prepared with the Nuclear Extract Kit (cat. no. ab113474; Abcam). After harvesting the cells, the extraction buffer was prepared by adding DTT solution and a protease inhibitor cocktail at a 1:1,000 dilution. The cell pellet was treated with the prepared extraction buffer at a volume of 10 µl/10⁶ cells, and then incubated on ice for 15 min, briefly vortexing for ~5 sec every 3 min for mixing. Subsequently, the mixture was centrifuged at 18,500 × g for 10 min at 4°C. The resulting supernatant was carefully transferred to a new microcentrifuge tube; this contained the nuclear protein extract. The nuclear extract was then used for western blot analysis iof NF-κB. Equal amounts of proteins (150 µg/well) were separated by SDS-PAGE on 10% gels and were then transferred onto nitrocellulose membranes. Subsequently, he blots were blocked for 1 h at room temperature with 5% skim milk. The membranes were then incubated with NF-κB and TBP antibodies diluted in 5% skim milk overnight at 4°C with agitations. The membranes were then washed with TBS-T and incubated for 1 h with HRP-conjugated secondary antibodies at room temperature. Detection was performed as aforementioned.

All experiments were performed in triplicate. Data are presented as the mean ± SEM of three independent experiments conducted in triplicate (n=3). The control group was set to 100 for RT-qPCR results. A one-way ANOVA followed by Tukey's post hoc test was used for the statistical analysis. The analyses were performed using SAS 9.3 software (SAS Institute, Inc.). P<0.05 was considered to indicate a statistically significant difference.

We previously demonstrated that LPS-induced inflammation enhances iron metabolism through increased iron production and transport, which is responsible for iron homeostasis (5). To determine the effect of sulfur compounds on LPS-induced iron metabolism, the amount of Fe²⁺ was estimated following the treatment of THP-1 cells with NTS, MSM or TLR4-C34, a specific TLR4 inhibitor (Fig. 1A). Flow cytometry revealed increased Fe²⁺ levels after LPS treatment, whereas NTS and MSM exhibited decreased Fe²⁺ levels. A slightly different phenomenon was observed in TLR4-C34-treated cells, indicating the activity of sulfur compounds to inhibit LPS-induced inflammation by attenuating TLR4-independent iron metabolism. Next, western blot analysis was performed to identify the proteins responsible for iron transport and the molecular mechanisms associated with sulfur compound-regulated iron homeostasis. The results indicated upregulation of the TfR and FPN proteins by LPS, which was suppressed by treatment with NTS, MSM or TLR4-C34 (Fig. 1B). In addition, the expression levels of DMT1 and STEAP3, which are key factors in iron metabolism, were increased by LPS treatment. The regulation of iron metabolism may lead to heme synthesis. To confirm the induction of heme biosynthesis by LPS, the expression levels of proteins responsible for heme synthesis were examined through western blot analysis (Fig. 1C). The results indicated notable LPS-induced upregulation of HO-1, MFRN, ABCB6, ABCB10, ALAS1, FECH and FLVCR in THP-1 cells; however, these protein levels were markedly decreased following NTS or MSM treatment, but increased by TLR4-C34 treatment. These results indicated that iron/heme metabolism is pivotal in the anti-inflammatory activity of sulfur compounds, independent of TLR4.

TLRs act as receptors for the inflammatory response in immune cells. Thus, the current study analyzed the interaction of sulfur compounds with TLRs upon LPS-induced inflammation. First, the expression pattern of TLRs (TLR2/4) was examined in response to two sulfur compounds during LPS-induced inflammation. The results indicated that LPS increased the expression levels of TLR2/4 proteins, which were markedly downregulated by the addition of 3 µg/ml NTS, 200 mM MSM or 40 µM TLR4-C34 (Fig. 2A). The present study also analyzed the mRNA expression levels of TLR2/4 after LPS treatment in the presence of NTS, MSM or TLR4-C34 in THP-1 cells. The results confirmed the inhibition of LPS-induced expression of TLR2/4 by NTS, MSM and the TLR4 inhibitor (Fig. 2B). Flow cytometric analysis also confirmed that TLR2/4 was downregulated by NTS, MSM or TLR4-C34 (Fig. 2C). These results suggested that the anti-inflammatory effects of natural sulfurs depend on the inhibition of TLR2/4 during LPS-induced inflammation.

Generally, LPS is known to induce inflammation by generating ROS. The present study analyzed the effects of sulfur compounds on ROS generation in LPS-induced inflammation. First, the expression levels of the iNOS protein, which serves an important role in ROS generation, were measured. Western blot analysis revealed increased expression levels of iNOS in response to LPS, which were inhibited by NTS, MSM, and TLR4-C34 (Fig. 3A). These effects were also confirmed at the transcriptional level; increased iNOS mRNA expression levels were observed following LPS treatment, which were suppressed by NTS, MSM, and TLR4-C34 treatment (Fig. 3B). These results suggested that sulfur molecules exhibit similar effects to TLR4-C34, thus indicating the anti-inflammatory effects of NTS and MSM against LPS-induced inflammation. Finally, ROS generation was measured in response to treatment with sulfur compounds at the cellular and mitochondrial levels (Fig. 3C). Increased ROS levels were observed following LPS treatment, which were significantly reduced by treatment with natural sulfur compounds and a TLR4 inhibitor. These results indicated the cytoprotective nature of NTS and MSM against inflammation.

It was hypothesized that NF-κB signaling may be responsible for the anti-inflammatory response induced by NTS and MSM; therefore, the PKC-mediated upstream targets of NF-κB were examined. Increased expression levels of p-PKCα, p-ERK, and p-p38 were observed in LPS-treated THP-1 cells (Fig. 4A). By contrast, NTS, MSM and TLR4-C34 markedly reduced LPS-induced expression of PKC-mediated signaling factors, suggesting the possible involvement of NF-κB activity in the inflammatory response. Next, the present study assessed the expression levels of canonical NF-κB pathway elements. Treatment with LPS upregulated the expression levels of p-IKKα/β and p-IκBα, which were decreased by NTS, MSM, and TLR4-C34 treatment (Fig. 4B). These results indicated the involvement of NF-κB in the anti-inflammatory effects of sulfur compounds against LPS-induced inflammation.

DNA damage is a possible outcome of prolonged ROS production (32). In the present study, LPS-induced ROS generation was inhibited by treatment with sulfur compounds. Subsequently, a comet assay was performed to determine whether LPS induced DNA damage and if sulfur compounds could revert such an effect or induce a DNA damage response (DDR). The analysis of THP-1 cells by fluorescence microscopy revealed an increase in comet length and the number of comet-positive cells following LPS exposure, whereas these effects were reversed by NTS, MSM, and TLR4-C34 treatment (Fig. 5A). These results suggested the possible induction of DDR by sulfur compounds. Next, the current study analyzed the expression levels of several molecular signaling proteins responsible for DDR. LPS induced the expression of DNA damage markers, including p-ATR, p-ATM, p-Chk2, p-BRCA1 and p-p53, and DNA damage by LPS stabilized p53 and p-p53 by inhibiting MDM2-mediated degradation (Fig. 5B); however, NTS, MSM, and TLR4-C34 notably decreased their expression levels, indicating the ability of natural sulfurs to induce DDR in response to LPS-induced inflammation by regulating TLR4 expression.

Two sulfur compounds downregulated the upstream targets of NF-κB during LPS-induced inflammation; therefore, the current analyzed the regulation of NF-κB under the same conditions. The results indicated increased protein expression levels of COX-2 and NF-κB, but not COX-1, in response to LPS, whereas treatment with MSM, NTS, and TLR4-C34 markedy reduced their expression levels (Fig. 6A). The mRNA expression levels of NF-κB and COX-2 were consistent with their protein levels; sulfur molecules inhibited the LPS-induced expression of NF-κB and COX-2, but not COX-1 (Fig. 6B). The present study also analyzed the expression of proinflammatory cytokines, including IL-6, IL-1β and TNF-α, at the protein and mRNA levels (Fig. 6C and D); these were consistently upregulated by LPS and downregulated by NTS, MSM and the TLR4 inhibitor. Next, the current study evaluated the binding of NF-κB to the promoter region of proinflammatory cytokines using ChIP assay, and a significant inhibition of NF-κB binding to proinflammatory cytokines was observed by natural sulfur compounds (Fig. 6E). The inhibition of the LPS-induced nuclear translocation of NF-κB by NTS or MSM also strongly supported the inhibitory mechanism against the LPS-induced NF-κB-dependent inflammatory response (Fig. 6F). These results suggested that sulfur compounds may suppress LPS-induced inflammatory responses by inhibiting iron homeostasis and heme biosynthesis in THP-1 human monocytes (Fig. 7).