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Introduction

Iron is a major nutrient and an essential component of hemoglobin, which is present in red blood cells and carries oxygen from the lungs to the rest of the body. Iron is also involved in metabolism redox regulation and reactive oxygen species (ROS) production. To support these mechanisms, a comprehensive association between iron transport and homeostasis is required (1). The balance between iron as a nutritional source and a toxicant is sensitive and delicate, and iron transport and homeostasis are closely associated with inflammation (2,3). Macrophages are essential in balancing iron transport and inflammation. M1 macrophages are involved in iron uptake when iron is accumulated through ferroportin (FPN) and heme oxygenase (HO)-1 activities; this directs M2 macrophages, along with ferritin, to export excess iron and to inhibit proinflammatory cytokines (4).

Iron is a major contributor in the biosynthesis of heme, which is a porphyrin molecule containing a ferrous ion (Fe2+). Notably, iron-dependent ferritin, carbon monoxide and bilirubin are formed through heme degradation (5), and heme is involved in various physiological processes, including inflammation that is induced by excess heme production. However, heme is an iron-containing complex that is essential for a number of biological processes, including oxygen transport and storage, electron transfer and protein synthesis. Therefore, heme is considered a double-edged sword because it can be toxic by mediating inflammation (6). It also participates in oxygen transport, signal transduction and mitochondrial function, and an imbalance in iron/heme metabolism may cause tumorigenesis (7). Free heme molecules and heme-containing proteins are generated under various pathological conditions, such as ischemia/reperfusion, hemorrhage and muscle injuries (8). An excess amount of heme also contributes to renal failure because of local inflammation and promotes the expression of proinflammatory cytokines, such as IL-1β and TNF-α (9).

Lipopolysaccharide (LPS) is a vital component of the outer layer of gram-negative bacteria, which induces innate immunity and initiates the inflammatory response in other cells (10). LPS is a pathogenic stimulator and can induce an inflammatory response by regulating pathogen-associated molecular patterns (11). Toll-like receptor (TLR)4 recognizes innate LPS and controls the innate immune response by neutralizing proinflammatory factors (12). LPS also activates various inflammatory signaling molecules, such as inducible nitric oxide synthase (iNOS), COX-2, NF-κB and p38-MAP kinase pathways (13). When exposed to LPS, TLRs activate innate immunity by activating NF-κB through canonical or noncanonical pathways and proinflammatory cytokines, such as TNF-α and various ILs, in response to inflammation (14).

Sulfur is an essential element in amino acids, such as cysteine and methionine, as well as in some sulfur-containing natural compounds that exert anti-inflammatory activities (15). Sulfur intake is possible through the diet, for example via the consumption of garlic, onion and duck meat (16). Methylsulfonylmethane (MSM) is a natural sulfur compound with various capabilities, such as anti-ketotic (17), antioxidant (18,19) and anticancer activities (20,21). Studies on MSM have demonstrated its anti-inflammatory activity against various inflammatory conditions (22,23). MSM is also found in cardiac cells, where it inhibits the inflammatory response by mediating TNF-α (24). In some instances, sulfur can be toxic; however, it can be coated with various nontoxic substances to enhance the effect of sulfur compounds against various conditions, including inflammation (25). Nontoxic sulfur (NTS) is a sulfur compound coated with a nontoxic substance, which when used to enrich livestock feed can enhance meat quality and boost immunity (26). In a previous study, rats orally administered NTS did not experience cell death (27). Furthermore, our previous study on NTS indicated that it can enhance the signaling of growth hormone in C2C12 mouse myoblasts (28). In addition, NTS inhibits inflammatory responses in C2C12 cells by mediating TLR4 and JAK2/STAT3 signaling to regulate IL-6 expression (29). Our previous study also demonstrated the anti-inflammatory effects of MSM and NTS against high glucose-induced inflammatory conditions by regulating NF-κB signaling in THP-1 human monocytes (30).

The present study aimed to determine the effects of NTS and MSM on LPS-induced inflammation, and to assess the role of iron/heme metabolism in the anti-inflammatory activity of these sulfur compounds. Molecular analyses of the TLR and NF-κB signaling pathways, iron homeostasis and heme biosynthesis were also performed under LPS-induced conditions.

Materials and methods

Reagents and antibodies

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.

Cell culture and treatment

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).

Fe2+ determination assay

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.

Western blotting

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).

Reverse transcription-quantitative PCR (RT-qPCR) assay

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.

Table I. Primer sequences used for reverse transcription-quantitative PCR analysis.

Table I.

Primer sequences used for reverse transcription-quantitative PCR analysis.

Gene	Sequence
TLR2	Sense: 5′-TGCAAGTACGAACTGGACTTCT-3′
	Antisense: 5′-CCAGGTAGGTCTTGGTGTTCATT-3′
TLR4	Sense: 5′-TAGCCATTGCTGCCAACATCAT-3′
	Antisense: 5′-AAGATACACCAACGGCTCTGAA-3′
iNOS	Sense: 5′-TGCTCAGCTCATCCGCTATG-3′
	Antisense: 5′-GATGTTCCATGGCCACCTCA-3′
NF-κB	Sense: 5′-GAAATTCCTGATCCAGACAAAAAC-3′
	Antisense: 5′-ATCACTTCAATGGCCTCTGTGTAG-3′
COX-1	Sense: 5′-GGCAGCAGAGTTGGAGGAAT-3′
	Antisense: 5′-CTTCTTCAGTGTGGCCGTCT-3′
COX-2	Sense: 5′-TTGCATTCTTTGCCCAGCAC-3′
	Antisense: 5′-ACCGTAGATGCTCAGGGACT-3′
IL-1β	Sense: 5′-ATTGCCTCTTCCAGCAGCTT-3′
	Antisense: 5′-GGCTTCACTGAGGTTGCCTT-3′
IL-6	Sense: 5′-GCTGATCCTGCCTCTGCC-3′
	Antisense: 5′-GACCCTCAAACCCACCCG-3′
TNF-α	Sense: 5′-TGGTGAGACAGAAAGAGCGG-3′
	Antisense: 5′-AGCCCTGAGGTGTCTGGT-3′
GAPDH	Sense: 5′-ACCCACTCCTCCACCTTTGA-3′
	Antisense: 5′-CATACCAGGAAATGAGCTTGACAA-3′

[i] iNOS, inducible nitric oxide synthase; TLR, Toll-like receptor.

Flow cytometric analysis

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.

Comet assay

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).

Chromatin immunoprecipitation (ChIP) assay

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 extraction assay and NF-κB detection

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.

Statistical analyses

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.

Results

Sulfur compounds inhibit LPS-induced iron metabolism and heme biosynthesis in THP-1 human monocytes

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.

Figure 1. Sulfur compounds inhibit LPS-induced inflammation and iron/heme metabolism. (A) Flow cytometry showing Fe2+ levels in THP-1 cells following treatment with LPS (10 ng/ml) + NTS (3 µg/ml), MSM (200 mM) or TLR-C34 (40 µM) for 48 h. Western blot analysis of (B) transferrin receptor, ferroportin, DMT1 and STEAP3; and (C) MFRN, ABCB6, ABCB10, ALAS1, HO-1, FECH and FLVCR proteins in THP-1 cells following treatment with LPS (10 ng/ml) + NTS (3 µg/ml), MSM (200 mM) or TLR-C34 (40 µM) for 48 h. ALAS1, 5′-aminolevulinate synthase 1; DMT1, divalent metal transporter 1; Fe2+, ferrous ion; FECH, ferrochelatase; FPN, ferroportin; HO-1, heme oxygenase-1; LPS, lipopolysaccharide; MFRN, mitoferrin; MSM, methylsulfonylmethane; NTS, nontoxic sulfur; TfR, transferrin receptor; TLR, Toll-like receptor.

Sulfur compounds downregulate the LPS-induced expression of TLRs in THP-1 cells

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.

Figure 2. Sulfur compounds inhibit LPS-induced TLR expression. (A) Western blot analysis of TLR2 and TLR4 expression in THP-1 cells treated with LPS (10 ng/ml) + NTS (3 µg/ml), MSM (200 mM) or TLR-C34 (40 µM) for 48 h. (B) Reverse transcription-quantitative PCR analysis of the relative expression levels of TLR2 and TLR4 normalized to GAPDH following treatment with LPS (10 ng/ml) + NTS (3 µg/ml), MSM (200 mM) or TLR-C34 (40 µM) for 48 h. ***P<0.001; #P<0.001 vs. non-treated control (one-way ANOVA and Tukey's test). (C) Flow cytometry showing the inhibition of LPS-induced TLR4 expression by 48-h treatment with NTS (3 µg/ml), MSM (200 mM) and TLR-C34 (40 µM). LPS, lipopolysaccharide; MSM, methylsulfonylmethane; NTS, nontoxic sulfur; TLR, Toll-like receptor.

Sulfur molecules decrease LPS-induced ROS generation in THP-1 cells

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.

Figure 3. Sulfur compounds inhibit LPS-induced ROS. (A) Western blot analysis of iNOS expression in THP-1 cells treated with LPS (10 ng/ml) + NTS (3 µg/ml), MSM (200 mM) or TLR-C34 (40 µM) for 48 h. (B) Reverse transcription-quantitative PCR analysis of iNOS mRNA expression in cells treated with LPS (10 ng/ml) + NTS (3 µg/ml), MSM (200 mM) or TLR-C34 (40 µM) for 48 h. Data are normalized to GAPDH. ***P<0.001; #P<0.001 vs. non-treated control (one-way ANOVA and Tukey's test). (C) Flow cytometric analysis of cellular and mitochondrial ROS in THP-1 cells following treatment with LPS (10 ng/ml) + NTS (3 µg/ml), MSM (200 mM) or TLR-C34 (40 µM) for 48 h. iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; MSM, methylsulfonylmethane; NTS, nontoxic sulfur; TLR, Toll-like receptor; ROS, reactive oxygen species.

Sulfur molecules inhibit PKC-mediated inflammation and canonical NF-κB pathways against LPS-induced inflammation in THP-1 cells

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.

Figure 4. Sulfur compounds inhibit the LPS-induced PKC-dependent and canonical NF-κB pathways. Western blot analysis of THP-1 cells treated with LPS (10 ng/ml) + NTS (3 µg/ml), MSM (200 mM) or TLR-C34 (40 µM) for 48 h showing the inhibition of LPS-induced (A) p-PKC-α, p-ERK and p-p38 expression; and (B) p-IKKα/β and p-IκBα expression. LPS, lipopolysaccharide; MSM, methylsulfonylmethane; NTS, nontoxic sulfur; p-, phosphorylated; TLR, Toll-like receptor.

Sulfur molecules alleviate LPS-induced DNA damage in THP-1 cells

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.

Figure 5. Sulfur compounds induce DNA damage response following LPS-induced DNA damage. (A) Images of the comet assay were captured by fluorescence microscopy at ×10 and ×40 magnification levels, showing the fragmented DNA migrating out of the nucleoid body, which formed a comet tail following treatment with LPS (10 ng/ml) + NTS (3 µg/ml), MSM (200 mM) or TLR-C34 (40 µM) for 48 h. *P<0.05 and ***P<0.001; $P<0.05 vs. non-treated control; and $$$P<0.001 vs. non-treated control (one-way ANOVA and Tukey's test). (B) Western blot analysis of THP-1 cells; NTS (3 µg/ml) and MSM (200 mM) inhibited the LPS-induced expression of p-ATM, p-ATR, p-Chk2, p-BRCA1, and p-p53. However, the expression levels of p-MDM2 were suppressed by LPS treatment, and were increased by NTS (3 µg/ml), MSM (200 mM) or TLR4-C34 (40 µM). LPS, lipopolysaccharide; MSM, methylsulfonylmethane; NTS, nontoxic sulfur; p-, phosphorylated; TLR, Toll-like receptor.

Sulfur molecules suppress the expression of LPS-induced NF-κB and proinflammatory cytokines in THP-1 cells

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).

Figure 6. Sulfur compounds inhibit LPS-induced NF-κB, COX-2 and proinflammatory cytokine expression. (A) Western blot analysis of the expression levels of NF-κB, COX-1 and COX-2 in THP-1 cells treated with LPS (10 ng/ml) + NTS (3 µg/ml), MSM (200 mM) or TLR-C34 (40 µM) for 48 h. (B) RT-qPCR analysis of the relative expression levels of NF-κB, COX-1 and COX-2 normalized to GAPDH following treatment with LPS (10 ng/ml) + NTS (3 µg/ml), MSM (200 mM) or TLR-C34 (40 µM) for 48 h. ***P<0.001; #P<0.001 vs. non-treated control (one-way ANOVA and Tukey's test). (C) Western blot analysis of the expression levels of IL-1β, IL-6 and TNF-α in THP-1 cells treated with LPS (10 ng/ml) + NTS (3 µg/ml), MSM (200 mM) or TLR-C34 (40 µM) for 48 h. (D) RT-qPCR analysis of the relative expression levels of IL-1β, IL-6 and TNF-α normalized to GAPDH in cells following treatment with LPS (10 ng/ml) + NTS (3 µg/ml), MSM (200 mM) or TLR-C34 (40 µM) for 48 h. ***P<0.001; #P<0.001 vs. non-treated control (one-way ANOVA and Tukey's test). (E) Chromatin immunoprecipitation assay of THP-1 cells treated with LPS (10 ng/ml) + NTS (3 µg/ml), MSM (200 mM) or TLR-C34 (40 µM) for 48 h showing the relative binding of NF-κB to the promoters of IL-1β, IL-6 and TNF-α. ***P<0.001; #P<0.001 vs. non-treated control (one-way ANOVA and Tukey's test). (F) Nuclear protein extract analysis of THP-1 cells treated with LPS (10 ng/ml) + NTS (3 µg/ml), MSM (200 mM) or TLR-C34 (40 µM) for 48 h showing the protein expression levels of nuclear NF-κB. TBP was used as the housekeeping protein for the nuclear extract and β-actin was used to show the efficacy of nuclear protein extraction. LPS, lipopolysaccharide; MSM, methylsulfonylmethane; NTS, nontoxic sulfur; RT-qPCR, reverse transcription-quantitative PCR; TBP, TATA-binding protein; TLR, Toll-like receptor.

Figure 7. Molecular mechanism of LPS-induced regulation of the inflammatory response through iron/heme metabolism and TLR4/NF-κB expression through the canonical NF-κB and PKC-mediated inflammatory pathways. The anti-inflammatory activities of NTS and MSM were achieved by inhibiting iron/heme metabolism and suppressing the expression of TLR4/NF-κB signaling molecules, thus blocking the binding of NF-κB to the gene promoters of proinflammatory cytokines. DMT1, divalent metal transporter 1; Fe2+, ferrous ion; FPN, ferroportin; TfR, transferrin receptor; TLR4, Toll-like receptor 4.

Discussion

Natural compounds can effectively ameliorate inflammatory responses as they generally lack side effects; they also exhibit anti-inflammatory properties against LPS-induced inflammation (33). MSM and NTS exert anti-inflammatory effects, and these compounds may be suitable candidates for anti-inflammatory treatment, similar to artemisinin or artesunate, if they can inhibit the effects of LPS-induced inflammation. In the present study, it was demonstrated that NTS and MSM inhibited LPS-induced inflammation in THP-1 human monocytes and that iron/heme metabolism may serve an important role in this mechanism.

Iron metabolism is important to the inflammatory process and is essential for human health. Notably, an imbalance in iron transport can cause inflammation (34). LPS-treated THP-1 cells exhibit elevated Fe2+ levels, suggesting an increased amount of iron that must be oxidized. A failure in converting Fe2+ into Fe3+ for oxidation results in inflammation (35). The present study observed decreased Fe2+ levels following NTS and MSM treatment, suggesting that these sulfur compounds can regulate iron transport and inflammation. Generally, iron balance depends on FPN and TfR, which mediate iron homeostasis through iron transport; whereas FPN exports iron, TfR takes up Fe3+ ion for metabolism (36). DMT1 is another membrane transporter that transports iron from the endosomal system to the cytosol and functions in intestinal iron absorption by oxidizing Fe3+ before the translocation of Fe2+ (37,38). Inside the endosome, STEAP3 controls iron transport by reducing Fe3+ to Fe2+ (39). Therefore, dysregulation in the expression of these receptors may contribute to inflammation. The present study observed the increased expression of TfR, FPN, DMT1 and STEAP3 following LPS treatment, suggesting increased iron transport. NTS and MSM inhibited the expression of these receptors, which in turn, ameliorated iron transport and exerted anti-inflammatory effects.

Iron is an important molecule for heme biosynthesis and in the mitochondria, where it is essential for energy production, antioxidant defense and signal transduction (40). Generally, ALAS1 is important for heme synthesis, and combines glycine and succinyl-CoA to form aminolaevulinic acid. The FECH enzyme is also involved in heme synthesis by combining Fe2+ and protoporphyrin IX (41). ABCB6 is a putative promoter that contributes to the transition of ALA, and with the help of ALAS1, FECH and MFRN, promotes iron uptake for its interaction with FECH and ABCB10, which leads to heme synthesis (42,43). The resulting heme is then transferred to the cytoplasm with the help of a heme exporter, FLVCR1 (44). Similar to the present study, in a previous study, LPS has been shown to upregulate the expression of HO-1, which catalyzes the degradation of heme, and the expression of the proteins responsible for heme synthesis, including ALAS1, FECH, ABCB6, MFRN, ABCB10 and FLVCR (5). These results indicated that LPS-induced inflammation may promote heme production. NTS and MSM inhibited the expression of these proteins, which indicated an inhibition of heme synthesis by these sulfur compounds. The TLR4 inhibitor did not alter the expression of these proteins, indicating that heme synthesis may be independent of TLR4 expression.

TLR4 is a transmembrane receptor that mediates a signaling response to inflammation and represents a major part of the LPS-induced inflammatory response (45,46). TLR4 transduces signals to the major downstream target NF-κB, which is translocated into the nucleus upon activation to upregulate proinflammatory cytokines (47,48). The current study observed increased expression levels of TLR2 and TLR4 during LPS treatment, which were significantly reduced by NTS or MSM. These findings indicated that the anti-inflammatory effect of these sulfur compounds may be mediated by regulating TLR expression. A similar effect was also observed on NF-κB expression. Furthermore, the inhibition of NF-κB expression by TLR4-C34 treatment indicated that these sulfur compounds act through TLR4/NF-κB signaling.

Activation of NF-κB occurs through a canonical pathway (49) or a PKC-dependent pathway (50). The canonical pathway depends on IκBα and IKKα/β, whereas the PKC-dependent pathway signals through the ERK/p38 signaling pathway (11). Similar to the present study, in a previous study, LPS has been reported to upregulate p-IKKαβ and IκBα levels in the canonical pathway, whereas NTS or MSM downregulated the expression of these molecules without affecting the expression of total IκBα (11). In the PKC-dependent pathway, LPS-induced inflammation increased the expression of p-PKCα, p-ERK and p-p38 without altering the expression levels of their total forms. By contrast, the natural sulfur compounds NTS and MSM suppressed the LPS-induced expression of p-PKCα, p-ERK and p-p38 during TLR4-independent signaling. Upon inflammation, NF-κB is translocated into the nucleus and induces the transcription of genes encoding proinflammatory cytokines to elicit an immune response (51). The current study demonstrated that LPS-induced inflammation promoted the translocation of NF-κB into the nucleus, where it may bind to the promoters of the proinflammatory cytokines, IL-6, IL-1β and TNF-α. Notably, NTS and MSM successfully inhibited the translocation of NF-κB into the nucleus and blocked its binding to these proinflammatory cytokine gene promoters. Furthermore, the LPS-induced COX-2 expression was reversed by the two sulfur compounds without affecting the expression of COX-1, indicating the inhibition of the inflammatory response by NTS and MSM in a COX-1-independent and TLR4-dependent manner.

LPS-induced inflammation generates ROS that leads to oxidative damage (52). iNOS induction also results in ROS generation, and LPS can induce iNOS expression and ROS generation (53). The present results confirmed the increase in iNOS mRNA and protein expression, as well as cellular and mitochondrial ROS generation. Moreover, NTS and MSM suppressed the expression levels of iNOS and ROS generation at the cellular and mitochondrial levels. These results indicated the role of oxidative stress in LPS-dependent inflammation and the effect of sulfur compounds, which markedly reduced the inflammatory response by suppressing oxidative stress. This prolonged oxidative stress can cause DNA damage, and LPS has been shown to induce DNA double-strand breaks to promote tumorigenesis (32). Anti-inflammatory drugs may inhibit DNA damage by inducing DDR. In the present study, NTS and MSM suppressed the formation of DNA strand breaks, as determined using the comet assay, suggesting the induction of DDR by these sulfur molecules. The downregulation of LPS-induced expression levels of the proteins responsible for DDR by NTS or MSM also strongly supported the anti-inflammatory effects of these sulfur molecules by inhibiting LPS-induced oxidative stress. The present study also observed that the natural sulfur molecules could decrease the expression levels of MFRN, ABCB6, ABCB10, ALAS1, FECH and FLVCR, which are associated with the iron/heme metabolism in THP1 cells, whereas TLR4-C34 (TLR4 inhibitor) did not affect these expression levels. In addition, it was confirmed that natural sulfur molecules inhibited the TLR4/NF-κB-mediated inflammatory response. Although the direct interaction between sulfurs and TLR4-C34 has not yet been assessed, it may be hypothesized that sulfur molecules decrease the inflammatory response by inhibiting iron/heme metabolism prior to suppressing the TLR4/NF-κB pathway.

In conclusion, the natural sulfur compounds NTS and MSM inhibited the LPS-induced inflammatory response in THP-1 human monocytes. Iron/heme metabolism is important to the anti-inflammatory activity of these sulfur compounds, which includes the inhibition of LPS-induced expression of TLR4 and NF-κB through canonical and PKC-dependent pathways, thus suppressing the production of the proinflammatory cytokines, COX-2, IL-1β and IL-6. In addition, NTS and MSM suppressed LPS-induced ROS production and induced DDR in THP-1 human monocytes. Therefore, NTS and MSM may have potential as therapeutic candidates for inflammatory diseases caused by LPS, similar to artemisinin or artesunate.

Acknowledgements

Not applicable.

Funding

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (Ministry of Science and ICT) to KJJ (grant no. RS-2024-00450676). This work was also supported by the Basic Science Research Program to the Research Institute for Basic Sciences of Jeju National University through the NRF funded by the Ministry of Education to SWB (grant no. 2019R1A6A1A10072987). Additionally, this research was supported by a Korea Basic Science Institute (National Research Facilities and Equipment Center) grant funded by the Ministry of Education (grant no. 2023R1A6C101A045).

Availability of data and materials

The data generated in the present study may be requested from the corresponding author.

Authors' contributions

KJJ designed the experiments and wrote the manuscript. DYK and SWB performed all the experiments and analyzed the data. KJJ, DYK and SWB confirm the authenticity of all the raw data. All authors helped to revise the manuscript, and read and approved the final version of the manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References