Biol. pharm. bull. 32(4): 651-656 (2009)

Biol. Pharm. Bull. 32(4) 651—656 (2009)
The Anti-inflammatory Effects of Methylsulfonylmethane on
Lipopolysaccharide-Induced Inflammatory Responses in Murine
Macrophages
Yoon Hee KIM,a Dae Hwan KIM,a Hwan LIM,b Doo-Yeon BAEK,b Hyun-Kyung SHIN,a,c and Jin-Kyung KIM*,a a Center for Efficacy Assessment and Development of Functional Foods and Drugs, Hallym University; c Department ofFood Chemistry and Nutrition, Hallym University; Chuncheon 200–702, Republic of Korea: and b AD Biotech Co., Ltd.;857–4 Toegye-dong, Chuncheon 200–170, Republic of Korea.
Received December 16, 2008; accepted January 6, 2009; published online January 15, 2009 Methylsulfonylmethane (MSM), also known as dimethyl sulfone and methyl sulfone, is an organic sulfur-
containing compound that occurs naturally in a variety of fruits, vegetables, grains, and animals, including hu-
mans. In the present study, we demonstrated the anti-inflammatory effects of MSM in lipopolysaccharide (LPS)-
stimulated murine macrophages, RAW264.7 cells. MSM significantly inhibited the release of nitric oxide and
prostaglandin E by alleviating the expression of inducible nitric oxide synthase and cyclooxygenase-2 in LPS-
stimulated RAW264.7 cells. Furthermore, the levels of interleukin-6 and tumor necrosis factor-a were decreased
by MSM treatment in cell culture supernatants. Further study indicated that the translocation of the p65 subunit
of nuclear factor (NF)-kB to the nucleus was inhibited by MSM treatment in LPS-stimulated RAW264.7 cells, in
which it helped block degradation of inhibitor of NF-kB. In addition, in vivo studies demonstrated that topical
administration of MSM at 500—1250 mg/ear resulted in similar inhibitory activities in 12-O-tetradecanoylphor-
bol 13-acetate-induced mouse ear edema. Collectively, theses results indicate that MSM inhibits LPS-induced re-
lease of pro-inflammatory mediators in murine macrophages through downregulation of NF-kB signaling.
Key words
methylsulfonylmethane; inflammation; nuclear factor-k B Inflammation is a beneficial host response to foreign There is currently a strong interest in developing new pathogens or tissue injury, and it eventually leads to the anti-inflammatory agents from natural products. Methylsul- restoration of normal tissue structure and function. A normal fonylmethane (MSM) is found in small amounts in many inflammatory response is self-limiting and involves down- foods, including unpasteurized milk, grains, meat, eggs, and regulation of pro-inflammatory protein expression, increased fish.13—15) It is also found in a popular dietary supplement expression of anti-inflammatory proteins, and reversal of the with a molecular weight of 94.16) Health claims associated vascular changes that facilitated the initial immune cell re- with MSM include relief of pain, inflammation, arthritis, al- cruitment process.1—3) Macrophages play a salient role in the lergies, certain parasitic infections, and asthma.17—19) In the inflammatory response and serve as an essential interface be- present study, we investigated the anti-inflammatory effects tween innate and adaptive immunity. Following activation, and underlying mechanisms of action of MSM using LPS-in- macrophages modulate the expression of accessory mole- duced inflammatory responses.
cules such as CD14 and toll-like receptor (TLR) 4.4,5) Stimu-lation of TLR4 by lipopolysaccharide (LPS) triggers the re- MATERIALS AND METHODS cruitment of the cytoplasmic adaptor protein MyD88 andsubsequently culminates in the activation of downstream sig- Cell line and Reagents
RAW264.7 murine macrophages naling pathways: the transcription factor nuclear factor-kB were obtained from the Korean Cell Bank (Seoul, Korea).
(NF-k B) pathway. These pathways induce the expression of LPS derived from Escherichia coli, 12-O-tetradecanoylphor- various inflammatory mediators, including nitric oxide (NO), bol 13-acetate (TPA), dexamethasone and acetone were pur- prostaglandins (PGs), and inflammatory cytokines.6—9) chased from Sigma-Aldrich (St. Louis, MO, U.S.A.). The NF-k B, the key molecule in the inflammatory response, Dulbecco's modified Eagle's medium (DMEM), fetal bovine is a dimeric transcription factor that is formed by the di- serum (FBS), penicillin, and streptomycin used in this study merization of proteins in the Rel family.10) NF-k B activity were obtained from Hyclone (Logan, Utah, U.S.A.). MSMis effected through regulation of the expression of genes was supplied by AD Biotech, Co., Ltd. (Chuncheon, Korea).
that encode inflammatory cytokines, adhesion molecules, Cell Culture, Cell Viability, and Cytotoxicity Assay
chemokines, growth factors, and inducible enzymes such as RAW264.7 murine macrophages were cultured in DMEM cyclooxygenase (COX)-2 and inducible nitric oxide synthase containing 10% FBS, 100 U/ml penicillin, and 100 m g/ml (iNOS).10,11) NF-k B activity is usually inhibited in the cyto- streptomycin at 37 °C in 5% CO . The effects of MSM on plasm through its association with an endogenous inhibitory cell viability and cytotoxicity were tested using CellTiter 96® protein of the Ik -B (inhibitor of NF-k B) family. Upon acti- One Solution Assay of cell proliferation (Promega, vation, Ik -B undergoes phosphorylation and degradation, Madison, WI, U.S.A.) and CytoTox 96® Non-Radioactive processes that facilitate the movement of NF-k B to the cell Cytotoxicity Assay (Promega), respectively. RAW264.7 cells nucleus. Once there, it binds to DNA and induces transcrip- were plated at a density of 1104 cells/well in a 96-well flat- bottom plate, and MSM was added to each well at a concen- ∗ To whom correspondence should be addressed. e-mail: [email protected] 2009 Pharmaceutical Society of Japan tration of 0—10 mg/ml in complete DMEM. After 24 h incu- above and control group was treated with TPA only. Mouse bation, viability and cytotoxicity were measured according to ears were excised 6 h after the last application and homo- the manufacturer's instructions.
geneized in 50 mM Tris–HCl buffer (pH 7.5) with 1 mM Measurement of Nitrite and PGE
The amount of ni- EDTA, and their homogenates were incubated on ice for trite and PGE produced by the mouse macrophages was 20 min in the presence of 0.1% Troton X-100. The ho- measured in RAW264.7 cell culture supernatant. RAW264.7 mogenates were centrifuged at 10000g for 15 min and su-
cells were plated at a density of 1105 cells in a 24-well cell pernatant was collected for cytokine measurement. Parts of culture plate with 500 m l of culture medium and incubated ear were fixed 10% buffered formalin solution, embedded in for 18 h. They were then treated with various concentrations paraffin by standard methods and stained with hematoxylin- (0—10 mg/ml) of MSM in the absence or presence of eosin (H & E).
500 ng/ml of LPS and incubated for another 24 h. The Values are expressed as means amount of nitrite was measured using the Griess reagent sys- S.E.M. of the results for at least three experiments. One-way tem (Promega). The amount of PGE produced was meas- analysis of variance (ANOVA) was used for comparison be- ured using an enzyme-linked immunosorbent assay (ELISA) tween the control and treatment groups. p-values 0.05 were kit (R&D systems, Minneapolis, MI, U.S.A.), according to considered statistically significant.
the manufacturer's instructions.
The amounts of tumor necrosis factor (TNF)-a and interleukin (IL)-6 in the cell culture super-natant were measured using an ELISA kit (eBioscience, San MSM Was Not Toxic to Murine Macrophages
Diego, CA, U.S.A.). RAW264.7 cells were plated in a 24- RAW264.7 macrophages were chosen for use in an investiga- well cell culture plate at a density of 1105 cells and incu- tion of the anti-inflammatory effects of MSM. We first exam- bated for 18 h. They were then treated with 0—10 mg/ml of ined whether MSM has cytotoxicity in RAW264.7 cells. No MSM in the absence or presence of 500 ng/ml of LPS and in- notable cytotoxicity was observed when the cells were ex- cubated for another 24 h. The culture supernatant was col- posed up to 10 mg/ml for 24 h (Fig. 1). Since MSM did not lected and assayed according to the manufacturer's instruc- show cytotoxic effects up to 10 mg/ml, we used MSM at a concentration of 0—10 mg/ml for the subsequent experi- Western Blot Analysis
Cells were washed with ice-cold PBS and scraped, and then whole cell lysates and cytoplas- MSM Inhibits the Release of NO and PGE by Reduc-
mic and nuclear proteins were extracted using PRO-PREP ing iNOS and COX-2 Expression in LPS-Stimulated
Protein Extraction Solution (iNtRON Biotechnology, FL, We initially determined the concentration U.S.A.) and a Nuclear Extract Kit (Active Motif, CA, of LPS that causes inflammatory reaction, such as production U.S.A.), respectively. Twenty micrograms of protein was of inflammatory mediators and Ik -Ba degradation. Both NO,resolved in a loading buffer for sodium dodecyl sulfate IL-6 production and Ik -Ba degradation were induced at con- (SDS)–PAGE, electrophoresed on SDS/polyacrylamide gels, centrations of 10—1000 ng/ml LPS (Fig. 2). Based on these and electroblotted onto polyvinylidene difluoride (PVDF) data, we used 500 or 1000 ng/ml of LPS in order to induce membranes. The membranes were then blocked in Tris- inflammatory responses in the rest of the experiments.
buffered saline (TBS)-Tween 20 solution containing 5% non- In order to assess the anti-inflammatory activities of fat dry milk and incubated overnight at 4 °C with specific an- MSM, RAW264.7 cells were exposed to LPS in the presence tibodies against iNOS (BD Biosciences, San Jose, CA, or absence of MSM, and the level of nitrite, a stable metabo- U.S.A.), COX-2 (BD Biosciences), Ik -Ba (BD Biosciences), lite of NO, was measured in the medium. As shown in Fig.
NF-k B p65 (Cell Signaling Technologies Inc., Beverly, MA, 3A, MSM inhibited LPS-induced NO production in a dose- U.S.A.), b -actin (Sigma), and lamin B (Santa Cruz Biotech- dependent manner.
nology, Santa Cruz, CA, U.S.A.). Proteins were visualized Since PGE is another key inflammatory mediator, we in- using goat anti-rabbit or mouse antibody conjugated to vestigated the effects of MSM on PGE production in LPS- horseradish peroxidase and a chemiluminescence Western stimulated RAW264.7 cells. Similar to the findings related blotting detection system (ECL PlusTM Western BlottingReagents, Amersham Biosciences, Boston, MA, U.S.A.).
Mouse Ear Inflammation Induced by Multiple Applica-
tions of TPA
We assessed the anti-inflammatory activity of MSM by measuring TPA-induced ear edema in mice. Six-week-old female ICR mice were purchased from ORIENTBIO Inc. (Seongnam, Korea) and maintained under specific-pathogen-free conditions at the animal facility of HallymUniversity (Chuncheon, Korea). Inflammation was inducedthrough topical application of TPA (2.5 m g/ear) dissolved inacetone (20 m l) on days 1, 3, 5, 7 and 10 to the inner andouter surfaces of each ear of mice (5 per group) with the aidof a micropipette. The MSM (50, 500, 1250 m g/ear) or dex- Effects of Methylsulfonylmethane (MSM) on Viability and Cyto- amethasone (0.05 mg/ear) was administered topically daily toxicity in Murine Macrophages for 10 consecutive days. The blank group was given topical RAW264.7 cells were treated with 0—10 mg/ml of MSM for 24 h. Cell viability and cytotoxicity were determined as described in Materials and Methods. The results are re- applications of acetone in the same regimen as described ported as meansS.E.M. for three independent experiments.
Effects of LPS on Production of Inflammatory Mediators and Degradation of Ik -Ba RAW264.7 cells were treated with 0—1000 ng/ml of LPS for 24 h. The NO (A) and IL-6 (B) concentrations in cell culture supernatants were measured as described in Materials and Methods. The results are reported as meansS.E.M. for three independent experiments. Significant differences from cells treated with 0 ng/ml LPS are indicated as follows: ∗∗∗ p0.001. (C) RAW264.7 cells were treated with 0—1000 ng/ml of LPS for 30 min. The 50 mg of protein obtained from whole cell lysates were resolved on 10% SDS-PAGE.
Western blot analysis was performed as described in Materials and Methods. b -Actin was used as a loading control.
Effects of Methylsulfonylmethane (MSM) on Production and Expression of Inflammatory Mediators RAW264.7 cells were treated with 0—10 mg/ml of MSM in the presence of LPS (500 ng/ml) for 24 h. The NO (A) and PGE (B) concentrations in cell culture supernatants were measured as described in Materials and Methods. The results are reported as meansS.E.M. for three independent experiments. Significant differences from cells treated with LPSin the absence of MSM are indicated as follows: ∗ p0.05, ∗∗ p0.01, ∗∗∗ p0.001. The 20 mg of protein obtained from cell lysates was resolved on 8% and 10% SDS-PAGE foriNOS and COX-2, respectively (C). Western blot analysis was performed as described in Materials and Methods. b -Actin was used as a loading control.
Effects of Methylsulfonylmethane (MSM) on the Production of LPS-Induced Interleukin (IL)-6 and Tumor Necrosis Factor (TNF)-a Production in Murine Macrophages RAW264.7 cells were treated with 0—10 mg/ml of MSM in the presence of LPS (500 ng/ml) for 24 h. The cell culture supernatants were then collected, and the amounts of re- leased IL-6 (A) and TNF-a (B) were measured as described in Materials and Methods. The results are reported as meansS.E.M. for three independent experiments. Significantdifferences from cells treated with LPS in the absence of MSM are indicated as follows: ∗∗∗ p0.001.
to nitrite accumulation, treatment of RAW264.7 cells with COX-2 was responsible for the inhibition of NO and PGE2 500 ng/ml of LPS led to a significant increase in PGE pro- duction. Consistent with the nitrite accumulation, PGE pro- MSM Reduces the Production of Pro-inflammatory
duction was blocked by MSM treatment (Fig. 3B).
Cytokines in LPS-Stimulated RAW264.7 Cells
In order to determine the mechanism by which MSM re- attempted to examine the potential effects of MSM on the duces LPS-induced NO and PGE production, we studied the production of the pro-inflammatory cytokines, IL-6 and effect of MSM on iNOS and COX-2 protein expression in TNF-a . RAW264.7 cells were incubated with MSM in the RAW264.7 cells using Western blot analysis. As shown in presence of 500 ng/ml of LPS for 24 h, and IL-6 and TNF-a Fig. 3C, iNOS and COX-2 protein expression were markedly levels were evaluated in the culture supernatants. Because of induced in RAW264.7 cells after treatment with 500 ng/ml of LPS exposure, the IL-6 and TNF-a level had significantly in- LPS for 24 h. This induction was suppressed by MSM treat- creased to 888.353.6 and 12224.0178.2 pg/ml, respec- ment in a dose-dependent manner. These results indicate that tively. MSM treatment (10 mg/ml) prevented significant in- MSM-induced reduction in the expression of iNOS and creases in IL-6 and TNF-a levels, holding them at 21.98.5 and 8528.0487.1 pg/ml, respectively (Fig. 4).
flammation. Ear edema was measured in the ears prior to and MSM Inhibits Degradation of Ik-Ba and Nuclear
at 6 h following treatments. As shown in Figs. 6A and B, ex- Translocation of NF-kB p65 in LPS-Stimulated
posure to TPA resulted in marked increases in skin thickness.
Finally, we examined NF-k B activation Topical application of acetone (vehicle) did not alter the skin to determine the molecular mechanisms by which MSM in- thickness significantly. However, MSM (500, 1250 m g/ear) or hibits LPS-induced inflammatory responses. NF-k B is a dexametasone significantly inhibited the TPA-induced in- major transcription factor involved in the release of proteinsthat mediate the inflammatory response, and the degradationand phosphorylation of Ik -B are necessary to release NF-k Bfrom the cytoplasmic NF-k B/Ik -B complex and to allow itssubsequent translocation to the cell nucleus. We evaluatedthe effect of MSM on NF-k B activation to determine if it ismediated by Ik -B degradation. We observed that LPS-in-duced Ik -B degradation was inhibited after 30 min of expo-sure to MSM (Fig. 5A).
In order to directly investigate the effect of MSM on nu- clear translocation, we determined NF-k B p65 levels amongboth cytosolic and nuclear proteins. The expression levels ofNF-k B p65 protein were decreased in the nuclear fractionsof cells when they were exposed to MSM, indicating thatMSM inhibits the translocation of NF-k B p65 protein fromthe cytosol to the nucleus (Fig. 5B). These findings suggestthat MSM exerts anti-inflammatory actions by blocking NF-kB signal.
In order to address whether MSM inhibits the activation of NF-k B induced by other stimuli other than LPS, we used re-combinant mouse TNF-a as a stimuli for NF-k B activation.
MSM also inhibited the degradation of Ik -Ba in TNF-astimulated RAW264.7 cells (Fig. 5C). This result suggests Effects of Methylsulfonylmethane (MSM) on LPS-Induced Degra- that MSM blocks NF-k B activation caused by LPS or TNF-a dation of Ik -Ba and Nuclear Translocation of NF-k B-p65 in Murine RAW264.7 cells were treated with 0—10 mg/ml of MSM in the presence of 1 m g/ml Effect of MSM on TPA-Induced Cutaneous Inflamma-
of LPS (A, B) or recombinant mouse TNF-a (C) for 30 min. The 50 m g of protein ob- We assessed the anti-inflammatory activity of MSM in tained from whole cell lysates, cytosolic and nuclear fractions of each cell were re- a TPA-induced ear inflammation model. Increased skin thick- solved on 10% SDS-PAGE. Western blot analysis was performed as described in Mate-rials and Methods. b -Actin and lamin B were used as loading controls of cytosolic pro- ening is often the first hallmark of skin irritation and local in- tein and nuclear protein, respectively.
Effects of Methylsulfonylmethane (MSM) on TPA-Induced Ear Inflammation Mice were treated with MSM (0, 50, 500, 1250 m g/ear) or dexametasone (50 m g/ear) with topical application of acetone (vehicle) or TPA in acetone. Ear thickness was measured at 6 h after TPA treatment (A). Representative micrographs of H&E-stained mouse ear cross-sections in TPA-induced ear inflammation model. Ears were harvested 6 h post-treat-ment with acetone vehicle, TPA plus MSM or dexametasone (B). Sections shown are representative of observations from five animals in each group (200 magnification). Serumand ear homogenates were taken 6 h after TPA treatment and examined for the production of the IL-6 using ELISA (C). The results are reported as meansS.E.M. for five mice pergroup. Significant differences from mice treated with TPA in the absence of MSM are indicated as follows: ∗∗ p0.01, ∗∗∗ p0.001.
creases in ear thickness, indicating the therapeutic effect of degradation and the nuclear translocation of p65. The nuclear this extract.
translocation and DNA binding of NF-k B is preceded by the In addition, topical application of TPA caused a dramatic degradation of Ik -Ba . Our study indicates that MSM inhib- increase in the production of IL-6 in plasma and ear ho- ited LPS-induced inflammatory responses via blocking the mogenates by 6 h after challenge. In contrast, treatment with degradation of Ik -Ba and the subsequent nuclear transloca- TPA plus MSM (1250 m g/ear) or dexamethasone reduced IL- tion of NF-k B p65. Previous studies have shown that the 6 levels significantly (Fig. 6C).
phosphorylation of Ik -B is regulated by a and b isoforms ofIk -B kinase complex (IKK).31) Although we did not observe the effect of MSM on IKK activity, MSM surely inhibitedIk -Ba degradation. It might represent MSM inhibits IKK ac- It has been reported that MSM has positive effects on a va- tivity, Ik -Ba degradation and nuclear translocation of NF- riety conditions, including osteoarthritis,20) allergic rhinitis,17) kB p65 in LPS-stimulated RAW264.7 cells.
and cancer.21) However, studies addressing the working MSM is used orally and topically. Although the optimum mechanisms of MSM are limited. In the present study, we dosage has not been clearly defined, the suggested oral thera- aimed to examine the anti-inflammatory effects of MSM peutic doses are 4—6 g/d.19,20) There is limited formal safetyand to elucidate its underlying mechanisms using murine data, and no long-term assessment has been performed.
macrophage cells and mouse ear inflammation induced by However, MSM is rapidly excreted from the body,32,33) and animal toxicity studies of MSM have shown only minor ad- We found that MSM dramatically inhibits LPS-induced in- verse effects with doses of 1.5 g/kg and 2.0 g/kg of MSM for creases in NO and PGE production through suppression of 90 d.34) This represents a human dose of 30—42 g/d, which is iNOS and COX-2 expression (Fig. 3). We also found that equivalent to 5—7 times the proposed maximum recom- MSM strongly inhibits IL-6 and TNF-a production in LPS- mended human dose of 6 g/d.34) Another study confirmed stimulated murine macrophages (Fig. 4). The expression of that MSM has no toxic effects on either pregnant rats or their inflammatory mediators such as NO, PGE , IL-6, and TNF-a fetuses.35) Based on these previous results and the present is regulated by the key transcription factor, NF-k B.22) NK- data, we believe MSM is relatively safe and effective for the kB is maintained in a latent form in the cytoplasm, where it treatment of inflammatory diseases.
is in complex with the inhibitory Ik -B proteins. The interac-tion of NF-k B with Ik -B masks the nuclear localization sig- This work was supported by the nal. Phosphorylation of Ik -B by Ik -B kinase leads to ubiqui- Regional Innovation Center (RIC) program of the Ministry of tination of the protein and its subsequent degradation. NF- Commerce, Industry, and Energy, Republic of Korea and kB is then free to translocate to the nucleus, where it binds to Korea Association of Small Business Innovation Research DNA and induces activation of a wide variety of target genes (lab-07-1-2), Republic of Korea.
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