Research Article Rebamipide, an Amino Acid Analog of 2(1H) … · 2019. 7. 30. · Research Article Rebamipide, an Amino Acid Analog of 2(1H)-Quinolinone, Inhibits the Formation of

Research ArticleRebamipide an Amino Acid Analog of 2(1H)-QuinolinoneInhibits the Formation of Human Osteoclasts

Yuki Nanke Tsuyoshi Kobashigawa Toru Yago Manabu KawamotoHisashi Yamanaka and Shigeru Kotake

Institute of Rheumatology Tokyo Womenrsquos Medical University 10-22 Kawada-cho Shinjuku-ku Tokyo 162-0054 Japan

Correspondence should be addressed to Yuki Nanke ynntwmuacjp

Received 3 August 2016 Accepted 20 October 2016

Academic Editor Yi-Ping Liu

Copyright copy 2016 Yuki Nanke et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Objectives Drug repositioning or drug reprofiling (DR) has recently been growing in importance DR has a significant advantageover traditional drug development because the repositioned drug has already passed toxicity tests its safety is known and therisk of adverse toxicology is reduced In the current study we investigated the role of rebamipide a mucosa-protecting agent withrecently reported anti-inflammatory function in human osteoclastogenesisMethodsPeripheral bloodmononuclear cells (PBMCs)were cultured in the presence of M-CSF and sRANKL Osteoclast formation was evaluated by immunohistological staining forCD5161 (vitronectin receptors) Osteoclast formation in the presence or absence of rebamipide (0 1 and 3mM) was observedby time-lapse photography and actin ring formation The number of absorption sites and area of absorption were calculated usingOsteologic plates Pit formation was studied by 3D-SEM Results Rebamipide inhibited human osteoclast formation at 3mM apharmacological concentration and inhibited resorbing activity dose-dependently Rebamipide induced the degradation of actinrings in mature osteoclastsThis mechanismmay involve inhibiting the osteoclast fusion pathway through reducing the expressionof DC-specific transmembrane protein (DC-STAMP) Conclusions The present study suggests that rebamipide would be useful asa novel agent for osteoporosis and rheumatoid arthritis

1 Introduction

Drug repositioning or drug reprofiling (DR) has recentlybeen growing in importance [1] DR has a significantadvantage over traditional drug development because therepositioned drug has already passed toxicity tests its safety isknown and the risk of adverse toxicology is reduced UsingDR pharmaceutical companies have achieved a number ofsuccesses The use of repurposed drugs can bypass much ofthe early cost and time needed to bring a drug to market Atthis point to investigate new pleiotropic effects of drugs isvery meaningful For example Mundy et al reported that thestatins drugswidely used for lowering serumcholesterol alsoenhance new bone formation and thus statins may be usedfor the treatment of osteoporosis [2]

Rheumatoid arthritis (RA) is a chronic inflammatorydisease characterized by synovitis and the destruction ofarticular cartilage and bone [3 4] RA causes osteoporosisOsteoclasts multinucleated giant cells contain calcitonin

receptors and have the ability to form resorption pits onbone or dentin slices Receptor activator of nuclear factor-120581B ligand (RANKL) induces osteoclastogenesis by binding toRANK Osteoprotegerin (OPG) is a soluble decoy receptor ofRANKL These three molecules OPG RANK and RANKLplay an important role in regulating osteoclastogenesisOsteoclasts play a crucial role in both osteoporosis and bonedestruction [5]Thus they are target cells to treat bothRAandosteoporosis In fact some agents such as anti-RANKLantibody denosumab which inhibits osteoclastogenesis arenow used as a drug for osteoporosis and RA

We recently demonstrated that geranylgeranylacetone(GGA) a nontoxic inducer of heat shock protein used asan antiulcer drug potently inhibits human osteoclastogenesisinduced by soluble RANKL [6] and also induces cell deathin fibroblast-like synoviocytes (FLS) from patients with RAby inhibiting protein geranylgeranylation [7] We showed thepleiotropic effects of GGA antiosteoclastogenesis and anti-inflammatory effects

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 6824719 7 pageshttpdxdoiorg10115520166824719

2 BioMed Research International

Rebamipide (2-(4-chlorobenzoylamino)-3-[2(1H)-qui-nolinon-4-yl]propionic acid OPC-12759 molecular weight37079) is a mucosal protective agent used for the treatmentof gastritis and gastric ulcer Rebamipide is derived from agastric mucosal stabilizer and gastric mucosal prostaglandininducer It was shown to increase the PGE

2content in

the gastric mucosa and exhibit a gastric cytoprotectiveeffect to prevent mucosal injury [8 9] This drug alsodirectly scavenged hydroxyl radicals suppressed superoxideproduction and prevented inflammatory cell infiltration [10]In addition it inhibited the activation of NF-120581B expressionof interleukin-8 mRNA and production of interleukin-8in epithelial cells [11] Also it was recently reported thatrebamipide works as an anti-inflammatory agent in bothacute and chronic inflammation and has an inhibitory effecton proinflammatory cytokines [12]

In the current study we investigated the effect of rebami-pide an amino acid analog of 2(1H)-quinolinone on humanosteoclastogenesis

2 Materials and Methods

21 Chemicals and Reagents Rebamipide was supplied byOtsuka Co Ltd (Tokyo Japan) Recombinant human mac-rophage colony-stimulating factor (M-CSF) was obtainedfrom Kyowa Hakko Kirin (Tokyo Japan) Recombinanthuman soluble RANKL (sRANKL) was purchased fromPeproTech (London UK) For in vitro experiments thesereagents were dissolved in double-distilled water andadjusted to appropriate concentrations for each experiment

22 Cells and Cell Culture for Osteoclastogenesis Humanperipheral blood was collected from healthy normal vol-unteers Peripheral blood mononuclear cells (PBMCs) wereisolated by centrifugation over Histopaque 1077 (Sigma StLouisMOUSA) density gradients washed and resuspendedat 13 times 106 cellsmL in a-minimal essential medium (GibcoBRL Gaithersburg MD USA) supplemented with 10 fetalbovine serum (JRH Biosciences Lenexa KS) PBMCs werethen cultured for 3 days in 48-well plates in the presence ofM-CSF (100 ngmL) Nonadherent cells were then removedAdherent PBMCs were cultured in the presence of var-ious concentrations of rebamipide with human sRANKL(30 ngmL) We used adherent PBMCs as monocytes inthe culture system Osteoclast formation was evaluated byimmunohistological staining for CD5161 (vitronectin recep-tors) after culture in the presence of sRANKL and M-CSFfor 7 days We identified vitronectin-receptor positive cellsincluding more than three nuclei as osteoclasts

23 Cell Proliferation Assay Cell proliferation was measuredusing a Cell Proliferation Assay Kit (XTT baser) (BiologicalIndustries Ltd Israel)The assay was performed according tothemanufacturerrsquos protocol Cells (3times 105well) were culturedin 96-well plates Before cells reached confluence we culturedPBMC in the presence of M-CSF (100 ngmL) and RANKL(30 ngmL) and rebamipide (0 1 and 3mM) After 72 hours

the cells were collected and cell proliferation was measuredusing the kit

24 Human Osteoclast Formation (Time-Lapse Photography)We cultured human osteoclasts in the presence of M-CSF(100 ngmL) and RANKL (30 ngmL) Osteoclast formationin the presence or absence of rebamipide (0 1 and 3mM)was determined by time-lapse photography every one hourfor 9 days

25 Activation of Human Osteoclasts (Osteologic) We cul-tured human osteoclasts in the presence of M-CSF (100 ngmL) and RANKL (30 ngmL) using Osteologic plates Num-ber of absorption instances and area of absorption werecalculated

26 Cellular Staining and Actin Structure Mature osteoclastsinduced by sRANKL as described above were cultured in thepresence or absence of rebamipide (0 1 and 3mM) for 2 daysOsteoclasts were fixed with 10 formaldehyde in phosphate-buffered saline (PBS) for 10minutes at room temperature andrinsedwith PBS Cells were thenwashedwith Triton-X (01)for 1minute and stainedwith 20UmL rhodamine-phalloidin(Molecular Probes Eugene OR) in PBS for 30 minutes atroom temperature

27 Activation of Human Osteoclasts (Dentin Slices) Humanosteoclasts were cultured on dentin slices and the absorptionarea was observed using SEM (JSM6610LA JEOL LtdTokyo Japan) Also the absorption formation was assessedusing 3D-SEM Mature osteoclasts induced by sRANKL asdescribed above were cultured in the presence or absence ofrebamipide (0 1 and 3mM) for 2 days

28 RNA Isolation and Quantitative Real-Time PCR (qPCR)Total RNA was extracted from human CD14+ cells usingthe NucleoSpin RNA kit (MACHEREY-NAGEL DurenGermany)

First-strand cDNA was synthesized from total RNA byreverse transcription using the ReverTra Ace qPCR RTKit (Toyobo Osaka Japan) according to the manufacturerrsquosinstructions

Each cDNA sample was analyzed for gene expression byquantitative real-time PCR (qPCR) using the fluorescent Taq-Man 51015840-nuclease assay with an Applied Biosystems 7900HTsequence detection system (Applied Biosystems Foster CityCA USA) The TaqMan real-time PCR was performed using2x TaqMan Master Mix and 20x premade TaqMan geneexpression assays (Applied Biosystems) Glyceraldehyde-3-phosphate dehydrogenase (GADPH) was used as an internalstandard of mRNA expression Analysis was performed withABI 7900HT SDS 22 Software and relative RNA quantifica-tion using the comparative Ct method as described by Livakand Schmittgen [13]

Analysis of mRNA of integrin alpha V (ITGAV) integrinbeta 3 (ITGB3) nuclear factor of activated T-cells cyto-plasmic calcineurin-dependent 1 (NFATc1) T-cell leukemiatranslocation-associated gene (TCTA) voltage-dependent

BioMed Research International 3

30m

Positive control Negative control

1mM 3mM(a)

Rebamipide (mM)

times105lowast

0 1 3N

(m

2 )

0

50

100

150

(b)

Rebamipide

Ncell+ cell+ cell+

1mM 3mM

22H

0

20

40

60

80

100

120

()

(c)

Figure 1 (a) Effect of rebamipide on human osteoclastogenesis Rebamipide (0 1 and 3mM) dose-dependently inhibited the formationof human osteoclasts induced in the presence of sRANKL (30 ngmL) and M-CSF (100 ngmL) Immunohistological staining for CD5161(vitronectin receptors) (b) Effect of rebamipide on human osteoclastogenesis lowast119901 = 0048 (c) XTT assayThe 119910-axis represents expressionof XTT assay compared with N (negative control without adding rebamipide) Each colored bar represents each level of normal volunteers

anion channel 1 (VDAC1) FBJ murine osteosarcoma viraloncogene homolog (FOS) tumor necrosis factor (TNFA)RANK Jun protooncogene (JUN) DC-specific transmem-brane protein (DC-STAMP) and colony-stimulating factor 1receptor (CSF120572R) on adding rebamipide was carried out

To investigate the molecular mechanism of the effectof rebamipide on human osteoclastogenesis mature humanosteoclasts were cultured with rebamipide and mRNA ofITGAV ITGB3NFATc1 TCTAVDAC1 FOS TNFARANKJUN DC-STAMP and CSF120572R was investigated

29 Statistical Analysis Data were analyzed with the MannndashWhitney test

This study was approved by the ethics committee of ourinstitution and carried out in compliance with the HelsinkiDeclaration

3 Results

(1) Rebamipide inhibited human osteoclast formation (48-well plate)

Rebamipide (0 1 and 3mM) dose-dependently inhibitedthe formation of human osteoclasts induced in the presenceof sRANKL (30 ngmL) andM-CSF (100 ngmL) (Figures 1(a)and 1(b))The effect of rebamipide was not a result of cytotox-icity because the concentrations were pharmacological [14]


1mM 3mMPositive control

30m

(a)

Rebamipide (mM) 0 1 3

times105lowast

lowastlowast

(m

2 )

0

100

200

300

(b)

Figure 2 (a) Rebamipide inhibits human osteoclast formation (Osteologic) Rebamipide inhibits resorbing activity dose-dependently (b)Effect of rebamipide on bone resorption by human osteoclastogenesis in vitro (Osteologic) lowast119901 lt 0001 lowastlowast119901 = 00013

(2) Rebamipide did not significantly inhibit the prolif-eration of cells although the cell proliferation was slightlyinhibited at 1 and 3mM (Figure 1(c))

(3) Rebamipide inhibited human osteoclast formation(time-lapse photography)

Rebamipide inhibited human osteoclast formation at3mM a pharmacological concentration from the early phaseof preosteoclasts (data not shown)

(4) Rebamipide inhibited human osteoclast formation(Osteologic plates)

Rebamipide inhibited the resorbing activity dose-dependently (Figures 2(a) and 2(b))

(5) Rebamipide degenerates the formation of actin ringsof mature osteoclasts

The degradation of actin rings as a part of mature osteo-clasts was promoted by adding rebamipide dose-dependently(Figure 3)

(6) Human osteoclasts (dentin slice) were inactivatedPit formationwas seen on dentin slices in positive control

Resorption pits on dentin were completely inhibited by

adding rebamipide (3mM) (Figure 4) We used 3D-SEM toobserve a single pit of each osteoclast It is impossible toobserve a low power view using 3D-SEM

(7) Rebamipide reduced the expression of DC-STAMPmRNA

The expression of only DC-STAMP mRNA was reducedby adding rebamipide (3mM) (119899 = 5) (Figure 5)

4 Discussion

In the present study we demonstrated that rebamipide dose-dependently inhibited human osteoclastogenesis induced bysRANKL Rebamipide inhibited human osteoclast formationat pharmacological concentrations (3mM) and inhibitedresorbing activity dose-dependently In addition it inducedthe degradation of actin rings in mature osteoclasts at a phar-macological concentration Thus rebamipide has inhibitoryeffects on the formation and function of osteoclasts

Rebamipide directly inhibited osteoclastogenesis fromhuman monocytes since we used human monocytes alone


Positive control

30m

1mM 3mM

Figure 3 Rebamipide degenerates the formation of actin rings of mature osteoclasts The formation of actin rings of a part of the matureosteoclasts was generated by adding rebamipide dose-dependently

M-CSF (ngmL)

sRANKL (ngmL)

Rebamipide (mM)

100 100 100

50 500

0 0 3

Figure 4 Activation of human osteoclasts (dentin slices)Middle panel pit formationwas seen on dentin slices by adding the positive controlRight panel resorption pits on dentin slices were completely inhibited by adding rebamipide (3mM)

from peripheral blood in our culture system In addition itreduced the expression of DC-STAMP mRNA thus theseeffects are suggested to inhibit the function of DC-STAMPin the osteoclast fusion pathway

DR has become very important over the last few years [1]DR has a significant advantage over traditional drug devel-opment because the repositioned drug has already passedtoxicity tests its safety is known and the risk of adversetoxicology is reduced Using DR pharmaceutical companieshave achieved a number of successes The use of repurposeddrugs can bypass much of the early cost and time needed tobring a drug to market Thus to investigate novel pleiotropiceffects of drugs is verymeaningfulWe recently demonstratedthe novel pleiotropic activity of GGA a nontoxic inducer ofheat shock protein as an anti-gastric-ulcer drug [6 7]

GGApotently inhibits human osteoclastogenesis inducedby soluble RANKL [6] and induced cell death in FLS frompatients with RA by inhibiting protein geranylgeranylation[7] In this study we have shown a novel pleiotropic effect ofrebamipide on human osteoclastogenesis

Rebamipide is a mucosal protective agent used for thetreatment of gastritis and gastric ulcer It is an optically active120572-amino acid derivative of 2(1H)-quinolinone The precisemechanisms are still unknown however it has a benefi-cial effect on the gastric mucosa through several mecha-nisms increased gastric mucosal prostaglandin production

[8] increased gastric mucus production [15] scavenging ofhydroxyl radicals [16] inhibition of neutrophil activation[10] and suppression of gastric mucosal inflammation [17]

In addition to these effects some other pharmacologicalactions of rebamipide have been reported such as the regu-lation of apoptosis-related genes [18] inhibition of tyrosinenitration [19] promotion of Sonic hedgehog (Shh) restora-tion [20] correctingMAPK signaling abnormalities counter-acting the downregulation of120573EGF expression inhibiting theexpression of apoptosis-related genes and maintaining tightjunctional complexes [21]

It was recently reported that rebamipide works as an anti-inflammatory agent in acute and chronic inflammation andit inhibited proinflammatory cytokines [12] Experimentaldata have shown the protective effect of rebamipide on theintestinal barrier and its immunoregulatory properties Itis also capable of regulating lymphocyte proliferation andcytokine secretion and is expected to provide a new strategyfor the treatment of ulcerative colitis patients [12]

As mentioned above rebamipide scavenged hydroxylradicals directly and suppressed superoxide production bypolymorphonuclear leukocytes and prevented inflamma-tory cell infiltration In addition to these pleiotropic phar-macological actions rebamipide suppressed inflammatorycytokines Choe et al [22] reported that rebamipide inhibitstumor necrosis factor-120572-induced interleukin-8 expression by


DC-STAMP

0

20

40

60

80

100

mRN

A ex

pres

sion

()

3mM0mMRebamipide

Wilcoxon p = 0043

Figure 5 Rebamipide reduced the expression of DC-STAMPmRNA The expression of DC-STAMP mRNA was reduced byadding rebamipide (3mM) (119899 = 5)

suppressing the NF-120581B signal pathway in human umbilicalvein endothelial cells Kim et al [23] reported that rebamipideinhibits endothelial adhesion to hypoxiareoxygenation-stimulated endothelial cells via the NF-120581B-dependent path-way

Some reports have demonstrated that rebamipide is effec-tive for autoimmune disorders Kohashi et al [24] demon-strated that the administration of rebamipide effectivelyinhibits the autoimmune pathology in the NFSsld mousemodel of Sjogrenrsquos syndrome (SS) Rebamipide treatmentsuppressed the activation of CD4+ T-cells and Th1 cytokines(interleukin-2 interferon-120574) associated with impaired NF-120581B activity and inhibited the expression of IRF-4B atranscription factor associated with B-cell activation anddifferentiation Kohashi et al identified decreased apoptosisof salivary gland epithelia in rebamipide-treated mice Sincerebamipide inhibited the production of serum autoantibod-ies IgM and IgG1 and induced a reduction in the transcrip-tional activity of IRF-4 via the downregulation of NF-120581Brebamipide may represent a novel therapeutic approach forSS

RA causes the inflammation of synovial membranesproduces IL-17 and RANKL [3] which infiltrate synovialmembranes and is involved in the formation of osteoclastsT-cells activate macrophages and synovial cells and produceinflammatory cytokines such as IL-1120573 IL-6 and TNF-120572Their stimulation promotes the expression of RANKL insynovial cells and osteoblasts thereby inducing osteoclasticdifferentiation [5] Since rebamipide treatment suppressedthe activation of CD4+ T-cells rebamipide may be useful totreat bone destruction caused by osteoclasts Recently Moonet al [25] reported that rebamipide suppresses collagen-induced arthritis through reciprocal regulation of Th17Tregcell differentiation and heme oxygenase 1 induction Inaddition Byun et al [26] reported that rebamipide attenuates

autoimmune arthritis severity in SKG mice via regulation ofB-cell and antibody production

The concentrations of rebamipide (1ndash3mM) used inthe current study are within the range of pharmacologylevels It has been reported that a mucosal concentrationof rebamipide is more than 02mM after the oral intake of10mgkg rebamipide in rats [27] In addition Suzuki et al[28] reported that rebamipide (1mM) attenuatesHelicobacterpylori-induced gastric mucosal cell injury associated withneutrophil derived oxidants It is also speculated that theconcentration of rebamipide reaches 3mM in the microenvi-ronment in vivoThus we performed our experiments using0 1 and 3mM of rebamipide

In summary this study clearly demonstrated that reba-mipide inhibited human osteoclastogenesis and induced thedegradation of actin rings in mature osteoclasts At leastin part this mechanism is due to inhibiting the osteoclastfusion pathway through reducing the expression of DC-STAMP Next we will examine whether the inhibitory effectsof rebamipide on osteoclastogenesis in vitro are accompaniedby inhibitory effects on bone loss In this study we showedanother pleiotropic effect of rebamipideThe findings suggestthat rebamipide may represent a novel therapeutic approachfor osteoporosis and RA

Competing Interests

The authors declare that they have no competing interests

References

[1] C Schubert ldquoMatchmaking service links up researchers towallflower drugsrdquo Nature Medicine vol 16 article 7 2010

[2] G Mundy R Garrett S Harris et al ldquoStimulation of boneformation in vitro and in rodents by statinsrdquo Science vol 286no 5446 pp 1946ndash1949 1999

[3] Y Nanke S Kotake H Akama and N Kamatani ldquoAlkalinephosphatase in rheumatoid arthritis patients possible contri-bution of bone-type ALP to the raised activities of ALP inrheumatoid arthritis patientsrdquo Clinical Rheumatology vol 21no 3 pp 198ndash202 2002

[4] S Kotake N Udagawa N Takahashi et al ldquoIL-17 in synovialfluids from patients with rheumatoid arthritis is a potent stimu-lator of osteoclastogenesisrdquoThe Journal of Clinical Investigationvol 103 no 9 pp 1345ndash1352 1999

[5] S Kotake Y NankeMMogi et al ldquoIFN-120574-producing human Tcells directly induce osteoclastogenesis from humanmonocytesvia the expression ofRANKLrdquoEuropean Journal of Immunologyvol 35 no 11 pp 3353ndash3363 2005

[6] Y Nanke S Kotake T Ninomiya T Furuya H Ozawa andN Kamatani ldquoGeranylgeranylacetone inhibits formation andfunction of human osteoclasts and prevents bone loss intail-suspended rats and ovariectomized ratsrdquo Calcified TissueInternational vol 77 no 6 pp 376ndash385 2005

[7] Y Nanke M Kawamoto T Yago J Chiba H Yamanaka andS Kotake ldquoGeranylgeranylacetone a non-toxic inducer of heatshock protein induces cell death in fibroblast-like synoviocytesfrom patients with rheumatoid arthritisrdquo Modern Rheumatol-ogy vol 19 no 4 pp 379ndash383 2009


[8] K Yamasaki T Kanbe T Chijiwa H Ishiyama and S MoritaldquoGastric mucosal protection by OPC-12759 a novel antiulcercompound in the ratrdquo European Journal of Pharmacology vol142 no 1 pp 23ndash29 1987

[9] A Kleine S Kluge and B M Peskar ldquoStimulation ofprostaglandin biosynthesis mediates gastroprotective effect ofrebamipide in ratsrdquo Digestive Diseases and Sciences vol 38 no8 pp 1441ndash1449 1993

[10] T Yoshikawa Y Naito T Tanigawa and M Kondo ldquoFree radi-cal scavenging activity of the novel anti-ulcer agent rebamipidestudied by electron spin resonancerdquo Drug Research vol 43 no3 pp 363ndash366 1993

[11] M Aihara A Azuma H Takizawa et al ldquoMolecular analysisof suppression of interleukin-8 production by rebamipide inHelicobacter pylori-stimulated gastric cancer cell linesrdquo Diges-tive Diseases and Sciences vol 43 no 9 pp 174ndash180 1998

[12] H Bamba S Ota A Kato et al ldquoEffect of rebamipide onprostaglandin receptors-mediated increase of inflammatorycytokine production by macrophagesrdquo Alimentary Pharmacol-ogy andTherapeutics vol 18 no 1 pp 113ndash118 2003

[13] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCRand the 2minusΔΔ119862TmethodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[14] Y Naito T Yoshikawa S Iinuma et al ldquoLocal gastric and serumconcentrations of rebamipide following oral administration topatients with chronic gastritisrdquoDrug Research vol 46 no 7 pp698ndash700 1996

[15] K Ishihara Y Komuro N Nishiyama K Yamasaki and KHotta ldquoEffect of rebamipide onmucus secretion by endogenousprostaglandin-independent mechanism in rat gastric mucosardquoDrug Research vol 42 no 12 pp 1462ndash1466 1992

[16] Y Naito T Yoshikawa T Tanigawa et al ldquoHydroxyl radi-cal scavenging by rebamipide and related compounds elec-tron paramagnetic resonance studyrdquo Free Radical Biology andMedicine vol 18 no 1 pp 117ndash123 1995

[17] N Yoshida T Yoshikawa S Iinuma et al ldquoRebamipide protectsagainst activation of neutrophils by Helicobacter pylorirdquo Diges-tive Diseases and Sciences vol 41 no 6 pp 1139ndash1144 1996

[18] Y Naito M Kuroda K Mizushima et al ldquoTranscriptomeanalysis for cytoprotective actions of rebamipide againstindomethacin-induced gastric mucosal injury in ratsrdquo Journalof Clinical Biochemistry and Nutrition vol 41 no 3 pp 202ndash210 2007

[19] N Kinjo H Kawanaka T Akahoshi et al ldquoSignificance of ERKnitration in portal hypertensive gastropathy and its therapeuticimplicationsrdquo American Journal of PhysiologymdashGastrointestinaland Liver Physiology vol 295 no 5 pp G1016ndashG1024 2008

[20] T Nishizawa H Suzuki I Nakagawa et al ldquoRebamipide-promoted restoration of gastric mucosal sonic hedgehogexpression after earlyHelicobacter pylori eradicationrdquoDigestionvol 79 no 4 pp 259ndash262 2009

[21] Y Naito and T Yoshikawa ldquoRebamipide a gastrointestinalprotective drug with pleiotropic activitiesrdquo Expert Review ofGastroenterology amp Hepatology vol 4 no 3 pp 261ndash270 2010

[22] J-Y Choe K-Y Park S-J Lee S-H Park and S-KKim ldquoRebamipide inhibits tumor necrosis factor-120572-inducedinterleukin-8 expression by suppressing the NF-120581B signal path-way in human umbilical vein endothelial cellsrdquo InflammationResearch vol 59 no 12 pp 1019ndash1026 2010

[23] C D Kim Y K Kim S H Lee and K W Hong ldquoRebamipideinhibits neutrophil adhesion to hypoxiareoxygenation-stimulated endothelial cells via nuclear factor-120581B-dependent

pathwayrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 294 no 3 pp 864ndash869 2000

[24] M Kohashi N Ishimaru R Arakaki and Y Hayashi ldquoEffectivetreatment with oral administration of rebamipide in a mousemodel of Sjogrenrsquos syndromerdquo Arthritis amp Rheumatism vol 58no 2 pp 389ndash400 2008

[25] S-J Moon J-S Park Y-J Woo et al ldquoRebamipide suppressescollagen-induced arthritis through reciprocal regulation ofTh17Treg cell differentiation and heme oxygenase 1 inductionrdquoArthritis and Rheumatology vol 66 no 4 pp 874ndash885 2014

[26] J-K Byun S-J Moon J-Y Jhun et al ldquoRebamipide attenuatesautoimmune arthritis severity in SKG mice via regulation ofB cell and antibody productionrdquo Clinical and ExperimentalImmunology vol 178 no 1 pp 9ndash19 2014

[27] Y Shioya E Kashiyama K Okada et al ldquoMetabolic fate of theanti-ulcer agent absorption distribution and excretion in ratsand dogsrdquo Iyakuhin Kenkyu vol 20 pp 522ndash533 1989

[28] M Suzuki SMiuraMMori et al ldquoRebamipide a novel antiul-cer agent attenuatesHelicobacter pylori induced gastricmucosalcell injury associated with neutrophil derived oxidantsrdquo Gutvol 35 no 10 pp 1375ndash1378 1994

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Rebamipide (2-(4-chlorobenzoylamino)-3-[2(1H)-qui-nolinon-4-yl]propionic acid OPC-12759 molecular weight37079) is a mucosal protective agent used for the treatmentof gastritis and gastric ulcer Rebamipide is derived from agastric mucosal stabilizer and gastric mucosal prostaglandininducer It was shown to increase the PGE

2content in

the gastric mucosa and exhibit a gastric cytoprotectiveeffect to prevent mucosal injury [8 9] This drug alsodirectly scavenged hydroxyl radicals suppressed superoxideproduction and prevented inflammatory cell infiltration [10]In addition it inhibited the activation of NF-120581B expressionof interleukin-8 mRNA and production of interleukin-8in epithelial cells [11] Also it was recently reported thatrebamipide works as an anti-inflammatory agent in bothacute and chronic inflammation and has an inhibitory effecton proinflammatory cytokines [12]

In the current study we investigated the effect of rebami-pide an amino acid analog of 2(1H)-quinolinone on humanosteoclastogenesis

2 Materials and Methods

21 Chemicals and Reagents Rebamipide was supplied byOtsuka Co Ltd (Tokyo Japan) Recombinant human mac-rophage colony-stimulating factor (M-CSF) was obtainedfrom Kyowa Hakko Kirin (Tokyo Japan) Recombinanthuman soluble RANKL (sRANKL) was purchased fromPeproTech (London UK) For in vitro experiments thesereagents were dissolved in double-distilled water andadjusted to appropriate concentrations for each experiment

22 Cells and Cell Culture for Osteoclastogenesis Humanperipheral blood was collected from healthy normal vol-unteers Peripheral blood mononuclear cells (PBMCs) wereisolated by centrifugation over Histopaque 1077 (Sigma StLouisMOUSA) density gradients washed and resuspendedat 13 times 106 cellsmL in a-minimal essential medium (GibcoBRL Gaithersburg MD USA) supplemented with 10 fetalbovine serum (JRH Biosciences Lenexa KS) PBMCs werethen cultured for 3 days in 48-well plates in the presence ofM-CSF (100 ngmL) Nonadherent cells were then removedAdherent PBMCs were cultured in the presence of var-ious concentrations of rebamipide with human sRANKL(30 ngmL) We used adherent PBMCs as monocytes inthe culture system Osteoclast formation was evaluated byimmunohistological staining for CD5161 (vitronectin recep-tors) after culture in the presence of sRANKL and M-CSFfor 7 days We identified vitronectin-receptor positive cellsincluding more than three nuclei as osteoclasts

23 Cell Proliferation Assay Cell proliferation was measuredusing a Cell Proliferation Assay Kit (XTT baser) (BiologicalIndustries Ltd Israel)The assay was performed according tothemanufacturerrsquos protocol Cells (3times 105well) were culturedin 96-well plates Before cells reached confluence we culturedPBMC in the presence of M-CSF (100 ngmL) and RANKL(30 ngmL) and rebamipide (0 1 and 3mM) After 72 hours

the cells were collected and cell proliferation was measuredusing the kit

24 Human Osteoclast Formation (Time-Lapse Photography)We cultured human osteoclasts in the presence of M-CSF(100 ngmL) and RANKL (30 ngmL) Osteoclast formationin the presence or absence of rebamipide (0 1 and 3mM)was determined by time-lapse photography every one hourfor 9 days

25 Activation of Human Osteoclasts (Osteologic) We cul-tured human osteoclasts in the presence of M-CSF (100 ngmL) and RANKL (30 ngmL) using Osteologic plates Num-ber of absorption instances and area of absorption werecalculated

26 Cellular Staining and Actin Structure Mature osteoclastsinduced by sRANKL as described above were cultured in thepresence or absence of rebamipide (0 1 and 3mM) for 2 daysOsteoclasts were fixed with 10 formaldehyde in phosphate-buffered saline (PBS) for 10minutes at room temperature andrinsedwith PBS Cells were thenwashedwith Triton-X (01)for 1minute and stainedwith 20UmL rhodamine-phalloidin(Molecular Probes Eugene OR) in PBS for 30 minutes atroom temperature

27 Activation of Human Osteoclasts (Dentin Slices) Humanosteoclasts were cultured on dentin slices and the absorptionarea was observed using SEM (JSM6610LA JEOL LtdTokyo Japan) Also the absorption formation was assessedusing 3D-SEM Mature osteoclasts induced by sRANKL asdescribed above were cultured in the presence or absence ofrebamipide (0 1 and 3mM) for 2 days

28 RNA Isolation and Quantitative Real-Time PCR (qPCR)Total RNA was extracted from human CD14+ cells usingthe NucleoSpin RNA kit (MACHEREY-NAGEL DurenGermany)

First-strand cDNA was synthesized from total RNA byreverse transcription using the ReverTra Ace qPCR RTKit (Toyobo Osaka Japan) according to the manufacturerrsquosinstructions

Each cDNA sample was analyzed for gene expression byquantitative real-time PCR (qPCR) using the fluorescent Taq-Man 51015840-nuclease assay with an Applied Biosystems 7900HTsequence detection system (Applied Biosystems Foster CityCA USA) The TaqMan real-time PCR was performed using2x TaqMan Master Mix and 20x premade TaqMan geneexpression assays (Applied Biosystems) Glyceraldehyde-3-phosphate dehydrogenase (GADPH) was used as an internalstandard of mRNA expression Analysis was performed withABI 7900HT SDS 22 Software and relative RNA quantifica-tion using the comparative Ct method as described by Livakand Schmittgen [13]

Analysis of mRNA of integrin alpha V (ITGAV) integrinbeta 3 (ITGB3) nuclear factor of activated T-cells cyto-plasmic calcineurin-dependent 1 (NFATc1) T-cell leukemiatranslocation-associated gene (TCTA) voltage-dependent


30m


1mM 3mM(a)

Rebamipide (mM)

times105lowast

0 1 3N

(m

2 )

0

50

100

150

(b)

Rebamipide

Ncell+ cell+ cell+

1mM 3mM

22H

0

20

40

60

80

100

120

()

(c)






3 Results





30m

(a)


times105lowast

lowastlowast

(m

2 )

0

100

200

300

(b)














4 Discussion




Positive control

30m

1mM 3mM


M-CSF (ngmL)

sRANKL (ngmL)

Rebamipide (mM)

100 100 100

50 500

0 0 3











DC-STAMP

0

20

40

60

80

100

mRN

A ex

pres

sion

()

3mM0mMRebamipide

Wilcoxon p = 0043








Competing Interests


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















30m


1mM 3mM(a)

Rebamipide (mM)

times105lowast

0 1 3N

(m

2 )

0

50

100

150

(b)

Rebamipide

Ncell+ cell+ cell+

1mM 3mM

22H

0

20

40

60

80

100

120

()

(c)






3 Results





30m

(a)


times105lowast

lowastlowast

(m

2 )

0

100

200

300

(b)














4 Discussion




Positive control

30m

1mM 3mM


M-CSF (ngmL)

sRANKL (ngmL)

Rebamipide (mM)

100 100 100

50 500

0 0 3











DC-STAMP

0

20

40

60

80

100

mRN

A ex

pres

sion

()

3mM0mMRebamipide

Wilcoxon p = 0043








Competing Interests


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















30m

(a)


times105lowast

lowastlowast

(m

2 )

0

100

200

300

(b)














4 Discussion




Positive control

30m

1mM 3mM


M-CSF (ngmL)

sRANKL (ngmL)

Rebamipide (mM)

100 100 100

50 500

0 0 3











DC-STAMP

0

20

40

60

80

100

mRN

A ex

pres

sion

()

3mM0mMRebamipide

Wilcoxon p = 0043








Competing Interests


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Positive control

30m

1mM 3mM


M-CSF (ngmL)

sRANKL (ngmL)

Rebamipide (mM)

100 100 100

50 500

0 0 3











DC-STAMP

0

20

40

60

80

100

mRN

A ex

pres

sion

()

3mM0mMRebamipide

Wilcoxon p = 0043








Competing Interests


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















DC-STAMP

0

20

40

60

80

100

mRN

A ex

pres

sion

()

3mM0mMRebamipide

Wilcoxon p = 0043








Competing Interests


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Research Article Rebamipide, an Amino Acid Analog of 2(1H) … · 2019. 7. 30. · Research Article Rebamipide, an Amino Acid Analog of 2(1H)-Quinolinone, Inhibits the Formation of