research article effect of 4-allyl-1-hydroxy-2-methoxybenzene...

Research ArticleEffect of 4-Allyl-1-hydroxy-2-methoxybenzene(Eugenol) on Inflammatory and Apoptosis Processes inDental Pulp Fibroblasts

Andrea Martiacutenez-Herrera1 Amaury Pozos-Guilleacuten12 Socorro Ruiz-Rodriacuteguez2

Arturo Garrocho-Rangel2 Antonio Veacutertiz-Hernaacutendez3 and Diana Mariacutea Escobar-Garciacutea12

1Pediatric Dentistry Postgraduate Program Faculty of Dentistry San Luis Potosi University 78290 San Luis Potosı SLP Mexico2Basic Sciences Laboratory Faculty of Dentistry San Luis Potosi University 78290 San Luis Potosı SLP Mexico3Coordinacion Academica Region Altiplano San Luis Potosi University 78700 Matehuala SLP Mexico

Correspondence should be addressed to Diana Marıa Escobar-Garcıa dianaescobarsipuaslpmx

Received 21 August 2016 Revised 1 November 2016 Accepted 6 November 2016

Academic Editor Tania Silvia Frode

Copyright copy 2016 Andrea Martınez-Herrera et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Eugenol (mixed with zinc oxide powder) is widely used as direct capping material during pulp therapy in primary teeth The aimof the present study was to evaluate the effect of eugenol on diverse genes involved in inflammatory and cell apoptosis processesThe regulatory effect of eugenol on the expression of inflammation and apoptotic genes was evaluated in dental pulp fibroblastsfrom extracted third molars cultured under concentration of eugenol of 13120583M Eugenol allowed the expression of inflammatoryand apoptotic genes when compared with positive and negative controls Eugenol is a proinflammatory agent when it is in directcontact with healthy tissues and behaves as an anti-inflammatory agent in tissues undergoing inflammatoryapoptotic processes asin cases of pulp inflammation in primary teeth These findings are relevant for dentistry when considering the application of saferpulp treatments to grossly carious childrenrsquos teeth

1 Introduction

Dental caries is the most common chronic disease in child-hood caused mainly by a poor long-term oral hygieneIn its initial stages the disease can be preventable andreversible however without appropriate and timely controlcariogenic bacteria may seriously destroy the hard dentaltissues and consequently affect the dental pulp [1] Dentalpulp is a connective tissue composed mainly of fibroblastsand odontoblasts with an inherent capability to producereparative dentin in a favorable environment [2] Hence itis imperative to preserve the pulp vitality and function ofprimary teeth whenever possible

In cases of pulp tissue inflammation by carious pulpexposure there is a penetration of cariogenic Gram-positiveand Gram-negative bacteria the predominant microbiotain the oral cavity It is well known that the cell wallstructure of cariogenic bacteria is predominantly constituted

of lipopolysaccharides (LPS) LPS can activate the pulpimmune system inducing the segregation of proinflamma-tory chemokines and cytokines with an increase in capillarypermeability [3] Additionally the pulprsquos role during theinflammatory response is to fight against infection and tissuedamage through immune cells such as fibroblasts and othercells that recognize damage from cariogenic bacteria [4]fibroblasts activate the recognition of bacterial surface recep-tors which initiates the inflammatory response by stimulat-ing the production of cytokines including proinflammatoryanti-inflammatory and regulatory cytokines [5] Further-more recruitment of neutrophils and polymorphonuclearcells occurs which increases expression of the interleukins[6]

Maintaining the integrity and health of primary teeth infunctional status until their natural exfoliation is one of thefundamental objectives of pediatric dentistry It is essentialfor achieving normal oral function and facial growth [7]

Hindawi Publishing CorporationMediators of InflammationVolume 2016 Article ID 9371403 7 pageshttpdxdoiorg10115520169371403

2 Mediators of Inflammation

For this purpose pediatric dentistry practitioners employdiverse restorative or endodontic procedures for treatingdeep carious lesions with or without pulp tissue exposure inprimary teeth One of themost common and widely acceptedclinical endodontic treatments in pediatric dentistry is thepulpotomy technique [8] The objective of pulpotomy is toremove only the affected coronal pulp tissue so that the unaf-fected radicular pulp tissue remains in place and functioningnormally until the tooth is ready to exfoliate [9] Becauseof its sedative or anodyne properties low cost simplicityand efficacy the zinc oxideeugenol combination (ZOE) isfrequently used as capping material over the radicular pulpwhen pulpotomies are performed in primary teeth [9]

Eugenol (4-allyl-1-hydroxy-2-methoxybenzene) is anessential oil extracted from the plant Eugenia caryophyllata[10] popularly known as ldquoclovesrdquo which grows in differentregions worldwide [11] This oil has demonstrated to possessdifferent biological properties such as antimicrobial anal-gesic anti-inflammatory antioxidant antimutagenic andanticarcinogenic effects [3 12] Recent in vivo studies haveexplored the potential anticancer property of eugenol dem-onstrating the induction of apoptosis in several cancer celllines from animal tumor models [4 13] On the other handthrough different experimental models eugenol has provento reduce Oxidative Stress (OS) by preventing oxidativedamage [14ndash16] Paradoxically ZOE has also proven topossess harmful effects when placed directly on dentin tissueor when employed as pulpotomy material in primary teeth[17 18] Eugenol is cytotoxic for several types of human cellincluding pulp fibroblasts it reduces not only the growth andsurvival of these cells but also their collagen synthesis andbone sialoprotein expression which play a critical role inreparative dentine formation [18] Therefore it is necessaryto obtain new information concerning why when and underwhich circumstances eugenol provides either beneficial orharmful effects in order to procure safer employment of ZOEin clinical pediatric dentistry when treating deeply cariousprimary teeth

The aim of this study was to assess the anti-inflammatoryeffects of eugenol on cultured dental pulp fibroblastsunder inflammatory conditionsThese effects were measuredthrough the production of several inflammatory cytokinesand the expression of inflammation related-genes

2 Materials and Methods

21 Cell Cultures The whole culture process was based ona previous reported method by Escobar-Garcıa et al [19]Fibroblasts of dental pulp were obtained from extractedcaries-free third molars The extracted molars were placedin a transport medium (Phosphate-Buffered Saline (PBS)solution with a 3 antibiotic mixture (1000UmL Penicillin1mgmL Streptomycin and Amphotericin B 25mg120583L))Then the pulp chamber was exposed with a diamond discand the pulp tissue was removed washed with transportmedium and incubated in a collagenase solution (2mgmL)for 3 h at 37∘C 5 CO2 and 95 humidity After cen-trifuging the supernatant was removed The remainingtissue was cultured for explant (tissue dissected into small

1ndash3mm pieces) together with 3mL of Dulbeccorsquos ModifiedEagle Medium (DMEM) culture medium enriched with 10Fetal Bovine Serum (FBS) and an antibiotic combination(1000UmL Penicillin 1mgmL Streptomycin and Ampho-tericin B 25mg120583L) the mixture was incubated at 37∘C5 CO2 and 95 humidity and the culture medium wasreplaced every other day

22 Treatment and Control Groups Once the cell culturesreached a confluence of gt70 fibroblasts were assignedto different study groups Five groups were designated asfollows (1) fibroblasts placed in an enriched FBS culturemedium (negative control group) (2) fibroblasts treatedonly with LPS 10 120583gmL (lipopolysaccharide from Escherichiacoli 0127B8) without eugenol (positive control of inflam-mation) for 48 h (3) fibroblasts treated for 24 h with LPS10 120583gmL and eugenol 13120583M for 24 h (experimental groupA) (4) fibroblasts treated with eugenol for 24 h withoutLPS (experimental group B) and (5) fibroblasts treated onlywith H2O2 for 24 h without eugenol (positive control of cellapoptosis) In experimental groups (A and B) the mediumwas modified according to the concentration of eugenol of13 120583M(this concentration is based on previous work reportedby Escobar-Garcıa et al [19]) and then incubated for anadditional 24 h Each experiment was carried out in triplicateAfter the experiments were conducted cells were detachedfrom the culture box employing trypsin ethylenediaminete-traacetic acid (EDTA) 0025 and collected for subsequentRNA isolation

23 RNA Isolation RNA was isolated from cultured fibrob-lasts through the method of Tri Reagent (Sigma-Aldrich Bio-Sciences St Louis MO USA) following the manufacturerrsquosinstructions The extracted RNA was then suspended inRNase-free water The concentration of genetic material wasquantified using the NanoDrop reagent (Thermo ScientificFC Multiskan Thermo Scientific Vantaa Finland)

24 cDNA Synthesis Complementary DNA (cDNA) synthe-sis was performedwith 1 120583g of RNA utilizing the reverse tran-scription long range reverse transcriptase (QIAGEN GmbHD-40724Hilden Germany) reagent and was incubated twicethat is for 60min at 37∘C and for 5min at 85∘C in order toinactivate the transcriptase

25 Amplification of the PCR Products Specific amplificationfor inflammation (NF-120581B IL-1120573 TNF-120572 and VEGFA) andapoptotic (p53 and Apaf-1) genes was performed separatelyutilizing primer pairs under the amplifications (Table 1)Forward and reverse PCR reaction was carried out in 25 120583Lof green GoTaq (Promega Co Madison WI USA) MasterMix consisting of cDNA 250 ng and primer 05 120583M Fordesigning the primers the Amplifix 154 program wasemployment alignment and temperatures were confirmedusing Oligocalculator online software The sequences of theprimers and the annealing temperatures are described inTable 1 Finally the PCR products underwent electrophoresison agarose gels 1 90 volts for 1 h The agarose gels were

Mediators of Inflammation 3

Table 1 Primers and conditions employed for evaluation of gene expression

Gene symbol Primer sequence (51015840-31015840) Annealing temperature (∘C) Size of PCR product (bp)

Apaf-1 Fw ATGAGGCTCTAGACGAAGCCATGT 557 490Rv TGAATCAGATGAGCAGGGCCTACA

P53 Fw TTGCTATCTGGGACAGCCAAGT 591 491Rv CAGGCACAAACATGCACCTCAAAG

TNF-120572 Fw CGTCTCCTACCAGACCAAGGTCAA 588 401Rv TTCCTGAATCCCAGGTTTCGAAGTG

NF-120581B Fw CCTTGCACTTGGCAGTGATCACTA 593 294Rv ACTTCTGCTCCTGAGCATTGACG

VEGFA Fw ATCACCGAGCCCGGAAGATTAGA 55 384Rv CGGTGTTCCCAAAACTGGGTCAT

GAPDH Fw CCATCAATGACCCCTTCATTGACC 652 435Rv TGGTCATGAGTCCTTCCACGAT

IL1-120573 Fw CGATGCACCTGTACGATCACTGAA 569 223Rv CAACACGCAGGACAGGTACAGATT

Apaf-1-apoptotic peptidase activation factor 1 P53-tumor suppressor P53 TNF-120572-tumor necrosis factor-alfa NF-120581B-nuclear factor kappa B VEGFA-vascularendothelial growth factor A GAPDH-glyceraldehyde 3-phosphate dehydrogenase IL-1120573-interleukin 1 beta

analyzed in a Quantity One BioRad (BioRad LaboratoriesHercules CA USA) Photo Register in which the relativeexpression of each genewas comparedwith the control groupglyceraldehyde 3-phosphate dehydrogenase (GAPDH) wasutilized as housekeeping gene

26 Statistical Analyses Results from all study groups werecontinuous variables and are therefore expressed as meansand standard deviations (SD) three data sets per controlgroup and six data sets from each treatment group Meanswere compared by the one-way analysis of variance (ANOVA)followed by Tukey test for multiple comparisons employingSigmaPlot ver 110 statistical software (Systat Software Inc)A 119901 value of lt005 was taken as statistically significant

3 Results

31 Eugenol and Inflammation To assess whether there weredifferences among fibroblast gene expressions comparisonswere made as follows (a) negative control versus cell groupstreated with eugenol and (b) positive control (LPS) versus cellgroups treated with eugenol The first comparison (negativecontrol versus cell groups treated with eugenol) demon-strated that nuclear factor kappa B (NF-120581B) is expressed 116-timesmore than the negative control corresponding to a 16increase (Figure 1(a)) Interleukin 1 beta (IL1-120573) is expressed24-fold above the control corresponding to an increase inexpression of 145 (Figure 1(b)) Tumor necrosis factor alpha(TNF-120572) is expressed 13 times that of control cells represent-ing a 30 increase in expression (Figure 1(c)) The treatedcells of the vascular endothelial growth factor A (VEGFA)gene expressed eugenol at 108 times the negative controlrepresenting a 65 increase in gene expression (Figure 1(d))By performing a second analysis in which the expressionwas compared to inflammatory genes in eugenol-treated cellstreated versus cells that had previously undergone treatment

with LPS at a concentration of 10 120583gmL the NF-120581B genewas expressed 09 times that of the control representing 85inhibition in expression (Figure 1(a)) TNF-120572 is expressedonly 07-fold representing 254 inhibition (Figure 1(b))while IL1-120573 and VEGFA continued to exhibit proinflam-matory behavior expressed as 18-fold and 108-fold that ofcontrol inflammation respectively corresponding to 812IL1-120573 and 72 of VEGFA induction in increased expression(Figures 1(c) and 1(d))

32 Effect of Eugenol on the Apoptosis Process Two genesand their expressions were involved in the apoptosis processtumor suppressor p53 (p53) and the apoptotic peptidase acti-vation factor 1 (Apaf-1)Thep53 genewas expressed 104 timesthat of the control or a 4 increase in induction (Figure 2(a))which is statistically significant (119901 lt 005) Likewise Apaf-1gene-treated cells inhibited eugenol expression in 32 (068times) with regard to the control (Figure 2(b)) this inhibitionwas also significant (119901 lt 005)

4 Discussion

Premature loss of primary teeth can lead to malocclusionand phonetic or functional problems in the growing childTherefore it is necessary that pediatric dentistry practitionersalways intend to preserve primary tooth pulp vitality free ofinflammationinfection [20] Pathogenic bacteria and theirproducts are involved in severe forms of inflammation whenthe pulp is exposed by caries However it has been proventhat pulpal inflammation occurs even in the absence of bac-teria and that other factors participate in the inflammatoryprocess for example considering that ZOE-based materialsrelease small amounts of eugenol when placed in contact withdentin tissue eugenol can diffuse through dentinal tubulescausing potential injury to the pulp [21] After mixing ZOE achelation reaction occurs and zinc eugenolate is formedThis


Negativecontrol

LPScontrol

Eugenol Eugenol+ LPS

NF-120581B

lowast lowast

lowast

0

5

10

15

20

25

30

Relat

ive e

xpre

ssio

n

(a)

lowast

lowastlowast

IL-1120573

Negativecontrol

LPScontrol


02468

10121416

Relat

ive e

xpre

ssio

n

(b)

TNF-120572

lowast

lowast

lowast

Negativecontrol

LPScontrol


0

5

10

15

20

25

30

Relat

ive e

xpre

ssio

n

(c)

VEGFA

0

5

10

15

20

25

30

35

Relat

ive e

xpre

ssio

n

lowast lowast

Negativecontrol

LPScontrol


(d)

Figure 1 Relative expression of genes involved in the inflammatory process LPS-lipopolysaccharides NF-120581B-nuclear factor kappa B IL-1120573-interleukin 1 beta TNF-120572-tumor necrosis factor-alfa VEGFA-vascular endothelial growth factor A lowastThe differences in the mean valuesamong the treatment groups with respect to negative control are greater than what would be expected by chance there is a statisticallysignificant difference (119901 lt 005)

Apaf-1

Negativecontrol

EugenolcontrolH2O2

lowast lowast

0

5

10

15

20

25

30

Relat

ive e

xpre

ssio

n

(a)

p53

Negativecontrol

EugenolcontrolH2O2

lowast

lowast

0

5

10

15

20

25

Relat

ive e

xpre

ssio

n

(b)

Figure 2 Relative expression of genes involved in the apoptotic process Apaf-1-apoptotic peptidase activation factor 1 p53-tumor suppressorp53 lowastThe differences in themean values among the treatment groups with respect to negative control are greater thanwhat would be expectedby chance there is a statistically significant difference (119901 lt 005)


Healthy pulpal tissue

Coactivator P50

P50

P65

P65

IKB

DNA

mRNA

mRNA

Nucleus

Protein

P P

P50 P65

IKBP P Proteomic

degradationInactive NF-120581Bcomplex

ActiveNF-120581Bcomplex

Plasmatic membrane

+Eugenol

(a)

Inflamed pulpal tissue

Coactivator

P50 P65

IKB

DNANucleus

P P

P50 P65

IKBP PInactive

NF-120581Bcomplex

InactiveNF-120581Bcomplex

Plasmatic membrane

Eugenol +

(b)

Figure 3 A possible translocation of NF-120581B for healthy pulp tissue (a) and inflamed pulpal tissue (b) (a) Healthy pulpal tissue Eugenolallowed interaction with the IKB complex that mediates phosphorylation and degradation of inhibitor protein IKB in turn activating theNF-120581B transcription-B factor and translocation of NF-120581B into the nucleus where it participates in the activation of other genes involved inboth inflammation and apoptosis (b) Inflamed pulpal tissue NF-120581B expression is inhibited when there is preexisting inflammation becausethey do not carry out IKB phosphorylation inhibitor activities P-phosphorylation IKB-complex NF-120581B inhibitor P50-homodimer P50P65-homodimer P65 NF-120581B-nuclear factor kappa B

compound is readily hydrolyzed when exposed to dentinaltubule fluids to yield eugenol and zinc hydroxide free eugenolcan diffuse through the tubules up to reaching the pulp tissueand its cell components such as fibroblasts [17 21 22]

The present results strongly suggest that eugenol inducesthe expression of diverse genes involved in the inflammatoryprocess it was observed here that gene expression wassignificantly increased when fibroblasts were treated witheugenol as compared with untreated fibroblasts (Figure 3(a))However eugenol inhibited the expression of NF-120581B whenthe preexisting inflammatory-inducted process was present(eg with LPS) in this case the preexistence of inflammationsuch as the real condition of the dental pulp in primaryteeth (Figure 3(b)) Eugenol allowed interaction with the NF-120581B complex inhibitor (IKB) the latter mediates protein IKBphosphorylation and degradation in turn activating the NF-120581B transcription factor to promote nonapoptotic signalingpathways and gene expression via antiapoptotic pathwaysensuring nonapoptotic signaling activation [23] Further-more eugenol has been considered as anti-inflammatoryagent due to its well-known participation in the inhibition ofcyclooxygenase 2 (COX2) expressionThis protein is respon-sible for the production of inflammatory prostaglandinsUpregulation of COX2 is also associated with increased cell

adhesion phenotypic changes resistance to apoptosis andtumor angiogenesis [24 25]

Fibroblast cultures constitute an adequate model forstudying the mechanisms of inflammation and cell apoptosisat a molecular level [26] It has been reported that NF-120581Bcomprises a transcription factor regulating the inflamma-tory process and participating in the transcription of manycytokines such as TNF-120572 IL1-120573 and IL-6 [27] thus theinhibitors of this factor can be considered anti-inflammatoryagents [28] This gene also plays a significant role in otherprocesses including cell development growth and pro-liferation [29] TNF-120572 is an important proinflammatorycytokine secreted by many cell types such as macrophageslymphocytes fibroblasts and keratocytes in response to aninflammatory reaction infection or environmental changes[30]Within the same context IL1-120573 is an importantmediatorof the inflammatory response as a proinflammatory cytosineinvolved in different cellular activities including proliferationdifferentiation and apoptosis [31] it acts during the acuteresponse phase in antimicrobial defense VEGFA is a growthfactor expressed primarily in endothelial cells with variouseffects such as mediating increased vascular permeabilityangiogenesis promotion cell migration and apoptosis inhi-bition [32]


Apoptosis or programmed cell death is a complex phe-nomenon comprising the delicate regulation of signalingproteins via gene expression andor protein activity Apop-tosis participates in various physiological events involvedin many diseases including cancer and neurodegenerativedisorders [33 34] The process can be initiated intrinsicallyor extrinsically depending on the nature of the cell deathsignal After receiving intrinsic apoptotic stimuli severalproapoptotic proteins are released from the mitochondriainto the cytosol involving a large number of genes suchas p53 and Apaf-1 [23] p53 is a protein encoding a tumorsuppressive gene thus the encoded protein responds todiverse stressing mechanisms in the cell which regulatesthe expression of target genes and induces cell cycle arrestapoptosis senescence DNA repair and changes in cellmetabolism [35] Apaf-1 is a gene encoding a cytoplasmicprotein which promotes the apoptosis process mediatescytochrome C breaking and activates procaspase 9 thisprocess finally activates caspase 3 responsible for the onsetof apoptosis [36]

NF-120581B is a heterodimer complex that binds to kappa-B DNA sites of their target genes Individual dimers havepreferences for different kappa-B sites to which they canbind with distinguishable affinity and specificity This dimercomplex is localized in the cytoplasm in an inactive statebonded to the kappa-B inhibitor (I-kappa-B) Activation con-ventionally occurs by phosphorylation of I-kappa-throughthe I-kappa-B kinase (IKK) in response to various activa-tors and is subsequently degraded thereby releasing the B-complex once activated NF-120581B is then translocated intothe nucleus This gene participates in the regulation of gt500genes involved in some inflammatory process cell survivalproliferation angiogenesis and metastasis [37] Because NF-120581B is a regulator of the inflammatory process inhibitionof its expression is considered an anti-inflammatory effect[23]

Apaf-1 is a cytoplasmic protein and one of the axescontrolling the apoptosis regulatory network Eugenol is ableto significantly suppress the expression of Apaf-1 suggest-ing that eugenol does not yield a programmed cell deathalthough it is able to significantly induce the expression ofp53 which may indicate that the cell is in contact withthis oil If there are any alterations in the layers a genome-increased expression of p53 is motivated in order to repairdamage to genetic material but without achieving the finalapoptosis process Therefore apoptosis is a normal processof many multicellular organisms however alterations in thisprocess are associated with the pathogenesis of differentdiseases

5 Conclusions

According to the present studyrsquos results low-concentratedeugenol possesses the property of an anti-inflammatory agentwhen the pulp tissue is found in an inflamed state as isthe case of reversible pulpitis in primary teeth due to beingcapable of inhibiting gene expression such as that of NF-120581Band TNF-120572

Ethical Approval

This study was registered and approved by the InstitutionalEthics Committee of the Faculty of Dentistry at San LuisPotosi University San Luis Potosi Mexico (approval code noCEIFE-006-2016)

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This study was conducted with support of Project UASLP-C15-FAI-04-101101 The English-language reviewing of themanuscript by Maggie Brunner is particularly appreciated

References

[1] A Gonzalez-Lara M S Ruiz-Rodrıguez M Pierdant-PerezJ A Garrocho-Rangel and A J Pozos-Guillen ldquoZinc Oxidendasheugenol pulpotomy in primary teeth a 24-month follow-uprdquoJournal of Clinical Pediatric Dentistry vol 40 no 2 pp 107ndash1122016

[2] A Garrocho-Rangel H Flores D Silva-Herzog F Hernandez-Sierra P Mandeville and A J Pozos-Guillen ldquoEfficacy ofEMD versus calcium hydroxide in direct pulp capping ofprimary molars a randomized controlled clinical trialrdquo OralSurgery Oral Medicine Oral Pathology Oral Radiology andEndodontology vol 107 no 5 pp 733ndash738 2009

[3] R C Laughlin M Mickum K Rowin L G Adams and R CAlaniz ldquoAltered host immune responses to membrane vesiclesfrom Salmonella and Gram-negative pathogensrdquo Vaccine vol33 no 38 pp 5012ndash5019 2015

[4] K Newton and V M Dixit ldquoSignaling in innate immunity andinflammationrdquo Cold Spring Harbor Perspectives in Biology vol4 no 3 pp 1ndash9 2012

[5] T F Bachiega J P B De Sousa J K Bastos and J M SforcinldquoClove and eugenol in noncytotoxic concentrations exertimmunomodulatory anti-inflammatory action on cytokineproduction by murine macrophagesrdquo The Journal of Pharmacyand Pharmacology vol 64 no 4 pp 610ndash616 2012

[6] J Modaresi A Davoudi H Badrian and R Sabzian ldquoIrre-versible pulpitis and achieving profound anesthesia complex-ities and managementsrdquo Anesthesia Essays and Researches vol10 no 1 pp 3ndash6 2016

[7] G M Knight J M McIntyre G G Craig Mulyani P S Zilmand N J Gully ldquoAn in vitro investigation of marginal dentinecaries abutting composite resin and glass ionomer cementrestorationsrdquo Australian Dental Journal vol 52 no 3 pp 187ndash192 2007

[8] I Parisay J Ghoddusi andM Forghani ldquoA review on vital pulptherapy in primary teethrdquo Iranian Endodontic Journal vol 10no 1 pp 6ndash15 2015

[9] A Fuks and B Perets Pediatric EndodonticsmdashCurrent Conceptsin Pulp Therapy for Primary and Young Permanent TeethSpringer Berlin Germany 2016

[10] ANDaniel SM SartorettoG Schmidt SMCaparroz-AssefC A Bersani-Amado and R K N Cuman ldquoAnti-inflammatory


and antinociceptive activities of eugenol essential oil in exper-imental animal modelsrdquo Revista Brasileira de Farmacognosiavol 19 no 1 pp 212ndash217 2009

[11] M D F Agra K N Silva I J L D Basılio P F De Freitasand JM Barbosa-Filho ldquoSurvey ofmedicinal plants used in theregionNortheast of BrazilrdquoBrazilian Journal of Pharmacognosyvol 18 no 3 pp 472ndash508 2008

[12] C F Estevao-Silva R Kummer F C Fachini-Queiroz et alldquoAnethole and eugenol reduce in vitro and in vivo leukocytemigration induced by fMLP LTB4 and carrageenanrdquo Journalof Natural Medicines vol 68 no 3 pp 567ndash575 2014

[13] A Sarkar S Bhattacharjee and D P Mandal ldquoInduction ofapoptosis by eugenol and capsaicin in human gastric cancerAGS cells - elucidating the role of p53rdquo Asian Pacific Journal ofCancer Prevention vol 16 no 15 pp 6753ndash6759 2015

[14] M AMorsy and A A Fouad ldquoMechanisms of gastroprotectiveeffect of eugenol in indomethacin-induced ulcer in ratsrdquo Phy-totherapy Research vol 22 no 10 pp 1361ndash1366 2008

[15] E Nagababu and N Lakshmaiah ldquoInhibitory effect of eugenolon non-enzymatic lipid peroxidation in rat liver mitochondriardquoBiochemical Pharmacology vol 43 no 11 pp 2393ndash2400 1992

[16] H Kabuto M Tada and M Kohno ldquoEugenol [2-methoxy-4-(2-propenyl)phenol] prevents 6-hydroxydopamine-induceddopamine depression and lipid peroxidation inductivity inmouse striatumrdquo Biological amp Pharmaceutical Bulletin vol 30no 3 pp 423ndash427 2007

[17] W R Hume ldquoThe pharmacologic and toxicological propertiesof zinc oxide-eugenolrdquo The Journal of the American DentalAssociation vol 113 no 5 pp 789ndash791 1986

[18] M Anpo K Shirayama and T Tsutsui ldquoCytotoxic effect ofeugenol on the expression of molecular markers related tothe osteogenic differentiation of human dental pulp cellsrdquoOdontology vol 99 no 2 pp 188ndash192 2011

[19] M Escobar-Garcıa K Rodrıguez-Contreras S Ruiz-Rodrıguez M Pierdant-Perez B Cerda-Cristerna and APozos-Guillen ldquoEugenol toxicity in human dental pulpfibroblasts of primary teethrdquo Journal of Clinical PediatricDentistry vol 40 no 4 pp 312ndash318 2016

[20] S Koshy and RM Love ldquoEndodontic treatment in the primarydentitionrdquo Australian Endodontic Journal vol 30 no 2 pp 59ndash68 2004

[21] C A De Souza Costa J Hebling D L S Scheffel D G SSoares F G Basso and A P D Ribeiro ldquoMethods to eval-uate and strategies to improve the biocompatibility of dentalmaterials and operative techniquesrdquo Dental Materials vol 30no 7 pp 769ndash784 2014

[22] K Markowitz M Moynihan M Liu and S Kim ldquoBiologicproperties of eugenol and zinc oxide-eugenol A clinicallyoriented reviewrdquo Oral Surgery Oral Medicine Oral Pathologyvol 73 no 6 pp 729ndash737 1992

[23] J Chhibber-Goel C Coleman-Vaughan V Agrawal et alldquo120574-Secretase activity is required for regulated intramembraneproteolysis of tumor necrosis factor (TNF) receptor 1 and TNF-mediated pro-apoptotic signalingrdquo The Journal of BiologicalChemistry vol 291 no 11 pp 5971ndash5985 2016

[24] Y Murakami A Kawata Y Seki et al ldquoComparative inhibitoryeffects of magnolol honokiol eugenol and bis-eugenol oncyclooxygenase-2 expression and nuclear factor-kappa B acti-vation in RAW2647 macrophage-like cells stimulated withfimbriae of Porphyromonas gingivalisrdquo In Vivo vol 26 no 6 pp941ndash950 2012

[25] LHassanA Pinon Y Limami et al ldquoResistance to ursolic acid-induced apoptosis through involvement of melanogenesis andCOX-2PGE2 pathways in human M4Beu melanoma cancercellsrdquoExperimental Cell Research vol 345 no 1 pp 60ndash69 2016

[26] J F Pereira N T Awatade C A Loureiro P Matos M DAmaral and P Jordan ldquoThe third dimension new develop-ments in cell culture models for colorectal researchrdquo Cellularand Molecular Life Sciences vol 73 no 21 pp 3971ndash3989 2016

[27] R Yang L Yang X Shen et al ldquoSuppression of NF-120581B pathwayby crocetin contributes to attenuation of lipopolysaccharide-induced acute lung injury in micerdquo European Journal of Phar-macology vol 674 no 2-3 pp 391ndash396 2012

[28] R Di M-T Huang and C-T Ho ldquoAnti-inflammatory activ-ities of mogrosides from Momordica grosvenori in murinemacrophages and a murine ear edema modelrdquo Journal ofAgricultural and Food Chemistry vol 59 no 13 pp 7474ndash74812011

[29] XHuang Y Liu Y Lu andCMa ldquoAnti-inflammatory effects ofeugenol on lipopolysaccharide-induced inflammatory reactionin acute lung injury via regulating inflammation and redoxstatusrdquo International Immunopharmacology vol 26 no 1 pp265ndash271 2015

[30] V Baud and M Karin ldquoSignal transduction by tumor necrosisfactor and its relativesrdquo Trends in Cell Biology vol 11 no 9 pp372ndash377 2001

[31] P Piccioli and A Rubartelli ldquoThe secretion of IL-1120573 and optionsfor releaserdquo Seminars in Immunology vol 25 no 6 pp 425ndash4292013

[32] M-S Lee J Ghim S-J Kim et al ldquoFunctional interactionbetween CTGF and FPRL1 regulates VEGF-A-induced angio-genesisrdquo Cellular Signalling vol 27 no 7 pp 1439ndash1448 2015

[33] T Yun K Yu S Yang et al ldquoAcetylation of p53 proteinat lysine 120 up-regulates Apaf-1 protein and sensitizes themitochondrial apoptotic pathwayrdquo The Journal of BiologicalChemistry vol 291 no 14 pp 7386ndash7395 2016

[34] R Franco and J A Cidlowski ldquoApoptosis and glutathionebeyond an antioxidantrdquo Cell Death and Differentiation vol 16no 10 pp 1303ndash1314 2009

[35] R Vijayakumaran K H Tan P J Miranda S Haupt and YHaupt ldquoRegulation of mutant p53 protein expressionrdquo Frontiersin Oncology vol 5 article 284 2015

[36] D-I KohH AnM-Y Kim et al ldquoTranscriptional activation ofAPAF1 byKAISO (ZBTB33) and p53 is attenuated by RelAp65rdquoBiochimica et Biophysica ActamdashGene Regulatory Mechanismsvol 1849 no 9 pp 1170ndash1178 2015

[37] B Sung S Prasad V R Yadav and B B Aggarwal ldquoCancer cellsignaling pathways targeted by spice-derived nutraceuticalsrdquoNutrition and Cancer vol 64 no 2 pp 173ndash197 2012

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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EndocrinologyInternational Journal of



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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


For this purpose pediatric dentistry practitioners employdiverse restorative or endodontic procedures for treatingdeep carious lesions with or without pulp tissue exposure inprimary teeth One of themost common and widely acceptedclinical endodontic treatments in pediatric dentistry is thepulpotomy technique [8] The objective of pulpotomy is toremove only the affected coronal pulp tissue so that the unaf-fected radicular pulp tissue remains in place and functioningnormally until the tooth is ready to exfoliate [9] Becauseof its sedative or anodyne properties low cost simplicityand efficacy the zinc oxideeugenol combination (ZOE) isfrequently used as capping material over the radicular pulpwhen pulpotomies are performed in primary teeth [9]

Eugenol (4-allyl-1-hydroxy-2-methoxybenzene) is anessential oil extracted from the plant Eugenia caryophyllata[10] popularly known as ldquoclovesrdquo which grows in differentregions worldwide [11] This oil has demonstrated to possessdifferent biological properties such as antimicrobial anal-gesic anti-inflammatory antioxidant antimutagenic andanticarcinogenic effects [3 12] Recent in vivo studies haveexplored the potential anticancer property of eugenol dem-onstrating the induction of apoptosis in several cancer celllines from animal tumor models [4 13] On the other handthrough different experimental models eugenol has provento reduce Oxidative Stress (OS) by preventing oxidativedamage [14ndash16] Paradoxically ZOE has also proven topossess harmful effects when placed directly on dentin tissueor when employed as pulpotomy material in primary teeth[17 18] Eugenol is cytotoxic for several types of human cellincluding pulp fibroblasts it reduces not only the growth andsurvival of these cells but also their collagen synthesis andbone sialoprotein expression which play a critical role inreparative dentine formation [18] Therefore it is necessaryto obtain new information concerning why when and underwhich circumstances eugenol provides either beneficial orharmful effects in order to procure safer employment of ZOEin clinical pediatric dentistry when treating deeply cariousprimary teeth

The aim of this study was to assess the anti-inflammatoryeffects of eugenol on cultured dental pulp fibroblastsunder inflammatory conditionsThese effects were measuredthrough the production of several inflammatory cytokinesand the expression of inflammation related-genes

2 Materials and Methods

21 Cell Cultures The whole culture process was based ona previous reported method by Escobar-Garcıa et al [19]Fibroblasts of dental pulp were obtained from extractedcaries-free third molars The extracted molars were placedin a transport medium (Phosphate-Buffered Saline (PBS)solution with a 3 antibiotic mixture (1000UmL Penicillin1mgmL Streptomycin and Amphotericin B 25mg120583L))Then the pulp chamber was exposed with a diamond discand the pulp tissue was removed washed with transportmedium and incubated in a collagenase solution (2mgmL)for 3 h at 37∘C 5 CO2 and 95 humidity After cen-trifuging the supernatant was removed The remainingtissue was cultured for explant (tissue dissected into small

1ndash3mm pieces) together with 3mL of Dulbeccorsquos ModifiedEagle Medium (DMEM) culture medium enriched with 10Fetal Bovine Serum (FBS) and an antibiotic combination(1000UmL Penicillin 1mgmL Streptomycin and Ampho-tericin B 25mg120583L) the mixture was incubated at 37∘C5 CO2 and 95 humidity and the culture medium wasreplaced every other day

22 Treatment and Control Groups Once the cell culturesreached a confluence of gt70 fibroblasts were assignedto different study groups Five groups were designated asfollows (1) fibroblasts placed in an enriched FBS culturemedium (negative control group) (2) fibroblasts treatedonly with LPS 10 120583gmL (lipopolysaccharide from Escherichiacoli 0127B8) without eugenol (positive control of inflam-mation) for 48 h (3) fibroblasts treated for 24 h with LPS10 120583gmL and eugenol 13120583M for 24 h (experimental groupA) (4) fibroblasts treated with eugenol for 24 h withoutLPS (experimental group B) and (5) fibroblasts treated onlywith H2O2 for 24 h without eugenol (positive control of cellapoptosis) In experimental groups (A and B) the mediumwas modified according to the concentration of eugenol of13 120583M(this concentration is based on previous work reportedby Escobar-Garcıa et al [19]) and then incubated for anadditional 24 h Each experiment was carried out in triplicateAfter the experiments were conducted cells were detachedfrom the culture box employing trypsin ethylenediaminete-traacetic acid (EDTA) 0025 and collected for subsequentRNA isolation

23 RNA Isolation RNA was isolated from cultured fibrob-lasts through the method of Tri Reagent (Sigma-Aldrich Bio-Sciences St Louis MO USA) following the manufacturerrsquosinstructions The extracted RNA was then suspended inRNase-free water The concentration of genetic material wasquantified using the NanoDrop reagent (Thermo ScientificFC Multiskan Thermo Scientific Vantaa Finland)

24 cDNA Synthesis Complementary DNA (cDNA) synthe-sis was performedwith 1 120583g of RNA utilizing the reverse tran-scription long range reverse transcriptase (QIAGEN GmbHD-40724Hilden Germany) reagent and was incubated twicethat is for 60min at 37∘C and for 5min at 85∘C in order toinactivate the transcriptase

25 Amplification of the PCR Products Specific amplificationfor inflammation (NF-120581B IL-1120573 TNF-120572 and VEGFA) andapoptotic (p53 and Apaf-1) genes was performed separatelyutilizing primer pairs under the amplifications (Table 1)Forward and reverse PCR reaction was carried out in 25 120583Lof green GoTaq (Promega Co Madison WI USA) MasterMix consisting of cDNA 250 ng and primer 05 120583M Fordesigning the primers the Amplifix 154 program wasemployment alignment and temperatures were confirmedusing Oligocalculator online software The sequences of theprimers and the annealing temperatures are described inTable 1 Finally the PCR products underwent electrophoresison agarose gels 1 90 volts for 1 h The agarose gels were














3 Results




4 Discussion



Negativecontrol

LPScontrol


NF-120581B

lowast lowast

lowast

0

5

10

15

20

25

30

Relat

ive e

xpre

ssio

n

(a)

lowast

lowastlowast

IL-1120573

Negativecontrol

LPScontrol


02468

10121416

Relat

ive e

xpre

ssio

n

(b)

TNF-120572

lowast

lowast

lowast

Negativecontrol

LPScontrol


0

5

10

15

20

25

30

Relat

ive e

xpre

ssio

n

(c)

VEGFA

0

5

10

15

20

25

30

35

Relat

ive e

xpre

ssio

n

lowast lowast

Negativecontrol

LPScontrol


(d)


Apaf-1

Negativecontrol

EugenolcontrolH2O2

lowast lowast

0

5

10

15

20

25

30

Relat

ive e

xpre

ssio

n

(a)

p53

Negativecontrol

EugenolcontrolH2O2

lowast

lowast

0

5

10

15

20

25

Relat

ive e

xpre

ssio

n

(b)




Coactivator P50

P50

P65

P65

IKB

DNA

mRNA

mRNA

Nucleus

Protein

P P

P50 P65

IKBP P Proteomic



Plasmatic membrane

+Eugenol

(a)


Coactivator

P50 P65

IKB

DNANucleus

P P

P50 P65

IKBP PInactive

NF-120581Bcomplex


Plasmatic membrane

Eugenol +

(b)










5 Conclusions


Ethical Approval


Competing Interests


Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























3 Results




4 Discussion



Negativecontrol

LPScontrol


NF-120581B

lowast lowast

lowast

0

5

10

15

20

25

30

Relat

ive e

xpre

ssio

n

(a)

lowast

lowastlowast

IL-1120573

Negativecontrol

LPScontrol


02468

10121416

Relat

ive e

xpre

ssio

n

(b)

TNF-120572

lowast

lowast

lowast

Negativecontrol

LPScontrol


0

5

10

15

20

25

30

Relat

ive e

xpre

ssio

n

(c)

VEGFA

0

5

10

15

20

25

30

35

Relat

ive e

xpre

ssio

n

lowast lowast

Negativecontrol

LPScontrol


(d)


Apaf-1

Negativecontrol

EugenolcontrolH2O2

lowast lowast

0

5

10

15

20

25

30

Relat

ive e

xpre

ssio

n

(a)

p53

Negativecontrol

EugenolcontrolH2O2

lowast

lowast

0

5

10

15

20

25

Relat

ive e

xpre

ssio

n

(b)




Coactivator P50

P50

P65

P65

IKB

DNA

mRNA

mRNA

Nucleus

Protein

P P

P50 P65

IKBP P Proteomic



Plasmatic membrane

+Eugenol

(a)


Coactivator

P50 P65

IKB

DNANucleus

P P

P50 P65

IKBP PInactive

NF-120581Bcomplex


Plasmatic membrane

Eugenol +

(b)










5 Conclusions


Ethical Approval


Competing Interests


Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Negativecontrol

LPScontrol


NF-120581B

lowast lowast

lowast

0

5

10

15

20

25

30

Relat

ive e

xpre

ssio

n

(a)

lowast

lowastlowast

IL-1120573

Negativecontrol

LPScontrol


02468

10121416

Relat

ive e

xpre

ssio

n

(b)

TNF-120572

lowast

lowast

lowast

Negativecontrol

LPScontrol


0

5

10

15

20

25

30

Relat

ive e

xpre

ssio

n

(c)

VEGFA

0

5

10

15

20

25

30

35

Relat

ive e

xpre

ssio

n

lowast lowast

Negativecontrol

LPScontrol


(d)


Apaf-1

Negativecontrol

EugenolcontrolH2O2

lowast lowast

0

5

10

15

20

25

30

Relat

ive e

xpre

ssio

n

(a)

p53

Negativecontrol

EugenolcontrolH2O2

lowast

lowast

0

5

10

15

20

25

Relat

ive e

xpre

ssio

n

(b)




Coactivator P50

P50

P65

P65

IKB

DNA

mRNA

mRNA

Nucleus

Protein

P P

P50 P65

IKBP P Proteomic



Plasmatic membrane

+Eugenol

(a)


Coactivator

P50 P65

IKB

DNANucleus

P P

P50 P65

IKBP PInactive

NF-120581Bcomplex


Plasmatic membrane

Eugenol +

(b)










5 Conclusions


Ethical Approval


Competing Interests


Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Coactivator P50

P50

P65

P65

IKB

DNA

mRNA

mRNA

Nucleus

Protein

P P

P50 P65

IKBP P Proteomic



Plasmatic membrane

+Eugenol

(a)


Coactivator

P50 P65

IKB

DNANucleus

P P

P50 P65

IKBP PInactive

NF-120581Bcomplex


Plasmatic membrane

Eugenol +

(b)










5 Conclusions


Ethical Approval


Competing Interests


Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















5 Conclusions


Ethical Approval


Competing Interests


Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article effect of 4-allyl-1-hydroxy-2-methoxybenzene...

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