Research Article The Effects of Brazilian Green Propolis against …downloads.hindawi.com/journals/ecam/2013/238279.pdf · 2019. 7. 31. · The Effects of Brazilian Green Propolis

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 238279 11 pageshttpdxdoiorg1011552013238279

Research ArticleThe Effects of Brazilian Green Propolis against ExcessiveLight-Induced Cell Damage in Retina and Fibroblast Cells

Hiromi Murase1 Masamitsu Shimazawa1 Mamoru Kakino12 Kenji Ichihara2

Kazuhiro Tsuruma1 and Hideaki Hara1

1 Molecular Pharmacology Department of Biofunctional Evaluation Gifu Pharmaceutical University1-25-4 Daigaku-nishi Gifu 501-1196 Japan

2Nagaragawa Research Center Api Co Ltd 692-3 Nagara Gifu 502-0071 Japan

Correspondence should be addressed to Masamitsu Shimazawa shimazawagifu-puacjp

Received 12 August 2013 Revised 20 November 2013 Accepted 25 November 2013

Academic Editor Seung-Heon Hong

Copyright copy 2013 Hiromi Murase et alThis 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

Background We investigated the effects of Brazilian green propolis and its constituents against white light- or UVA-induced celldamage inmouse retinal cone-cell line 661Wor human skin-derived fibroblast cells (NB1-RGB)Methods Cell damage was inducedby 3000lx white light for 24 h or 410 Jcm2 UVA exposure Cell viability was assessed by Hoechst33342 and propidium iodidestaining or by tetrazolium salt (WST-8) cell viability assay The radical scavenging activity of propolis induced by UVA irradiationin NB1-RGB cells was measured using a reactive-oxygen-species- (ROS-) sensitive probe CM-H

2DCFDA Moreover the effects of

propolis on theUVA-induced activation of p38 and extracellular signal-regulated kinase (ERK) were examined by immunoblottingResults Treatment with propolis and two dicaffeoylquinic acids significantly inhibited the decrease in cell viability induced by whitelight in 661W Propolis and its constituents inhibited the decrease in cell viability induced by UVA in NB1-RGB Moreover propolissuppressed the intracellular ROS production by UVA irradiation Propolis also inhibited the levels of phosphorylated-p38 and ERKby UVA irradiation Conclusion Brazilian green propolis may become amajor therapeutic candidate for the treatment of AMD andskin damage induced by UV irradiation

1 Introduction

People are exposed to visible light or ultraviolet (UV) on adaily basis When exposed excessively they will experienceserious effects in their eyes or skin Skin is the only organ thatis directly exposed to UV irradiation The skin coexists withmany environmental pollutants that are oxidants themselvesor can catalyze the formation of reactive oxygen species(ROS) Oxidative damage to the skin induced by severalexogenous and endogenous factors such as ultraviolet (UV)irradiation tobacco smoke infrared radiation transitionmetal ions and enzymatic and nonenzymatic antioxidantimpairment has been acknowledged as a key factor ofintrinsic and photoinduced skin aging Ultimately it inducesactinic elastosis and skin cancer [1]

We can classify the skin aging process into intrinsicaging and photoaging Damage to human skin resulting fromrepeated exposure to UV irradiation (photoaging) and dam-age caused by the passage of time cell replication and aerobicmetabolism (intrinsic aging) are considered to be distinctentities rather than similar skin aging processes [2] Sunlightconsists of the infrared visible and ultraviolet regions ofthe spectrum Ultraviolet radiation can be classified underUVA UVB and UVC wavelengths [3] UVC (200ndash280 nm)is blocked by the ozone layer UVA (320ndash400 nm) and UVB(280ndash320 nm) can pass through the ozone layer cross theepidermis and reach the dermis UVA waves have manybiological efficacies on living organisms UVA induces theproduction of matrix metalloproteinases that deteriorate theextracellular matrix UVA also induces the production of

2 Evidence-Based Complementary and Alternative Medicine

singlet oxygen which causes eliminations or point mutationsin mitochondrial DNA and is involved in DNA damagewhich activates the DNA damage response system finallyleading to cell senescence Therefore UVA is considered afundamental cause of aging

High levels of visible light or UV may cause oculardamage especially later in life It has been noted that long-term light exposure results in photoreceptor degradationand it may be among the most relevant damaging factorsinvolved in age-related macular degeneration (AMD) [4]Excessive light exposure can be a risk factor for the onsetand progression of AMD [5] and it leads to photoreceptordegeneration in animals [6 7] Both external and internalfactors are thought to be a pathogenesis of AMD [8 9] andexposure to sunlight or ultraviolet radiation is also a well-established risk factor for AMD

Propolis is made from a sticky substance that honeybeesproduce by mixing their own waxes with resinous sapobtained from the bark and leaf-buds of certain trees andother flowering plants Propolis is used as a sealant andsterilant in honeybee nestsThe color of propolis can be greenyellow brown or almost black depending on the plants fromwhich the resinous substance is collected [10]The propertiesand constituents of propolis also differ with its geographicalorigin [11] Brazilian green propolis is made of aromaticacids (cinnamic acid derivatives ferulic acid and caffeicacid) diethyl methyl succinate isobutylquinoline generalacetal patchouli alcohol menthol amyrins and flavonoidsBrazilian propolis has been the subject of many studies dueto its biological activities such as its antibacterial [12 13]antifungal [11 14ndash17] antiviral [18 19] anti-inflammatory[20] antioxidative [21] hepatoprotective [22] tumoricidal[23] and antiangiogenesis activities [24] as well as its neuro-protective activities against oxygen-glucose deprivation stress[25] Furthermore propolis and its compounds caffeic acidphenethyl ester (CAPE) and chrysin may restrain cell cycleproliferation or induce apoptosis in tumor cells [26]

The purpose of the present study was to clarify the effectsof Brazilian green propolis and its constituents against visiblelight- or UVA-induced cell damage in 661W photoreceptorcells or human skin-derived fibroblasts

2 Materials and Methods

21 Materials The drugs and sources used were as followsDulbeccorsquos modified Eaglesrsquo medium (DMEM) phenol red-free DMEM with sodium pyruvate without L-Glutamineand dimethyl sulfoxide (DMSO) and were purchased fromNacalai Tesque Inc (Kyoto Japan) Penicillin and strepto-mycin were purchased from Meiji Seika Kaisha Ltd (TokyoJapan) Fetal bovine serum (FBS) was purchased fromVALEANT (Costa Mesa CA USA) The cell counting Kit-8 (WST-8) was purchased from Dojin Kagaku (KumamotoJapan) Hoechst 33342 propidium iodide (PI) and 5-(and-6-)chloromethyl-2101584071015840-dichlorodihydrofluorescein diacetateacetyl ester (CM-H

2DCFDA) were purchased from Molec-

ular Probes (Eugene OR USA) 34-Di-O-caffeoylquinic

acid (34-CQA) 35-di-O-caffeoylquinic acid (35-CQA) p-coumaric acid and chlorogenic acidwere kindly gifted byApiCo Ltd (Gifu Japan)The propolis used in the present studywas Brazilian green propolis (Minas Gerais State Brazil)which originates mainly from Baccharis dracunculifolia TheBaccharis propolis was extracted with water at 50∘C to yieldthe extract used here (water extract of Brazilian green propo-lis WEP) The main constituents of WEP were previouslyreported

22 Cell Cultures The mouse retinal cone-cell line 661Wa transformed mouse cone-cell line derived from mouseretinal tumors was a gift from Dr Muayyad R Al-Ubaidi(University of Oklahoma Health Sciences Center OklahomaCity OK USA) The 661W cells were maintained in DMEMcontaining 10 fetal bovine serum (FBS) 100UmL peni-cillin and 100 120583gmL streptomycin Normal human skinfibroblast cells (NB1-RGB) were purchased from the RIKENBioresourceCenter Cell Bank (Tsukuba Ibaraki Japan) Cellswere cultured in phenol red-free DMEM with sodium pyru-vate without L-Glutamine containing 10 FBS 100UmLpenicillin and 100 120583gmL streptomycin Both cultures weremaintained at 37∘C in a humidified atmosphere of 95 airand 5 CO

2 The 661W and NB1-RGB cells were passaged by

trypsinization every 3 to 4 days respectively

23 Exposure of 661W to White Light The origin of the661W cell line is a mouse retinal tumor 661W has beencharacterized as a cone-specific cell line that expresses coneblue opsin or green opsin transducin and arrestin [27]The 661W cultures are useful for the estimation of light-induced stress in cone photoreceptors because they are ableto respond to light [28] The 661W mouse retinal cone-cellline cells were seeded at a density of 1 times 103 cells per wellinto a 96-well plate and the cells were then incubated ina humidified atmosphere of 95 air and 5 CO

2at 37∘C

for 24 h The entire medium was then replaced with phenolred-free DMEM containing 1 FBS After replacement ofthe medium propolis and its constituents were added to theculture One h after the addition of reagents the cultures wereexposed to 3000lx of white fluorescent light (C-FPS115DNikon Tokyo Japan) for 24 h at 37∘C The luminance wasmeasured using an LM-332 light meter (As One OsakaJapan)

24 Exposure of 661W or NB1-RGB to UVA Irradiation The661W and NB1-RGB cultures were seeded at a density of 3 times103 and 1 times 103 cells per well into 96-well plates respectivelyand the cells were then incubated in a humidified atmosphereof 95 air and 5 CO

2at 37∘C for 24 h To induce UVA

stress the 661W andNB1-RGB cells were washedwith phenolred-free DMEM containing 1 FBS After replacement ofthe medium propolis and its constituents were added tothe culture One h after the addition of reagents the 661Wcultures were exposed to 4 Jcm2 of UVA light (365 nmUVA light source CL-1000L UV Crosslinkers UltravioletProducts Ltd Cambridge UK) while the NB1-RGB cultures

Evidence-Based Complementary and Alternative Medicine 3

were exposed to 10 Jcm2 The UVA light was above the 96-well plate at a fixed distance of 115 cm Control cells wereincubated under the same conditions as experimental cellsbut were not exposed to UVA because they were covered withaluminum foil

25 Cell Proliferation Assay To evaluate cell survival weexamined the change in fluorescence intensity that followedthe cellular reduction ofWST-8 to formazan All experimentswere performed in phenol red-free DMEM at 37∘C Cellviability was assessed by culturing cells in a culture mediumcontaining 10 WST-8 (cell counting Kit-8) for 0 to 6 h at37∘C and was obtained by scanning with a microplate readerat 492 nm This absorbance was expressed as a percentageof that in the control cells (which were in phenol red-freeDMEM containing 1 FBS) after subtraction of backgroundabsorbance

26 Cell Death Assay (Hoechst 33342 and PI Staining) Celldeath was observed by using combination staining with twofluorescent dyes Hoechst 33342 and PI To examine theeffects of propolis on cell death induced by UVA irradiationNB1-RGB cells were seeded at a density of 1000 cells perwell into 96-well plates After pretreatment with propolisthe cells were irradiated with UVA 10 Jcm2 At the endof this culture period Hoechst 33342 (excitationemissionwavelengths 360490 nm) or PI (excitationemission wave-lengths 535617 nm) was added to the culture medium for15min at final concentrations of 8 and 15 120583M respectivelyImages were collected using an epifluorescence microscope(IX70 Olympus Tokyo Japan) fitted with a charge-coupleddevice camera (DP30BW Olympus) and fluorescence filtersfor Hoechst 33342 (U-MWU Olympus) and PI (U-MWIGOlympus)

27 Antioxidant Capacity Assay NB1-RGB cells and 661Wcells were seeded at a density of 1 times 103 cells and 2 times 103cells per well into 96-well plates and then incubated in ahumidified atmosphere of 95 air and 5 CO

2at 37∘C

respectively 24 h later the cell culture medium was replacedbefore treatment with propolis or its vehicle (phenol red-freeDMEMcontaining 1FBS) After pretreatment with propolisor its vehicle for 1 h we added the radical probe 5-(and-6-)chloromethyl-2101584071015840-dichlorodihydrofluorescein diacetateand acetyl ester (CM-H

2DCFDA) (10 120583M) by incubation for

20min at 37∘C Then the cell-culture medium was replacedto remove the extra probe CM-H

2DCFDA (inactive for

ROS) is converted to dichlorofluorescein (DCFH) (activefor ROS) by being taken into the cell and acted upon byan intracellular enzyme (esterase) To generate the ROSwe irradiated UVA 10 Jcm2 and 3000lx of white fluores-cent light for 24 h respectively Fluorescence was measuredafter the ROS-generating compounds had been present for6 h after the UVA or white light irradiation using SkanIt RE for Varioskan Flash 24 (Thermo Fisher ScientificWaltham MA USA) at excitationemission wavelengths of485535 nm

28 Western Blot Analysis NB1-RGB cells and 661W cellswere washed with PBS harvested and lysed using a cell-lysis buffer (RIPAbuffer R0278 Sigma-Aldrich)with protease(P8340 Sigma-Aldrich) and phosphatase inhibitor cocktails(P2850 and P5726 Sigma-Aldrich) The lysates were cen-trifuged at 12000timesg or 15min at 4∘C The supernatantswere collected and boiled for 5min in SDS sample buffer(Wako)The protein concentrationwasmeasured by compar-ison with a known concentration of bovine serum albuminusing a bicinchoninic acid (BCA) protein assay kit (PierceBiotechnology Rockford IL USA) A mixture of equalparts of an aliquot of protein and sample buffer with 102-mercaptoethanol was subjected to 10 sodium dodecylsulfate-polyacrylamide gel electrophoresis The separatedprotein was then transferred onto a polyvinylidene difluoridemembrane (Immobilon-P Millipore Corporation BedfordMA USA) The membranes were incubated with the fol-lowing primary antibodies phosphorylated p38mouse mon-oclonal antibody (Promega Madison WI USA) (1 1000)phosphorylated ERK rabbit polyclonal antibody (Cell Sig-naling Technology Inc Danvers MA USA) (1 1000) p38mousemonoclonal antibody (Santa Cruz Biotechnology IncSanta Cruz CA USA) (1 1000) ERK rabbit polyclonalantibody (Cell Signaling) (1 1000) and 120573-actin mouse mon-oclonal antibody (Sigma-Aldrich) (1 4000) After this incu-bation themembranewas incubatedwith the secondary anti-body HRP-conjugated goat anti-rabbit IgG (Pierce Biotech-nology) (1 2000) The immunoreactive bands were visual-ized using Super Signal West Femto Maximum SensitivitySubstrate (Pierce Biotechnology) andmeasured using GelPro(Media Cybernetics Silver Spring MD USA) To measurethe phosphorylation levels of ERK and p38 we normal-ized them with total ERK (t-ERK) and total p38 (t-p38)respectively

29 Effects of a MAPK Inhibitor on UVA-Induced CellularDamage NB1-RGB cells were seeded at a density of 1 times 103cells per well into a 96-well plate and then incubated ina humidified atmosphere of 95 air and 5 CO

2at 37∘C

24 h later the cell culture medium was replaced before treat-ment with propolis or its vehicle (phenol red-free DMEMcontaining 1 FBS) After pretreatment with propolis or itsvehicle for 1 h a MAPK inhibitor was added to the mediumseparately including SB203580 (a p38 MAPK inhibitor) andU0126 (an ERK inhibitor) at 5 120583M (both from CalbiochemSan Diego CA USA)

210 Statistical Analysis Data are presented as means plusmnSEM Statistical comparisons were made using Studentrsquos t-test or Dunnettrsquos test or Tukeyrsquos test by means of STAT VIEWversion 50 (SAS Institute Inc Cary NC USA) A value of119875 lt 005 was considered to indicate statistical significance

3 Results

31 Effects of Propolis and Its Constituents against VisibleLight-Induced Cell Damage in 661W Photoreceptor Cells We


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Figure 1 Effects of propolis and its constituent on cell damage induced by white light irradiation in a 661W culture ((a)ndash(c)) Representativephotographs at 24 h after light irradiation (a) Nonirradiated cells showed a normal shape (b)White light-induced alteration of cell shape (c)Pretreatment with propolis and (d) pretreatment with 34-di-O-caffeoylquinic acid at 1 h before the white light irradiation recovered the cellshape respectively (e) Cell viability was assessed by immersing cells in WST-8 solution for 6 h at 37∘C with absorbance recorded at 492 nmWhite light induced a decrease in cell viability Propolis (30 120583gmL) and 34-di-O-caffeoylquinic acid (3120583gmL) inhibited white light-inducedcell damage Data are shown as means plusmn SEM (119899 = 6) lowastlowast119875 lt 001 versus light exposure plus the vehicle-treated group and

119875 lt 001 versuscontrol CGA chlorogenic acid p-CA p-coumaric acid 35-CQA 35-di-O-caffeoylquinic acid 34-CQA 34-di-O-caffeoylquinic acid Scalebar represents 100 120583m

examined the effects of propolis and its constituents (chloro-genic acid p-coumaric acid 35-di-O-caffeoylquinic acidand 34-di-O-caffeoylquinic acid) on white light-induced661W cell damage Representative photographs of 661W cellsare shown in Figures 1(a)ndash1(d) As shown in Figures 1(a)ndash1(d) nontreated control cells displayed normal morphology(Figure 1(a)) whereas cells exposed to white light revealedshrinkage and condensation of their nuclei (Figure 1(b))After exposure to visible light plus propolis or 34-di-O-caffeoylquinic acid the nucleus morphology was similar tothat of the normal control cells (Figures 1(a) 1(c) and 1(d))To evaluate cell survival quantitatively we examined thechange in fluorescence intensity that occurred following thecellular reduction of WST-8 to formazan In the white light-irradiated vehicle group the cell viability was decreased to30of that of the control group Propolis (30120583gmL) and 34-di-O-caffeoylquinic acid (3 120583gmL) inhibited the decrease incell viability by light irradiation In contrast chlorogenic acidp-coumaric acid or 35-di-O-caffeoylquinic acid at 3120583gmLrespectively did not affect cell viability (Figure 1(e))

32 Effects of Propolis and Its Constituents against UVA-Induced Cell Damage and Phosphorylated p38MAPK in 661WPhotoreceptor Cells We studied the effects of propolis and its

constituents onUVA-induced 661Wcell damageUVA irradi-ation at 4 Jcm2 induced a 05-fold decrease in the cell viability(versus the control group) Pretreatment with propolis at 10ndash30 120583gmL concentration-dependently inhibited the decreasein cell viability (Figure 2(a)) The two dicaffeoylquinic acids(34- and 35-di-O-caffeoylquinic acid) reduced this celldamage (Figures 2(b) and 2(c)) The other chlorogenic acidand p-coumaric acid had no detectable effects (Figures 2(d)and 2(e)) To clarify the mechanism of action of propolisthe activities of mitogen-activated protein kinases (MAPKs)which are signals related to oxidative stress were measuredusing immunoblotting Phosphorylated p38 was markedlyincreased (versus nonirradiated cells) in the cells exposedto UVA against 120573-actin Propolis significantly reduced theUVA-induced phosphorylation of p38 (Figure 2(f))

33 The Effect of Propolis against UVA-Induced Cell Dam-age in Human Skin-Derived Fibroblasts Representative pho-tographs of Hoechst 33342 and PI staining after UVA irradi-ation to NB1-RGB fibroblast cells are shown in Figure 3(a)Hoechst 33342 stains all cells (live and dead cells) whereasPI stains only dead cells In the UVA 10 Jcm2-irradiatedgroup the PI positive cell numbers increased more than 10-fold (versus control) Propolis (3 10 and 30 120583gmL) added


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Figure 2 Effects of propolis and its constituents on cell damage or phosphorylated p38 induced by UVA irradiation in a 661W culture((a)ndash(e)) Cell viability was assessed by immersing cells in WST-8 solution for 6 h at 37∘C with absorbance recorded at 492 nm UVA induceda decrease in cell viability (a) Propolis at 10 and 30 120583gmL significantly inhibited UVA-induced cell damage in a 661W culture (b) 35-di-O-caffeoylquinic acid (c) 34-di-O-caffeoylquinic acid and (d) chlorogenic acid at 1 and 3120583gmL significantly inhibited cell damagerespectively (e) p-Coumaric acid at 1 120583gmL inhibited cell damage (f) Representative band images showing activation of p38 in the nontreatedUVA exposure plus vehicle-treated and UVA exposure plus propolis-treated cells UVA exposure plus vehicle-treated group had 2 lanes (g)Quantitative analysis of the band density of p38 Data are shown as means plusmn SEM (119899 = 6) lowast119875 lt 005 lowastlowast119875 lt 001 versus UVA exposure plusthe vehicle-treated group and

119875 lt 001 versus control CGA chlorogenic acid p-CA p-coumaric acid 35-CQA 35-di-O-caffeoylquinicacid and 34-CQA 34-di-O-caffeoylquinic acid


Control Vehicle

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Figure 3 Effects of propolis on cell damage induced by UVA irradiation in an NB1-RGB culture (a) Representative fluorescence microscopicimages show nuclear staining for Hoechst 33342 and PI after UVA 10 Jcm2 irradiation Upper photomicrographs show Hoechst 33342 andlower ones propidium iodide (PI) staining at 6 h after UVA irradiation (b) The number of cells exhibiting PI fluorescence was counted andpositive cells were expressed as the percentage of PI to Hoechst 33342 Pretreatment of cells with propolis (30120583gmL) significantly reducedthe amount of cell death (versus cells treated with UVA irradiation alone) (c) Cell viability was assessed by immersing cells inWST-8 solutionfor 6 h at 37∘C with absorbance recorded at 492 nm UVA induced a decrease in cell viability Propolis concentration-dependently inhibitedUVA-induced cell damage Data are shown as means plusmn SEM (119899 = 6) lowast119875 lt 005 lowastlowast119875 lt 001 versus UVA exposure plus the vehicle-treatedgroup and

119875 lt 001 versus control Scale bar represents 100 120583m

to the culture medium concentration-dependently decreasedthe number of cells showing PI staining after UVA irradiation(versus vehicle treatment) (Figure 3(b)) In the WST assaycell viability was found to be reduced to 07-fold after UVAirradiation (versus control) and this cell damage was reducedby treatment with propolis at 3ndash30120583gmL in a concentration-dependent manner (Figure 3(c))

34 Effects of Propolis Constituents against UVA-Induced CellDamage in Human Skin-Derived Fibroblasts As describedabove propolis has protective effects against UVA-induced

cell damage in NB1-RGB cells We next studied the effects offour constituents of propolis In the UVA 10 Jcm2-irradiatedgroup cell viability decreased 05-fold (versus control)(Figure 4) All four constituents suppressed this decreasein cell viability in a concentration-dependent manner itseffect being significant at concentrations of 3120583gmL or more(Figures 4(a)ndash4(d))

35 Effect of Propolis on UVA- or White Light-InducedIntracellular ROS Production in Human Skin-DerivedFibroblasts or 661W Photoreceptor Cells To investigate the


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Figure 4 Effects of constituents of propolis on cell damage induced by UVA irradiation in an NB1-RGB culture ((a)ndash(d)) Cell viability wasassessed by immersing cells in WST-8 solution for 6 h at 37∘C with absorbance recorded at 492 nm UVA induced a decrease in cell viability(a) 35-Di-O-caffeoylquinic acid (b) 34-di-O-caffeoylquinic acid (c) chlorogenic acid and (d) p-coumaric acid at 1 and 3120583gmL significantlyinhibited cell damage respectively (versus cells treated with UVA irradiation alone) Data are shown as means plusmn SEM (119899 = 6) lowastlowast119875 lt 001versus UVA exposure plus the vehicle-treated group and

119875 lt 001 versus control CGA chlorogenic acid p-CA p-coumaric acid 35-CQA35-di-O-caffeoylquinic acid and 34-CQA 34-di-O-caffeoylquinic acid

inhibitory effect of propolis on intracellular ROS productionby UVA or white light irradiation in NB1-RGB or 661Wcells we employed a radical scavenging-capacity assay usingthe ROS-sensitive probes 5-(and-6-)chloromethyl-2101584071015840-dichlorodihydrofluorescein diacetate (CM-H

2DCFDA)

In the 661W photoreceptor cells UVA 10 Jcm2 or 3000lxof white light-irradiated group the intracellular ROSproduction increased 25- or 23-fold respectively (versuscontrol) and it was concentration-dependently suppressedby the addition of propolis (Figures 5(a) and 5(b)) In thehuman skin-derived fibroblasts UVA 10 Jcm2-irradiatedgroup the intracellular ROS production increased 15-fold(versus control) and it was concentration-dependentlysuppressed by the addition of propolis (Figure 5(c))

36 Phosphorylations of p38 and ERK Induced by UVA andthe Effects of an MAPK Inhibitor in Human Skin-DerivedFibroblasts To investigate the mechanism by which propolissuppressed cell damage byUVA we evaluated the activities ofmitogen-activated protein kinase (MAPKs) which are signalsrelated to oxidative stress stimulated by UVA irradiation inNB1-RGB cells using Western blotting Phosphorylated-p38(p-p38) and phosphorylated-extracellular signal regulatedprotein kinases (p-ERK12) were markedly increased (versuscontrol) in NB1-RGB cells that were irradiated 10 Jcm2 UVAagainst total p38 and ERK respectively UVA irradiationincreased the levels of p-p38 and p-ERK by 20- and 30-fold respectively Propolis (30 120583gmL) treatment significantlyreduced the UVA-induced phosphorylation of p38 and ERK


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(c)

Figure 5 Effects of propolis on intracellular ROS production induced by UVA or white light irradiation in an NB1-RGB or 661W cultureThe NB1-RGB or 661W culture was treated with propolis for 1 h and this was supplemented with CM-H

2DCFDA at 10120583M for 20min ROS

production was stimulated with UVA 10 Jcm2 or 3000lx of white light and fluorescence was measured at 0ndash20min UVA or white lightirradiation induced oxidation of DCFH in NB1-RGB or 661W culture Intracellular ROS production was increased by UVA or white lightirradiation while it was concentration-dependently reduced by propolis treatment Data are shown as means plusmn SEM (119899 = 6) lowast119875 lt 005lowastlowast119875 lt 001 versus UVA or white light exposure plus the vehicle-treated group and

119875 lt 001 versus control

(Figures 6(a)ndash6(c)) Treatment with SB203580 (a p38 MAPKinhibitor) or U0126 (an ERK inhibitor) at 1 h before UVAirradiation inhibited the decrease of cellular viability inducedby UVA (Figure 6(d)) Although we have examined whetheran MAPK inhibitor could alter the effects of propolis anMAPK inhibitor did not affect the cell viability of a propolis-treated group

4 Discussion

In the present study we demonstrated that propolis and itsconstituents suppressed cell damage induced bywhite light orUVA in 661W cells and by UVA in NB1-RGB cells Treatmentwith propolis suppressed the intracellular ROS production

stimulated by UVA or white light irradiation Propolis alsoinhibited theUVA-induced phosphorylation of p38 and ERK

Brazilian green propolis (water-extract propolis WEP)exhibited potent antioxidant effects against a variety of ROSin our previous report [29] Moreover it has been reportedthat the main constituents of WEP (caffeoylquinic acidderivatives 34-CQA 35-CQA) were also found to haveantioxidant effects with similar efficacies to those of troloxwhich is a major antioxidant [29] These constituents maybe mainly responsible for the powerful antioxidative effectsof WEP In the present study propolis and its constituents(34-CQA and 35-CQA) suppressed cell damage induced byUVA irradiation via antioxidant effects Surprisingly CGAand p-CA also inhibited UVA-induced cell damage It issuggested that p-CA with IC

50values of more than 100120583M

did not scavenge any of the ROS [29] It has been suggested


Control Vehicle Propolis

UVA

p-p38

p38

p-ERK

ERK

120573-Actin

(a)

0

50

100

150

200

250

300

350

400


p38

Activ

atio

n (p

-p38

p38

)UVA

lowastlowast

(b)

0

50

100

150

200

250

300


UVA

ERK

Activ

atio

n(p

-ERK

12

ERK

12

)

lowastlowast

(c)

0

20

40

60

80

100

120

Control Propolis Vehicle Propolis SB203850 U0126UVA

lowast

lowast

lowastlowastC

ell v

iabi

lity

( o

f con

trol)

(d)

Figure 6 Effects of propolis on UVA-induced expression of phosphorylated p38 and ERK12 and MAPK inhibitors in an NB1-RGB cultureRepresentative Western blots (a) showing activation of p38 and ERK in the nontreated UVA 10 Jcm2 exposure plus vehicle-treated andUVA exposure plus propolis-treated (30120583gmL) groups Phosphorylation of p38 (b) and ERK12 (c) was determined by immunoblottingassay Quantitative analysis of the band density of p38 (b) and ERK (c) by densitometric analysisThese intensities were normalized with totalp38 and total ERK respectively (d) Cell viability was assessed by immersing cells inWST-8 solution for 6 h at 37∘C with absorbance recordedat 492 nm SB203580 and U0126 inhibited the decrease of cellular viability induced by UVA and the level was as high as of a propolis-treatedgroup Data are shown as means plusmn SEM (119899 = 6) lowastlowast119875 lt 001 versus UVA exposure plus vehicle-treated cells and


that antioxidant properties arise from complex mechanismsor synergistic interactions between constituents of propolisHowever further studies are necessary to understand theexact protective mechanism of CGA and p-CA

UV irradiation gives rise to the activation of multiplecell surface cytokine and growth factor receptors mitogen-activated protein kinases (MAPKs) signal modules such asextracellular-regulated protein kinase (ERK) and p38 kinase[30] Fibroblasts exposed toUV-induced oxidative stress havebeen shown to increase the expression of phosphorylated-MAPKs (p38 and ERK) [30 31] Various stresses includingischemia ultraviolet exposure and oxidative stress stimulate

p38 and JNK activation [32] They are involved in cell apop-tosis and differentiation [32] Rapid activation of p38 can beinduced by a variety of cellular stressors including UV irra-diation We investigated whether propolis could inhibit UV-induced phosphorylation of MAPKs The present data wereconsistent with studies by Syed et al [33] where it was shownthat the peak of the activated p38 in normal keratinocytesis 2ndash10min after UVA irradiation These results indicatethat UVA-induced ROS production may cause the activationof a p38 signaling cascade and propolis can reduce UVA-induced fibroblasts damage by an antioxidative mechanismMoreover oxidative stress mitogens and survival factors


activate ERK activation ERK is involved in cell proliferationand differentiation [34] ERK activation achieved a peakimmediately after UVA irradiation and propolis significantlyinhibited ERK activation Taken together the activationof ERK may provide a survival signal that allows humanfibroblasts to escape fromUVA-induced apoptosis AlthoughMAPKs (p38 and ERK) are activated by UVA irradiationthe effects of their inhibitors on UVA-induced human skin-derived fibroblasts cells damage are still unknown SB203580(a p38 inhibitor) and U0126 (an ERK inhibitor) inhibited thedecrease of cellular viability induced by UVA and the levelwas as high as of a propolis-treated group Our data indicatethat p38 and ERK signal pathways are involved in UVA-induced cellular damage and there is a potential link betweenpropolis and MAPK suggesting that MAPK signaling isinvolved in UVA damage and propolis shows the protectiveeffects by suppressing the MAPK activation

Activator protein-1 (AP-1) is an important factor forUVA-induced skin damage AP-1 is composed of heterodimersof members of the Fos (c-Fos FosB Fra-1 and Fra-2) andthe Jun (c-Jun JunB and JunD) families of proteins orof homodimers of members of the Jun family of proteins[35] AP-1 induces collagen degradation by promoting theexpression of matrix metalloproteinases MMP-1 MMP-3and MMP-9 [36 37] Furthermore AP-1 causes collagendegradation by preventing the expression of procollagen-1[38] Reports suggest that AP-1 activation through p38 andorERK andor JNK (c-Jun N-terminal kinase) is essential forUVA-induced skin damage Therefore there is a possibilitythat propolis may inhibit AP-1 activation by suppressing p38ERK and JNK activation However further studies will beneeded to clarify the precise mechanisms in UVA-inducedskin damage

5 Conclusion

Propolis may become a therapeutic candidate for the treat-ment of AMD and skin damage induced by visible light orUV irradiation

Acknowledgments

Masamitsu Shimazawa and Hideaki Hara received a financialsupport of Api Co Ltd (Gifu Japan) as a collaborativeresearch and the present study was conducted by thissupport Hiromi Murase and Kazuhiro Tsuruma declare thatthere is no conflict of interests regarding the publication ofthis paperMamoruKakino andKenji Ichihara are employeesof Api Co Ltd and the present study was conducted byusing Brazilian green propolis and its constituents of theircompany

References

[1] F Afaq and H Mukhtar ldquoBotanical antioxidants in the pre-vention of photocarcinogenesis and photoagingrdquo ExperimentalDermatology vol 15 no 9 pp 678ndash684 2006

[2] M Yaar and B A Gilchrest ldquoCellular and molecular mecha-nisms of cutaneous agingrdquo Journal of Dermatologic Surgery and

Oncology vol 16 no 10 pp 915ndash922 1990[3] R B Setlow ldquoThewavelengths in sunlight effective in producing

skin cancer a theoretical analysisrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 71 no9 pp 3363ndash3366 1974

[4] A J Chucair N P Rotstein J P SanGiovanni A During E YChew and L E Politi ldquoLutein and zeaxanthin protect photore-ceptors fromapoptosis induced by oxidative stress relationwithdocosahexaenoic acidrdquo Investigative Ophthalmology and VisualScience vol 48 no 11 pp 5168ndash5177 2007

[5] S Shahinfar D P Edward and M O M Tso ldquoA pathologicstudy of photoreceptor cell death in retinal photic injuryrdquoCurrent Eye Research vol 10 no 1 pp 47ndash59 1991

[6] W K Noell V S Walker B S Kang and S Berman ldquoRetinaldamage by light in ratsrdquo Investigative ophthalmology vol 5 no5 pp 450ndash473 1966

[7] S Imai Y Inokuchi S Nakamura K Tsuruma M Shimazawaand H Hara ldquoSystemic administration of a free radical scav-enger edaravone protects against light-induced photoreceptordegeneration in themouse retinardquoEuropean Journal of Pharma-cology vol 642 no 1ndash3 pp 77ndash85 2010

[8] J C Khan D A Thurlby H Shahid et al ldquoSmoking andage related macular degeneration the number of pack yearsof cigarette smoking is a major determinant of risk for bothgeographic atrophy and choroidal neovascularisationrdquo BritishJournal of Ophthalmology vol 90 no 1 pp 75ndash80 2006

[9] D D G Despriet C C W Klaver J C M Witteman et alldquoComplement factor H polymorphism complement activatorsand risk of age-related macular degenerationrdquo Journal of theAmericanMedical Association vol 296 no 3 pp 301ndash309 2006

[10] P G Pietta C Gardana and A M Pietta ldquoAnalytical methodsfor quality control of propolisrdquo Fitoterapia vol 73 supplement1 pp S7ndashS20 2002

[11] A Kujumgiev I Tsvetkova Y Serkedjieva V Bankova RChristov and S Popov ldquoAntibacterial antifungal and antiviralactivity of propolis of different geographic originrdquo Journal ofEthnopharmacology vol 64 no 3 pp 235ndash240 1999

[12] V Bankova M C Marcucci S Simova N Nikolova AKujumgiev and S Popov ldquoAntibacterial diterpenic acids fromBrazilian propolisrdquo Zeitschrift fur Naturforschung Section C vol51 no 5-6 pp 277ndash280 1996

[13] R M Souza M C de Souza M L Patitucci and J F MSilva ldquoEvaluation of antioxidant and antimicrobial activitiesand characterization of bioactive components of two Brazilianpropolis samples using a pK a-guided fractionationrdquo Zeitschriftfur Naturforschung C vol 62 no 11-12 pp 801ndash807 2007

[14] B Trusheva I Todorov M Ninova H Najdenski A Danesh-mand and V Bankova ldquoAntibacterial mono- and sesquiterpeneesters of benzoic acids from Iranian propolisrdquo Chemistry Cen-tral Journal vol 4 no 1 article 8 2010

[15] A Uzel K Sorkun O Oncag D Cogulu O Gencay andB Salih ldquoChemical compositions and antimicrobial activitiesof four different Anatolian propolis samplesrdquo MicrobiologicalResearch vol 160 no 2 pp 189ndash195 2005

[16] L Drago E De Vecchi L Nicola and M R Gismondo ldquoInvitro antimicrobial activity of a novel propolis formulation(Actichelated propolis)rdquo Journal of Applied Microbiology vol103 no 5 pp 1914ndash1921 2007

[17] K Ramanauskiene A M Inkeniene A Savickas R Mas-teikova and V Brusokas ldquoAnalysis of the antimicrobial activityof propolis and lysozyme in semisolid emulsion systemsrdquo ActaPoloniae Pharmaceutica vol 66 no 6 pp 681ndash688 2009


[18] M Amoros C M O Simoes L Girre F Sauvager and MCormier ldquoSynergistic effect of flavones and flavonols againstherpes simplex virus type 1 in cell culture Comparison with theantiviral activity of propolisrdquo Journal of Natural Products vol55 no 12 pp 1732ndash1740 1992

[19] M Amoros E Lurton J Boustie L Girre F Sauvager andM Cormier ldquoComparison of the anti-herpes simplex virusactivities of propolis and 3-methyl-but-2-enyl caffeaterdquo Journalof Natural Products vol 57 no 5 pp 644ndash647 1994

[20] N Paulino C Teixeira R Martins et al ldquoEvaluation of theanalgesic and anti-inflammatory effects of a Brazilian greenpropolisrdquo Planta Medica vol 72 no 10 pp 899ndash906 2006

[21] E W Teixeira D Message G Negri A Salatino and PC Stringheta ldquoSeasonal variation chemical composition andantioxidant activity of brazilian propolis samplesrdquo Evidence-based Complementary and AlternativeMedicine vol 7 no 3 pp307ndash315 2010

[22] P Basnet K Matsushige K Hase S Kadota and T NambaldquoFour di-O-caffeoyl quinic acid derivatives from propolispotent hepatoprotective activity in experimental liver injurymodelsrdquo Biological and Pharmaceutical Bulletin vol 19 no 11pp 1479ndash1484 1996

[23] T Mitamura T Matsuno S Sakamoto et al ldquoEffects of a newclerodane diterpenoid isolated from propolis on chemicallyinduced skin tumors in micerdquo Anticancer Research vol 16 no5 pp 2669ndash2672 1996

[24] H Izuta M Shimazawa K Tsuruma Y Araki S Mishima andH Hara ldquoBee products prevent VEGF-induced angiogenesis inhuman umbilical vein endothelial cellsrdquo BMC Complementaryand Alternative Medicine vol 9 article 45 2009

[25] Y Nakajima M Shimazawa S Mishima and H Hara ldquoNeu-roprotective effects of Brazilian green propolis and its mainconstituents against oxygen-glucose deprivation stress with agene-expression analysisrdquo Phytotherapy Research vol 23 no 10pp 1431ndash1438 2009

[26] D Sawicka H Car M H Borawska and J Niklinski ldquoThe anti-cancer activity of propolisrdquo Folia Histochemica et Cytobiologicavol 50 no 1 pp 25ndash37 2012

[27] E Tan X-Q Ding A Saadi N Agarwal M I Naash andM RAl-Ubaidi ldquoExpression of cone-photoreceptor-specific antigensin a cell line derived from retinal tumors in transgenic micerdquoInvestigative Ophthalmology and Visual Science vol 45 no 3pp 764ndash768 2004

[28] R R Krishnamoorthy M J Crawford M M Chaturvedi et alldquoPhoto-oxidative stress down-modulates the activity of nuclearfactor-120581B via involvement of caspase-1 leading to apoptosis ofphotoreceptor cellsrdquo Journal of Biological Chemistry vol 274no 6 pp 3734ndash3743 1999

[29] Y Nakajima K Tsuruma M Shimazawa S Mishima andH Hara ldquoComparison of bee products based on assays ofantioxidant capacitiesrdquo BMC Complementary and AlternativeMedicine vol 9 article 4 2009

[30] Z Assefa M Garmyn R Bouillon W Merlevede J R Van-denheede and P Agostinis ldquoDifferential stimulation of ERKand JNK activities by ultraviolet B irradiation and epidermalgrowth factor in human keratinocytesrdquo Journal of InvestigativeDermatology vol 108 no 6 pp 886ndash891 1997

[31] E Akasaka S Takekoshi Y Horikoshi et al ldquoProtein oxidativedamage and heme oxygenase in sunlight-exposed human skinroles of MAPK responses to oxidative stressrdquo Tokai Journal ofExperimental and Clinical Medicine vol 35 no 4 pp 152ndash1642010

[32] Z Xia M Dickens J Raingeaud R J Davis and M EGreenberg ldquoOpposing effects of ERK and JNK-p38 MAPkinases on apoptosisrdquo Science vol 270 no 5240 pp 1326ndash13311995

[33] D N Syed F Afaq and H Mukhtar ldquoDifferential activationof signaling pathways by UVA and UVB radiation in normalhuman epidermal keratinocytesrdquo Photochemistry and Photobi-ology vol 88 no 5 pp 1184ndash1190 2012

[34] Y Luo and D B DeFranco ldquoOpposing roles for ERK12 inneuronal oxidative toxicity distinct mechanisms of ERK12action at early versus late phases of oxidative stressrdquo Journal ofBiological Chemistry vol 281 no 24 pp 16436ndash16442 2006

[35] T Hai and T Curran ldquoCross-family dimerization of tran-scription factors FosJun and ATFCREB alters DNA bindingspecificityrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 88 no 9 pp 3720ndash3724 1991

[36] L Rittie and G J Fisher ldquoUV-light-induced signal cascades andskin agingrdquo Ageing Research Reviews vol 1 no 4 pp 705ndash7202002

[37] G J Fisher and J J Voorhees ldquoMolecular mechanisms ofphotoaging and its prevention by retinoic acid ultraviolet irra-diation induces MAP kinase signal transduction cascades thatinduce Ap-1-regulated matrix metalloproteinases that degradehuman skin in vivordquo Journal of Investigative DermatologySymposium Proceedings vol 3 no 1 pp 61ndash68 1998

[38] J H Chung S Kang J Varani J Lin G J Fisher and J JVoorhees ldquoDecreased extracellular-signal-regulated kinase andincreased stress-activatedMAP kinase activities in aged humanskin in vivordquo Journal of Investigative Dermatology vol 115 no2 pp 177ndash182 2000

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


singlet oxygen which causes eliminations or point mutationsin mitochondrial DNA and is involved in DNA damagewhich activates the DNA damage response system finallyleading to cell senescence Therefore UVA is considered afundamental cause of aging

High levels of visible light or UV may cause oculardamage especially later in life It has been noted that long-term light exposure results in photoreceptor degradationand it may be among the most relevant damaging factorsinvolved in age-related macular degeneration (AMD) [4]Excessive light exposure can be a risk factor for the onsetand progression of AMD [5] and it leads to photoreceptordegeneration in animals [6 7] Both external and internalfactors are thought to be a pathogenesis of AMD [8 9] andexposure to sunlight or ultraviolet radiation is also a well-established risk factor for AMD

Propolis is made from a sticky substance that honeybeesproduce by mixing their own waxes with resinous sapobtained from the bark and leaf-buds of certain trees andother flowering plants Propolis is used as a sealant andsterilant in honeybee nestsThe color of propolis can be greenyellow brown or almost black depending on the plants fromwhich the resinous substance is collected [10]The propertiesand constituents of propolis also differ with its geographicalorigin [11] Brazilian green propolis is made of aromaticacids (cinnamic acid derivatives ferulic acid and caffeicacid) diethyl methyl succinate isobutylquinoline generalacetal patchouli alcohol menthol amyrins and flavonoidsBrazilian propolis has been the subject of many studies dueto its biological activities such as its antibacterial [12 13]antifungal [11 14ndash17] antiviral [18 19] anti-inflammatory[20] antioxidative [21] hepatoprotective [22] tumoricidal[23] and antiangiogenesis activities [24] as well as its neuro-protective activities against oxygen-glucose deprivation stress[25] Furthermore propolis and its compounds caffeic acidphenethyl ester (CAPE) and chrysin may restrain cell cycleproliferation or induce apoptosis in tumor cells [26]

The purpose of the present study was to clarify the effectsof Brazilian green propolis and its constituents against visiblelight- or UVA-induced cell damage in 661W photoreceptorcells or human skin-derived fibroblasts

2 Materials and Methods

21 Materials The drugs and sources used were as followsDulbeccorsquos modified Eaglesrsquo medium (DMEM) phenol red-free DMEM with sodium pyruvate without L-Glutamineand dimethyl sulfoxide (DMSO) and were purchased fromNacalai Tesque Inc (Kyoto Japan) Penicillin and strepto-mycin were purchased from Meiji Seika Kaisha Ltd (TokyoJapan) Fetal bovine serum (FBS) was purchased fromVALEANT (Costa Mesa CA USA) The cell counting Kit-8 (WST-8) was purchased from Dojin Kagaku (KumamotoJapan) Hoechst 33342 propidium iodide (PI) and 5-(and-6-)chloromethyl-2101584071015840-dichlorodihydrofluorescein diacetateacetyl ester (CM-H

2DCFDA) were purchased from Molec-

ular Probes (Eugene OR USA) 34-Di-O-caffeoylquinic

acid (34-CQA) 35-di-O-caffeoylquinic acid (35-CQA) p-coumaric acid and chlorogenic acidwere kindly gifted byApiCo Ltd (Gifu Japan)The propolis used in the present studywas Brazilian green propolis (Minas Gerais State Brazil)which originates mainly from Baccharis dracunculifolia TheBaccharis propolis was extracted with water at 50∘C to yieldthe extract used here (water extract of Brazilian green propo-lis WEP) The main constituents of WEP were previouslyreported

22 Cell Cultures The mouse retinal cone-cell line 661Wa transformed mouse cone-cell line derived from mouseretinal tumors was a gift from Dr Muayyad R Al-Ubaidi(University of Oklahoma Health Sciences Center OklahomaCity OK USA) The 661W cells were maintained in DMEMcontaining 10 fetal bovine serum (FBS) 100UmL peni-cillin and 100 120583gmL streptomycin Normal human skinfibroblast cells (NB1-RGB) were purchased from the RIKENBioresourceCenter Cell Bank (Tsukuba Ibaraki Japan) Cellswere cultured in phenol red-free DMEM with sodium pyru-vate without L-Glutamine containing 10 FBS 100UmLpenicillin and 100 120583gmL streptomycin Both cultures weremaintained at 37∘C in a humidified atmosphere of 95 airand 5 CO

2 The 661W and NB1-RGB cells were passaged by

trypsinization every 3 to 4 days respectively

23 Exposure of 661W to White Light The origin of the661W cell line is a mouse retinal tumor 661W has beencharacterized as a cone-specific cell line that expresses coneblue opsin or green opsin transducin and arrestin [27]The 661W cultures are useful for the estimation of light-induced stress in cone photoreceptors because they are ableto respond to light [28] The 661W mouse retinal cone-cellline cells were seeded at a density of 1 times 103 cells per wellinto a 96-well plate and the cells were then incubated ina humidified atmosphere of 95 air and 5 CO

2at 37∘C

for 24 h The entire medium was then replaced with phenolred-free DMEM containing 1 FBS After replacement ofthe medium propolis and its constituents were added to theculture One h after the addition of reagents the cultures wereexposed to 3000lx of white fluorescent light (C-FPS115DNikon Tokyo Japan) for 24 h at 37∘C The luminance wasmeasured using an LM-332 light meter (As One OsakaJapan)

24 Exposure of 661W or NB1-RGB to UVA Irradiation The661W and NB1-RGB cultures were seeded at a density of 3 times103 and 1 times 103 cells per well into 96-well plates respectivelyand the cells were then incubated in a humidified atmosphereof 95 air and 5 CO

2at 37∘C for 24 h To induce UVA

stress the 661W andNB1-RGB cells were washedwith phenolred-free DMEM containing 1 FBS After replacement ofthe medium propolis and its constituents were added tothe culture One h after the addition of reagents the 661Wcultures were exposed to 4 Jcm2 of UVA light (365 nmUVA light source CL-1000L UV Crosslinkers UltravioletProducts Ltd Cambridge UK) while the NB1-RGB cultures






2at 37∘C








2at 37∘C



3 Results



Control

(a)

Vehicle

(b)

Propolis

(c)

34-CQA

(d)

0

20

40

60

80

100


Cel

l via

bilit

y (

of c

ontro

l)



p-CA

34-CQA35-CQA

(e)








120

0

20

40

60

80

100


UVA

lowastlowast

30 (120583gmL)

Cel

l via

bilit

y (

of c

ontro

l)

(a)

0

20

40

60

80

100

120


35-CQAUVA

lowastlowast

(120583gmL)

Cel

l via

bilit

y (

of c

ontro

l)

(b)

0

20

40

60

80

100

120


UVA

lowastlowastlowast

3 (120583gmL)

Cel

l via

bilit

y (

of c

ontro

l)

(c)

UVA

0

20

40

60

80

100

120


(120583gmL)

Cel

l via

bilit

y (

of c

ontro

l)

(d)


UVA

0

20

40

60

80

100

120

3 (120583gmL)

Cel

l via

bilit

y (

of c

ontro

l)

p-CA

(e)


0

50

100

150

200

250

300

350

400


p38

Activ

atio

n (p

-p38

act

in)

lowast

p-p38

120573-Actin

(f)




Control Vehicle

Propolis

Hoechst33342

PI

03

UVA

3 (120583gmL)

(a)

0

5

10

15

20

25

30


PI p

ositi

ve ce

lls(

of t

otal

cell

num

bers

)

UVAPropolis

lowastlowast

lowastlowast

lowastlowast

30 (120583gmL)

(b)

0

20

40

60

80

100

120

140


UVA

Propolis

lowastlowast

lowastlowast

30 (120583gmL)

Cel

l via

bilit

y (

of c

ontro

l)

(c)








0

20

40

60

80

100

120

Control Vehicle 1 3

35-CQA

UVA


(120583gmL)

Cel

l via

bilit

y (

of c

ontro

l)

(a)

35-CQA

UVA

Control Vehicle 10

20

40

60

80

100

120

lowastlowast

lowastlowast

3 (120583gmL)

Cel

l via

bilit

y (

of c

ontro

l)

(b)

Control Vehicle 1 3

UVA

CGA

0

20

40

60

80

100

120


(120583gmL)

Cel

l via

bilit

y (

of c

ontro

l)

(c)

UVA

0

20

40

60

80

100

120



Cel

l via

bilit

y (

of c

ontro

l)

p-CA

(d)




2DCFDA)




0

50

100

150

200

250

300


UVA

Intr

acel

lula

r RO

S pr

oduc

tion

( o

f con

trol)

lowast

lowast

(120583gmL)

(a)

0

50

100

150

200

250


White light

30

lowast

lowast

lowast

(120583gmL)

Intr

acel

lula

r RO

S pr

oduc

tion

( o

f con

trol)

(b)

0

20

40

60

80

100

120

140

160

180


UVA

Propolis

lowast

lowast

(120583gmL)

Intr

acel

lula

r RO

S pr

oduc

tion

( o

f con

trol)

(c)






4 Discussion








UVA

p-p38

p38

p-ERK

ERK

120573-Actin

(a)

0

50

100

150

200

250

300

350

400


p38

Activ

atio

n (p

-p38

p38

)UVA

lowastlowast

(b)

0

50

100

150

200

250

300


UVA

ERK

Activ

atio

n(p

-ERK

12

ERK

12

)

lowastlowast

(c)

0

20

40

60

80

100

120


lowast

lowast

lowastlowastC

ell v

iabi

lity

( o

f con

trol)

(d)









5 Conclusion


Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















2at 37∘C








2at 37∘C



3 Results



Control

(a)

Vehicle

(b)

Propolis

(c)

34-CQA

(d)

0

20

40

60

80

100


Cel

l via

bilit

y (

of c

ontro

l)



p-CA

34-CQA35-CQA

(e)








120

0

20

40

60

80

100


UVA

lowastlowast

30 (120583gmL)

Cel

l via

bilit

y (

of c

ontro

l)

(a)

0

20

40

60

80

100

120


35-CQAUVA

lowastlowast

(120583gmL)

Cel

l via

bilit

y (

of c

ontro

l)

(b)

0

20

40

60

80

100

120


UVA

lowastlowastlowast

3 (120583gmL)

Cel

l via

bilit

y (

of c

ontro

l)

(c)

UVA

0

20

40

60

80

100

120


(120583gmL)

Cel

l via

bilit

y (

of c

ontro

l)

(d)


UVA

0

20

40

60

80

100

120

3 (120583gmL)

Cel

l via

bilit

y (

of c

ontro

l)

p-CA

(e)


0

50

100

150

200

250

300

350

400


p38

Activ

atio

n (p

-p38

act

in)

lowast

p-p38

120573-Actin

(f)




Control Vehicle

Propolis

Hoechst33342

PI

03

UVA

3 (120583gmL)

(a)

0

5

10

15

20

25

30


PI p

ositi

ve ce

lls(

of t

otal

cell

num

bers

)

UVAPropolis

lowastlowast

lowastlowast

lowastlowast

30 (120583gmL)

(b)

0

20

40

60

80

100

120

140


UVA

Propolis

lowastlowast

lowastlowast

30 (120583gmL)

Cel

l via

bilit

y (

of c

ontro

l)

(c)








0

20

40

60

80

100

120

Control Vehicle 1 3

35-CQA

UVA


(120583gmL)

Cel

l via

bilit

y (

of c

ontro

l)

(a)

35-CQA

UVA

Control Vehicle 10

20

40

60

80

100

120

lowastlowast

lowastlowast

3 (120583gmL)

Cel

l via

bilit

y (

of c

ontro

l)

(b)

Control Vehicle 1 3

UVA

CGA

0

20

40

60

80

100

120


(120583gmL)

Cel

l via

bilit

y (

of c

ontro

l)

(c)

UVA

0

20

40

60

80

100

120



Cel

l via

bilit

y (

of c

ontro

l)

p-CA

(d)




2DCFDA)




0

50

100

150

200

250

300


UVA

Intr

acel

lula

r RO

S pr

oduc

tion

( o

f con

trol)

lowast

lowast

(120583gmL)

(a)

0

50

100

150

200

250


White light

30

lowast

lowast

lowast

(120583gmL)

Intr

acel

lula

r RO

S pr

oduc

tion

( o

f con

trol)

(b)

0

20

40

60

80

100

120

140

160

180


UVA

Propolis

lowast

lowast

(120583gmL)

Intr

acel

lula

r RO

S pr

oduc

tion

( o

f con

trol)

(c)






4 Discussion








UVA

p-p38

p38

p-ERK

ERK

120573-Actin

(a)

0

50

100

150

200

250

300

350

400


p38

Activ

atio

n (p

-p38

p38

)UVA

lowastlowast

(b)

0

50

100

150

200

250

300


UVA

ERK

Activ

atio

n(p

-ERK

12

ERK

12

)

lowastlowast

(c)

0

20

40

60

80

100

120


lowast

lowast

lowastlowastC

ell v

iabi

lity

( o

f con

trol)

(d)









5 Conclusion


Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Control

(a)

Vehicle

(b)

Propolis

(c)

34-CQA

(d)

0

20

40

60

80

100


Cel

l via

bilit

y (

of c

ontro

l)



p-CA

34-CQA35-CQA

(e)








120

0

20

40

60

80

100


UVA

lowastlowast

30 (120583gmL)

Cel

l via

bilit

y (

of c

ontro

l)

(a)

0

20

40

60

80

100

120


35-CQAUVA

lowastlowast

(120583gmL)

Cel

l via

bilit

y (

of c

ontro

l)

(b)

0

20

40

60

80

100

120


UVA

lowastlowastlowast

3 (120583gmL)

Cel

l via

bilit

y (

of c

ontro

l)

(c)

UVA

0

20

40

60

80

100

120


(120583gmL)

Cel

l via

bilit

y (

of c

ontro

l)

(d)


UVA

0

20

40

60

80

100

120

3 (120583gmL)

Cel

l via

bilit

y (

of c

ontro

l)

p-CA

(e)


0

50

100

150

200

250

300

350

400


p38

Activ

atio

n (p

-p38

act

in)

lowast

p-p38

120573-Actin

(f)




Control Vehicle

Propolis

Hoechst33342

PI

03

UVA

3 (120583gmL)

(a)

0

5

10

15

20

25

30


PI p

ositi

ve ce

lls(

of t

otal

cell

num

bers

)

UVAPropolis

lowastlowast

lowastlowast

lowastlowast

30 (120583gmL)

(b)

0

20

40

60

80

100

120

140


UVA

Propolis

lowastlowast

lowastlowast

30 (120583gmL)

Cel

l via

bilit

y (

of c

ontro

l)

(c)








0

20

40

60

80

100

120

Control Vehicle 1 3

35-CQA

UVA


(120583gmL)

Cel

l via

bilit

y (

of c

ontro

l)

(a)

35-CQA

UVA

Control Vehicle 10

20

40

60

80

100

120

lowastlowast

lowastlowast

3 (120583gmL)

Cel

l via

bilit

y (

of c

ontro

l)

(b)

Control Vehicle 1 3

UVA

CGA

0

20

40

60

80

100

120


(120583gmL)

Cel

l via

bilit

y (

of c

ontro

l)

(c)

UVA

0

20

40

60

80

100

120



Cel

l via

bilit

y (

of c

ontro

l)

p-CA

(d)




2DCFDA)




0

50

100

150

200

250

300


UVA

Intr

acel

lula

r RO

S pr

oduc

tion

( o

f con

trol)

lowast

lowast

(120583gmL)

(a)

0

50

100

150

200

250


White light

30

lowast

lowast

lowast

(120583gmL)

Intr

acel

lula

r RO

S pr

oduc

tion

( o

f con

trol)

(b)

0

20

40

60

80

100

120

140

160

180


UVA

Propolis

lowast

lowast

(120583gmL)

Intr

acel

lula

r RO

S pr

oduc

tion

( o

f con

trol)

(c)






4 Discussion








UVA

p-p38

p38

p-ERK

ERK

120573-Actin

(a)

0

50

100

150

200

250

300

350

400


p38

Activ

atio

n (p

-p38

p38

)UVA

lowastlowast

(b)

0

50

100

150

200

250

300


UVA

ERK

Activ

atio

n(p

-ERK

12

ERK

12

)

lowastlowast

(c)

0

20

40

60

80

100

120


lowast

lowast

lowastlowastC

ell v

iabi

lity

( o

f con

trol)

(d)









5 Conclusion


Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















120

0

20

40

60

80

100


UVA

lowastlowast

30 (120583gmL)

Cel

l via

bilit

y (

of c

ontro

l)

(a)

0

20

40

60

80

100

120


35-CQAUVA

lowastlowast

(120583gmL)

Cel

l via

bilit

y (

of c

ontro

l)

(b)

0

20

40

60

80

100

120


UVA

lowastlowastlowast

3 (120583gmL)

Cel

l via

bilit

y (

of c

ontro

l)

(c)

UVA

0

20

40

60

80

100

120


(120583gmL)

Cel

l via

bilit

y (

of c

ontro

l)

(d)


UVA

0

20

40

60

80

100

120

3 (120583gmL)

Cel

l via

bilit

y (

of c

ontro

l)

p-CA

(e)


0

50

100

150

200

250

300

350

400


p38

Activ

atio

n (p

-p38

act

in)

lowast

p-p38

120573-Actin

(f)




Control Vehicle

Propolis

Hoechst33342

PI

03

UVA

3 (120583gmL)

(a)

0

5

10

15

20

25

30


PI p

ositi

ve ce

lls(

of t

otal

cell

num

bers

)

UVAPropolis

lowastlowast

lowastlowast

lowastlowast

30 (120583gmL)

(b)

0

20

40

60

80

100

120

140


UVA

Propolis

lowastlowast

lowastlowast

30 (120583gmL)

Cel

l via

bilit

y (

of c

ontro

l)

(c)








0

20

40

60

80

100

120

Control Vehicle 1 3

35-CQA

UVA


(120583gmL)

Cel

l via

bilit

y (

of c

ontro

l)

(a)

35-CQA

UVA

Control Vehicle 10

20

40

60

80

100

120

lowastlowast

lowastlowast

3 (120583gmL)

Cel

l via

bilit

y (

of c

ontro

l)

(b)

Control Vehicle 1 3

UVA

CGA

0

20

40

60

80

100

120


(120583gmL)

Cel

l via

bilit

y (

of c

ontro

l)

(c)

UVA

0

20

40

60

80

100

120



Cel

l via

bilit

y (

of c

ontro

l)

p-CA

(d)




2DCFDA)




0

50

100

150

200

250

300


UVA

Intr

acel

lula

r RO

S pr

oduc

tion

( o

f con

trol)

lowast

lowast

(120583gmL)

(a)

0

50

100

150

200

250


White light

30

lowast

lowast

lowast

(120583gmL)

Intr

acel

lula

r RO

S pr

oduc

tion

( o

f con

trol)

(b)

0

20

40

60

80

100

120

140

160

180


UVA

Propolis

lowast

lowast

(120583gmL)

Intr

acel

lula

r RO

S pr

oduc

tion

( o

f con

trol)

(c)






4 Discussion








UVA

p-p38

p38

p-ERK

ERK

120573-Actin

(a)

0

50

100

150

200

250

300

350

400


p38

Activ

atio

n (p

-p38

p38

)UVA

lowastlowast

(b)

0

50

100

150

200

250

300


UVA

ERK

Activ

atio

n(p

-ERK

12

ERK

12

)

lowastlowast

(c)

0

20

40

60

80

100

120


lowast

lowast

lowastlowastC

ell v

iabi

lity

( o

f con

trol)

(d)









5 Conclusion


Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Control Vehicle

Propolis

Hoechst33342

PI

03

UVA

3 (120583gmL)

(a)

0

5

10

15

20

25

30


PI p

ositi

ve ce

lls(

of t

otal

cell

num

bers

)

UVAPropolis

lowastlowast

lowastlowast

lowastlowast

30 (120583gmL)

(b)

0

20

40

60

80

100

120

140


UVA

Propolis

lowastlowast

lowastlowast

30 (120583gmL)

Cel

l via

bilit

y (

of c

ontro

l)

(c)








0

20

40

60

80

100

120

Control Vehicle 1 3

35-CQA

UVA


(120583gmL)

Cel

l via

bilit

y (

of c

ontro

l)

(a)

35-CQA

UVA

Control Vehicle 10

20

40

60

80

100

120

lowastlowast

lowastlowast

3 (120583gmL)

Cel

l via

bilit

y (

of c

ontro

l)

(b)

Control Vehicle 1 3

UVA

CGA

0

20

40

60

80

100

120


(120583gmL)

Cel

l via

bilit

y (

of c

ontro

l)

(c)

UVA

0

20

40

60

80

100

120



Cel

l via

bilit

y (

of c

ontro

l)

p-CA

(d)




2DCFDA)




0

50

100

150

200

250

300


UVA

Intr

acel

lula

r RO

S pr

oduc

tion

( o

f con

trol)

lowast

lowast

(120583gmL)

(a)

0

50

100

150

200

250


White light

30

lowast

lowast

lowast

(120583gmL)

Intr

acel

lula

r RO

S pr

oduc

tion

( o

f con

trol)

(b)

0

20

40

60

80

100

120

140

160

180


UVA

Propolis

lowast

lowast

(120583gmL)

Intr

acel

lula

r RO

S pr

oduc

tion

( o

f con

trol)

(c)






4 Discussion








UVA

p-p38

p38

p-ERK

ERK

120573-Actin

(a)

0

50

100

150

200

250

300

350

400


p38

Activ

atio

n (p

-p38

p38

)UVA

lowastlowast

(b)

0

50

100

150

200

250

300


UVA

ERK

Activ

atio

n(p

-ERK

12

ERK

12

)

lowastlowast

(c)

0

20

40

60

80

100

120


lowast

lowast

lowastlowastC

ell v

iabi

lity

( o

f con

trol)

(d)









5 Conclusion


Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

20

40

60

80

100

120

Control Vehicle 1 3

35-CQA

UVA


(120583gmL)

Cel

l via

bilit

y (

of c

ontro

l)

(a)

35-CQA

UVA

Control Vehicle 10

20

40

60

80

100

120

lowastlowast

lowastlowast

3 (120583gmL)

Cel

l via

bilit

y (

of c

ontro

l)

(b)

Control Vehicle 1 3

UVA

CGA

0

20

40

60

80

100

120


(120583gmL)

Cel

l via

bilit

y (

of c

ontro

l)

(c)

UVA

0

20

40

60

80

100

120



Cel

l via

bilit

y (

of c

ontro

l)

p-CA

(d)




2DCFDA)




0

50

100

150

200

250

300


UVA

Intr

acel

lula

r RO

S pr

oduc

tion

( o

f con

trol)

lowast

lowast

(120583gmL)

(a)

0

50

100

150

200

250


White light

30

lowast

lowast

lowast

(120583gmL)

Intr

acel

lula

r RO

S pr

oduc

tion

( o

f con

trol)

(b)

0

20

40

60

80

100

120

140

160

180


UVA

Propolis

lowast

lowast

(120583gmL)

Intr

acel

lula

r RO

S pr

oduc

tion

( o

f con

trol)

(c)






4 Discussion








UVA

p-p38

p38

p-ERK

ERK

120573-Actin

(a)

0

50

100

150

200

250

300

350

400


p38

Activ

atio

n (p

-p38

p38

)UVA

lowastlowast

(b)

0

50

100

150

200

250

300


UVA

ERK

Activ

atio

n(p

-ERK

12

ERK

12

)

lowastlowast

(c)

0

20

40

60

80

100

120


lowast

lowast

lowastlowastC

ell v

iabi

lity

( o

f con

trol)

(d)









5 Conclusion


Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

50

100

150

200

250

300


UVA

Intr

acel

lula

r RO

S pr

oduc

tion

( o

f con

trol)

lowast

lowast

(120583gmL)

(a)

0

50

100

150

200

250


White light

30

lowast

lowast

lowast

(120583gmL)

Intr

acel

lula

r RO

S pr

oduc

tion

( o

f con

trol)

(b)

0

20

40

60

80

100

120

140

160

180


UVA

Propolis

lowast

lowast

(120583gmL)

Intr

acel

lula

r RO

S pr

oduc

tion

( o

f con

trol)

(c)






4 Discussion








UVA

p-p38

p38

p-ERK

ERK

120573-Actin

(a)

0

50

100

150

200

250

300

350

400


p38

Activ

atio

n (p

-p38

p38

)UVA

lowastlowast

(b)

0

50

100

150

200

250

300


UVA

ERK

Activ

atio

n(p

-ERK

12

ERK

12

)

lowastlowast

(c)

0

20

40

60

80

100

120


lowast

lowast

lowastlowastC

ell v

iabi

lity

( o

f con

trol)

(d)









5 Conclusion


Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















UVA

p-p38

p38

p-ERK

ERK

120573-Actin

(a)

0

50

100

150

200

250

300

350

400


p38

Activ

atio

n (p

-p38

p38

)UVA

lowastlowast

(b)

0

50

100

150

200

250

300


UVA

ERK

Activ

atio

n(p

-ERK

12

ERK

12

)

lowastlowast

(c)

0

20

40

60

80

100

120


lowast

lowast

lowastlowastC

ell v

iabi

lity

( o

f con

trol)

(d)









5 Conclusion


Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















5 Conclusion


Acknowledgments


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 The Effects of Brazilian Green Propolis against …downloads.hindawi.com/journals/ecam/2013/238279.pdf · 2019. 7. 31. · The Effects of Brazilian Green Propolis