research article propolis reduces phosphatidylcholine...

Research ArticlePropolis Reduces Phosphatidylcholine-SpecificPhospholipase C Activity and Increases Annexin a7 Level inOxidized-LDL-Stimulated Human Umbilical VeinEndothelial Cells

Hongzhuan Xuan12 Zhen Li1 Jiying Wang3 Kai Wang2

Chongluo Fu1 Jianlong Yuan1 and Fuliang Hu2

1 School of Life Science Liaocheng University Liaocheng 252059 China2 College of Animal Sciences Zhejiang University Hangzhou 310029 China3 Institute of Animal Science and Veterinary Medicine Shandong Academy of Agricultural Sciences Jinan 250100 China

Correspondence should be addressed to Fuliang Hu flhuzjueducn

Received 25 November 2013 Accepted 12 February 2014 Published 22 April 2014

Academic Editor Kashmira Nanji

Copyright copy 2014 Hongzhuan Xuan et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

To understand the mechanisms underlying the regulating dyslipidemia action of Chinese propolis and Brazilian green propolis weinvestigated their effects on phosphatidylcholine-specific phospholipase C (PC-PLC) activity and annexin a7 (ANXA7) level whichplay crucial roles in the control of the progress of atherosclerosis Furthermore active oxygen species (ROS) levels nuclear factor-KappaB p65 (NF-120581B p65) and mitochondrial membrane potential (MMP) were also investigated in oxidized-LDL- (ox-LDL-)stimulated human umbilical vein endothelial cells (HUVECs) Our data indicated that the treatment of both types of propolis125 120583gmL significantly increased cell viability and attenuated apoptosis rate increased ANXA7 level and decreased PC-PLCactivity Both types of propolis also inhibited ROS generation as well as the subsequent MMP collapse and NF-120581B p65 activationinduced by ox-LDL inHUVECsOur results also indicated that Chinese propolis and Brazilian green propolis had similar biologicalactivities and prevented ox-LDL induced cellular dysfunction in HUVECs

1 Introduction

Propolis is a brownish resinous material collected by workerbees from the leaf buds of numerous plants like birch poplarBaccharis dracunculifolia andDalbergia ecastaphyllum [1ndash4]It has been extensively used as a folk medicine since anc-ient time because of its special chemical components strongpharmacological and biological properties and low toxicity[5]

In recent years the regulation of dyslipidemia actions ofpropolis has beenwidely documented resulting in the genesisand progression of atherosclerosis A recent report indicatedethanolic extract of propolis inhibited atherosclerosis inApoE-knockout mice [6] Furthermore we also reported

that Chinese propolis regulated lipid metabolism of diabetesin vivo by regulating triglycerides total cholesterol high-density lipoprotein and low-density lipoprotein cholesterol[7 8] However the molecular mechanisms underlying suchprotect effects of propolis have not been fully elucidated

Phosphatidylcholine-specific phospholipase C (PC-PLC) an important member of phospholipase C family hasbeen implicated in several cellular signaling pathways suchas cell growth differentiation senescence apoptosis andautophagy of mammalian cells [9ndash13] Accumulating evide-nce demonstrated that PC-PLC was a key inducing elementof atherosclerosis and contributed to the progression ofatherosclerosis [14] Pharmacological blockade of PC-PLCinhibited the progression of atherosclerosis [15] And a recent

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 465383 9 pageshttpdxdoiorg1011552014465383

2 Evidence-Based Complementary and Alternative Medicine

study indicated that annexin a7 (ANXA7) a member of theannexin family of calcium-dependent phospholipid bindingproteins was an endogenous regulator of PC-PLC ANXA7also participated in the progression of atherosclerosis andtargeting ANXA7 inhibited atherosclerosis in apoEminusminus miceANXA7PC-PLC signaling pathway may represent a noveltarget for the treatment of atherosclerosis [16]

Chinese propolis affected PC-PLC activity Our previ-ous study indicated that Chinese propolis played an anti-inflammatory role partly through its inhibitory effect on theactivity of PC-PLC [17] Considering the important rolesof ANXA7 and PC-PLC in atherosclerosis and propolismodulated atherosclerosis and affected PC-PLC activity wehypothesized that propolis may also affect ANXA7 theendogenous regulator of PC-PLC In present study we testedthe hypothesis by investigating the effects of Chinese propo-lis and Brazilian green propolis on PC-PLC activity andANXA7 level in ox-LDL-stimulated HUVECs ox-LDL playscrucial role in triggering the development of atherosclerosisFurthermore we investigated the effects of both types ofpropolis on reactive oxygen species (ROS) levels nuclearfactor-KappaB p65 (NF-120581B p65) and mitochondrial mem-brane potential (MMP) which were regulated by PC-PLC inHUVECs

2 Materials and Methods

21 Chemicals and Reagents DMEMwas fromGibco (USA)Fetal bovine serum (FBS) was from Hyclone Lab Inc(USA) L-120572-phosphatidylcholine 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide (MTT) 2101584071015840-dichloro-dihydrofluorescein diacetate (DCFH) and JC-1 were fromSigmaCo (USA) Acridine orangewas fromAmresco (USA)Ox-LDL was from Beijing Union-Biology Co (China) Pri-mary antibodies against ANXA7 NF-120581B p65 GAPDH andsecondary antibody (horseradish peroxidase) were fromSanta Cruz Biotechnology (USA) Secondary antibody forimmunofluorescence donkey anti-rabbit IgG Alexa Fluor-488 was purchased from Life Technologies (USA) All otherreagents were of ultrapure grade

22 Preparation of Propolis Extracts Propolis used in presentstudy was Chinese propolis and Brazilian green propolisBoth types of propolis had been used in previous studies [1718] The extraction method was as before Briefly extractedChinese propolis was obtained from colonies of honeybeesA mellifera L in Shandong Province of North China andthe main plant origin was poplar (Populus sp) ExtractedBrazilian green propolis was collected in Minas Gerais Stateof Brazil where Baccharis dracunculifolia DC is the mainbotanical source Both types of propolis were stored at minus20∘Cuntil used Chinese propolis and Brazilian green propolissamples were extracted with ethanol and then filtered underreduced pressure and the filter liquid was concentratedunder reduced pressure at 40∘C until reaching a constantweight and then redissolved in ethanolThe ethanol-extractedChinese propolis (EECP) and ethanol-extracted Brazilian

green propolis (EEBP) had a brown colorTheprepared EECPand EEBP were stored under a dry condition at 4∘C

23 Cell Culture HUVECs were gifted by AtherosclerosisResearch Institute of Taishan Medical University of Chinapurchased from ATCC HUVECs were cultured in DMEM(high glucose) supplemented with 100UmL of penicillin100 120583gmL streptomycin and 10 heat-inactivated FBS at37∘C under humidified 95ndash5 (vv) air and CO

2

24 Exposure of HUVECs to EECP and EEBP When theHUVEC cultures reached 80 confluence then the cellswere divided for treatment (a) culture in 35 DMEMmedium with ethanol at lt01 (vv) (control) (b) culture inbasal DMEM medium with 45120583gmL ox-LDL with ethanolat lt01 (vv) and (c) culture in basal DMEM mediumwith 45 120583gmL ox-LDL with EECP and EEBP (125 120583gmL)respectively EECP and EEBP were dissolved in ethanol withfinal concentration of ethanol in the culture medium lt01(vv) Ethanol at 01 (vv) did not affect cell viability

25 Cell Viability Assay The viability of HUVECs was deter-mined by MTT assay HUVECs were seeded in 96-well cellculture plates and grown to 80 confluence and then treatedwith ox-LDL or EECP and EEBP for 12 and 24 h respectivelyMTT solution was added to each well and incubated for 4 hThe MTT-formazan product dissolved in DMSO to estimatebymeasuring absorbance at 570 nm in an ELISA plate readerThe viability () was expressed as (OD of treated groupODof ox-LDL group) times 100 The viability of the ox-LDL groupwas set at 100

26 Nuclear Fragmentation Assay The morphological chan-ges of nuclei were detected by acridine orange staining At24 h cells were stained with 5120583gmL acridine orange at roomtemperature for 5min and observed under a laser scanningconfocal microscopy (Olympus FV1200 Japan)

27 PC-PLC Activity Assay PC-PLC activity assay was per-formed as the described methods in [19 20] Briefly we usedL-120572-phosphatidylcholine as the substrate of PC-PLC Theoptical density wasmeasured at 660 nm Enzyme activity wasexpressed as nanomoles per minute per milligram protein

28 Immunofluorescence Microscopy After treatment cellswere fixed with 4 paraformaldehyde and blocked with5 normal donkey serum for 20min at room temperatureCells were incubated with ANXA7 and NF-120581B p65 primaryantibodies (1100) at 4∘C overnight After three rinses in01M phosphate-buffered saline cells were treated with acorresponding FITC-conjugated secondary antibody (1200)in a humid chamber at 37∘C for 1 h Cells were rinsed threetimes with 01M phosphate-buffered saline to eliminate theuncombined secondary antibody A laser scanning confocalmicroscope (Olympus FV1200 Japan) was used for fluores-cence detection Analysis used the Image-Pro Plus software(USA) Images are representative of three independent exper-iments

Evidence-Based Complementary and Alternative Medicine 3

020406080

100120140

Cel

l via

bilit

y

minus

minus minus

+ + ++ +

Ox-LDLOx-LDL

45120583gmLPropolis 125 120583gmL

24h12h

Control

lowastlowastlowastlowast

lowastlowast lowastlowast lowastlowast lowastlowast

EECP EEBP

(a)

Ox-LDL

24h

Control EECP EEBP

(b)

lowastlowast


minus

minus minus

+ + ++ +

Ox-LDLOx-LDL


Control EECP EEBP02468

101214

The r

atio

of a

popt

otic

cells

()

(c)

Figure 1 EECP and EEBP increased cell viability and inhibited apoptosis rate in ox-LDL-stimulated HUVECs (a) Effect of EECP and EEBPon cell viability induced by ox-LDL EECP and EEBP (125 120583gmL) and ox-LDL (45 120583gmL for HUVECs) were used (b) Effects of EECP andEEBP on nuclear fragment were detected by acridine orange staining (c) The ratio of apoptotic cells induced by ox-LDL (lowastlowast119875 lt 001 versusox-LDL group 119899 = 3) Data are means plusmn SEM

29 Western Blot Analysis Western blot assay of ANXA7level was performed as previously described [21] Thirtymicrograms of protein was separated by 12 SDS-PAGE andtransferred onto PVDF membrane The relative quantitiesof the proteins were evaluated by the use of Quantity Onesoftware

210 Measurement of ROS Production ROS production inHUVECs was determined by the use of a fluorescent probeDCFH which can be oxidized into fluorescent dichlorofluo-rescin (DCF) by intracellular ROS [22] After treating cellsfor 24 h cells were incubated with DCFH for 30min at37∘C Then cells were washed with basal DMEM medium 3times and observed on laser scanning confocal microscopy(Olympus FV1200 Japan)The level of ROSwas quantified byImage-Pro Plus software (USA) Results were shown as rela-tive fluorescence intensity of three independent experiments

211 Measurement ofMitochondrialMembrane Potential Thefluorescent dye JC-1 was used to measure mitochondrialmembrane potential JC-1 exists as a monomer at low mito-chondrial membrane potential and emits green fluorescencebut forms aggregates and emits red fluorescence at highmito-chondrial membrane potential [23] After treating cells for24 h cells were incubated with JC-1 for 15min at 37∘C Thencells were washed with basal DMEM medium 3 times andobserved on laser scanning confocal microscopy (OlympusFV1200 Japan) The mitochondrial membrane potential wasquantified by the use of the Image-Pro Plus software (USA)Results were shown as ratio of red to green fluorescence ofthree independent experiments

212 Statistical Analysis All experiments were performed induplicate and repeated at least 3 times Data are expressedas means plusmn SEM Statistical analyses were performed using


002040608

112

of P

C-PL

CTh

e rel

ativ

e lev

el lowastlowastlowast lowast

minus

minus minus

+ + ++ +

Ox-LDLOx-LDL


Control EECP EEBP

Figure 2 EECP and EEBP decreased PC-PLC activity in ox-LDL-stimulated HUVECs Cells were treated with EECP and EEBP125 120583gmL for 24 h respectively (lowast119875 lt 005 lowastlowast119875 lt 001 versus ox-LDL group 119899 = 3)

independent 119905-tests and analysis of variance (ANOVA)followed by theTukey post hoc test A119875 lt 005was consideredsignificant

3 Results

31 Effect of EECP and EEBP onHUVECViability Ox-LDL isamajor cause of endothelial dysfunctionMTT assay revealedthat ox-LDL significantly inhibited cell viability and aftertreatment with EECP and EEBP 125 120583gmL for 12 and 24 hthe cell viability obviously increased compared with ox-LDLgroup respectively (lowastlowast119875 lt 001 Figure 1(a))

32 Effect of EECP and EEBP on HUVEC Apoptosis We fur-ther examined the effects of EECP and EEBP on HUVECapoptosis induced by ox-LDL The results of AO stainingshowed that there were evidently condensation and fragmen-tation of chromosomes in ox-LDL group (Figure 1(b)) andcell apoptosis was significantly decreased by EECP and EEBPat 24 h (lowastlowast119875 lt 001 Figure 1(c))

33 Effect of EECPandEEBP onPC-PLCActivity Theactivityof PC-PLC in ox-LDL treated HUVECs was significantly inc-reased whereas EECP and EEBP evidently depressed PC-PLC activity at 24 h (lowast119875 lt 005 lowastlowast119875 lt 001 Figure 2)

34 Effect of EECP and EEBP on ANXA7 Level ANXA7 wasthe endogenous regulator of PC-PLC To further investigatethe relationship between PC-PLC and ANXA7 we investi-gated the effect of EECP andEEBPonANXA7 expression anddistribution in cells treated with ox-LDL Western blottingresults showed that EEBP obviously increased ANXA7 levelat 12 h and both EECP and EEBP significantly increasedANXA7 level at 24 h (lowastlowast119875 lt 001 Figure 3) And immunoflu-orescence assay results showed that cells treated with EECPand EEBP exhibited higher fluorescence intensity of ANXA7per cell in a noticeable punctate pattern compared with ox-LDL group (Figure 3(a))

35 Effect of EECP and EEBP on NF-120581B p65 Level Ox-LDL induced NF-120581B activation in HUVECs Both EECP and

EEBP 125 120583gmL significantly decreased NF-120581B p65 levelby immunofluorescence assay and both types of propolisinhibited translocation of NF-120581B p65 from cytoplasm tonucleus (lowastlowast119875 lt 001 Figure 4)

36 Effect of EECP and EEBP on ROS Level Both EECP andEEBP 125 120583gmL significantly decreased ROS generation inHUVECs at 24 h as compared with the ox-LDL group (lowast119875 lt005 Figure 5)

37 Effect of EECP and EEBP on Mitochondrial MembranePotential Ox-LDL damages mitochondria membrane pot-ential Both EECP and EEBP 125 120583gmL significantly incr-eased mitochondrial membrane potential compared with ox-LDL group (lowastlowast119875 lt 001 Figure 6)

4 Discussion

Atherosclerosis is considered to be a chronic inflammatorydisease Ox-LDL is believed to be a key step in endothelialcell injury and in the process of initiation and progression ofatherosclerotic disease [24 25] According to the documentson ox-LDL roles in HUVEC apoptosis [26] in current study45 120583gmLof ox-LDLwas usedThere aremore than 300 activecomponents in propolis Because of different plant sourcethe chemical constituents of Chinese propolis and Braziliangreen propolis are different Our previous researches sug-gested that both Chinese propolis and Brazilian green propo-lis 125 120583gmL averted apoptosis and protected HUVECswith nutrient deprivation [17 18] Munari et al (2010) alsosuggested that 125 120583gmL Baccharis dracunculifolia extractthe major plant resource of Brazilian green propolis wasthe most effective in antigenotoxic chemoprevention [27]Therefore we have chosen 125 120583gmL Chinese propolis andBrazilian green propolis used in current study and comparedtheir biological activity in ox-LDL-stimulated HUVECs

Accumulating evidence indicates that PC-PLC plays animportant role in progression of atherosclerosis and PC-PLCis an attractive target for antiatherosclerosis therapy [14] Arecent study showed that ANXA7 having different roles inautophagy tumor suppression and exocytosis [28ndash30] wasnegative regulation of PC-PLC in HUVECs and suggestedthat ANXA7PC-PLC signaling pathway may present a noveltarget to treat atherosclerosis [17] In present study the dataindicated that both Chinese propolis and Brazilian greenpropolis reduced PC-PLC activity and increased ANXA7level in ox-LDL-stimulated endothelial cells which suggestedthat ANXA7PC-PLC might be the targets of both types ofpropolis in modulating dyslipidemia

ROS play a critical role in vascular pathology as well as inthe maintenance of normal physiological vascular functionOverproduction of ROS will lead to oxidative stress whichcause the endothelial dysfunction and promote the develop-ment of many cardiovascular diseases such as atherosclerosisby activating downstream signal molecules such as NF-120581B[31]Our previous study also showed that elevatingROS levelstriggered apoptosis in HUVECs with nutrition deprivation[18] Ox-LDL is a potent inducer of ROS and this was con-


Ox-LDL

24 h

Control EECP EEBP

(a)

Ox-LDLControl EECP EEBP12 h

ANXA7

GAPDH

24 h

ANXA7

GAPDH

(b)

002040608

1121416

The r

elat

ive l

evel

of

AN

XA

7G

APD

H

lowastlowast

lowastlowast

lowastlowast lowastlowast lowastlowast

minus

minus minus

+ + ++ +

Ox-LDLOx-LDL


24h12h

Control EECP EEBP

(c)

Figure 3 EECP andEEBP increasedANXA7 level in ox-LDL-stimulatedHUVECs (a) Fluorescentmicrographs obtained at 24 h (b) ANXA7levels were detected bywestern blot analysis at 12 and 24 h (c)Thehemiquantification of ANXA7 level inHUVECs (lowastlowast119875 lt 001 versus ox-LDLgroup 119899 = 3)

firmed in the present study ROS level was increased inHUVECs treated with ox-LDL whereas both types of propo-lis could depress ROS level which lend support to the theorythat ROS scavenging could reduce the risk of cardiovasculardiseases [32] Furthermore transcription factor NF-120581B isactivated by high level of ROS [33] In present study ox-LDLinduced ROS increase and subsequent activation of NF-120581Bp65 were all attenuated by EECP and EEBP NF-120581B signalingpathway is involved inmultiple cell processes including apop-tosis proliferation and gene expression Moreover recentstudies have suggested that several natural products includingpropolis suppress inflammatory responses by regulating theNF-120581B pathway [34 35] Atherosclerosis is a kind of chronicinflammatory diseaseThese findings concurwith our finding

that the transcriptional inhibition of proinflammatory medi-ators by propolis is associated with the blockade of NF-120581Bsignaling pathway

Mitochondria are themost important intracellular sourceof ROS and elevated ROS levels can also decrease mitochon-drial membrane potential [36] Ox-LDL damages mitochon-drial membrane potential leading the cytochrome c releaseto induce apoptosis in HUVECs [37]We previously reportedthat high concentration of propolis damaged mitochondrialmembrane potential in endothelial cells with nutrition depri-vation Here we found that both types of propolis 125 120583gmLsignificantly protected mitochondrial membrane potentialTogetherwith these results we confirmed the protective effectof propolis on HUVECs induced by ox-LDL and it may be


Ox-LDLControl EECP EEBP

Mer

geN

ucle

iN

F-120581

B p65

(a)

NF-120581

B p65

012345

67

89

The r

elat

ive l

evel

of


lowastlowast lowastlowast

lowastlowast

minus

minus minus

+ + ++ +

Ox-LDL 45120583gmLPropolis 125 120583gmL

(b)

Figure 4 EECP and EEBP decreased NF-120581B p65 level and inhibited translocation of NF-120581B p65 from cytoplasm to nucleus in ox-LDL-stimulated HUVECs (a) Fluorescent micrographs obtained at 24 h Nuclei were counterstained with PI (b) The relative level of NF-120581B p65in HUVECs (lowastlowast119875 lt 001 versus ox-LDL group 119899 = 3)

the major mechanisms of propolis modulating atherosclero-sis

Many studies indicate that propolis from different areashas similar biological activity although the chemical con-stituents are different Hu et al (2011) reported that Chinesepropolis and Brazilian green propolis had similar biologicalactivities on streptozotocin-induced type 1 diabetes mellitusin rats [38] And we also previously found that Chinesepropolis and Brazilian green propolis had similar protectiveeffects on hepatocytes injury induced by hydrogen peroxide[39] In current study we confirmed that both Chinesepropolis and Brazilian green propolis had similar activity onANXA7 expression and PC-PLC activity and other signalmolecules inHUVECs induced by ox-LDLWe proposed thatit was not a simple chemical constituent in propolis playingcrucial role modulating dyslipidemia diseases it might be asynergy effect of various chemical constituents of propolis

However in other cells such as breast cancer MCF-7 andMDA-MB-231 cells we found that the cytotoxicity of Chinesepropolis and Brazilian green propolis was different So theactivities of propolis on different diseases and cells should befurther studied

Altogether the present findings indicated that both typesof propolis 125120583gmL significantly increased cell viabilityand attenuated apoptosis rate increased the expression ofANXA7 and decreased PC-PLC activity Both kinds ofpropolis also inhibited ROS generation as well as the subse-quent MMP collapse and NF-120581B p65 activation induced byox-LDL in HUVECs which may be the major mechanismsof propolis protecting endothelial injury and preventingatherosclerosis Our results also indicated that Chinesepropolis and Brazilian green propolis had similar biologicalactivities and prevented ox-LDL induced cellular dysfunctionin HUVECs Both types of propolis may be potent alternative


Ox-LDL

24h

Control EECP EEBP

(a)

012345678

The r

elat

ive l

evel

of R

OS

Ox-LDLControl EECP EEBPminus

minus minus

+ + ++ +


lowast

lowast lowast

(b)

Figure 5 EECP andEEBPdecreasedROS level in ox-LDL-stimulatedHUVECs (a) Fluorescentmicrographs obtained at 24 h (b)The relativequantity of ROS level in HUVECs (lowast119875 lt 005 versus ox-LDL group 119899 = 3)

Ox-LDL

24h

Control EECP EEBP

(a)


minus minus

+ + ++ +


0

02

04

06

08

1

12

14

16

18

The r

elat

ive l

evel

of

mito

chon

dria

mem

bran

epo

tent

ial

lowastlowastlowastlowast lowastlowast

(b)

Figure 6 EECP and EEBP increased mitochondria membrane potential in ox-LDL-stimulated HUVECs (a) Fluorescent micrographsobtained at 24 h (b) The relative quantity of mitochondrial membrane potential in HUVECs (lowastlowast119875 lt 001 versus ox-LDL group 119899 = 3)

drugs for the prevention of atherosclerosis However themechanism of propolis regulating dyslipidemia should befurther studied

Conflict of Interests

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

Acknowledgments

This work was supported by the Grant from the NationalNatural Science Foundation of China (nos 31201860 and31272512) Shandong Provincial Natural Science Foundationof China (no ZR2012CQ003) the Modern AgroindustryTechnology Research System from the Ministry of Agricul-ture of China (CARS-45) and the Doctor Foundation ofLiaocheng University of China (no 31805)


References

[1] A C de Groot ldquoPropolis a review of properties applicationschemical composition contact allergy and other adverse eff-ectsrdquo Dermatitis vol 24 no 6 pp 263ndash282 2013

[2] YK Park J F Paredes-GuzmanC LAguiar SMAlencar andF Y Fujiwara ldquoChemical constituents in Baccharis dracunculi-folia as the main botanical origin of Southeastern Brazilianpropolisrdquo Journal of Agricultural and Food Chemistry vol 52no 5 pp 1100ndash1103 2004

[3] A Daugsch C S Moraes P Fort and Y K Park ldquoBrazilian redpropolis chemical composition and botanical originrdquoEvidence-BasedComplementary andAlternativeMedicine vol 5 no 4 pp435ndash441 2008

[4] J M Sforcin and V Bankova ldquoPropolis is there a potential forthe development of new drugsrdquo Journal of Ethnopharmacologyvol 133 no 2 pp 253ndash260 2011

[5] V C Toreti H H Sato G M Pastore and Y K Park ldquoRecentprogress of propolis for its biological and chemical composi-tions and its botanical originrdquo Evidence-Based Complementaryand Alternative Medicine vol 2013 Article ID 697390 13 pages2013

[6] Y Fang H Sang N Yuan et al ldquoEthanolic extract of propolisinhibits atherosclerosis in ApoE-knockout micerdquo Lipids inHealth and Disease vol 12 no 1 article 123 2013

[7] F Hu Y Li M Chen and H Xuan ldquoEffects of encapsulatedpropolis on blood glycemic control lipid metabolism and ins-ulin resistance in type 2 diabetes mellitus ratsrdquo Evidence-BasedComplementary and Alternative Medicine vol 2012 Article ID981896 8 pages 2012

[8] H U Fuliang H R Hepburn H Xuan M Chen S Daya andS E Radloff ldquoEffects of propolis on blood glucose blood lipidand free radicals in rats with diabetesmellitusrdquo PharmacologicalResearch vol 51 no 2 pp 147ndash152 2005

[9] M Szumiło and I Rahden-Staron ldquoBiological role of phospha-tidylcholine-specific phospholipase C inmammalian cellsrdquoPos-tepy Higieny i Medycyny Doswiadczalnej vol 62 pp 593ndash5982008

[10] L Wang H Li J Zhang et al ldquoPhosphatidylethanolamine bin-ding protein 1 in vacular endothelial cell autophagy and athero-sclerosisrdquo Journal of Physiology vol 59 pp 5005ndash5015 2013

[11] E Iorio A Ricci M Bagnoli et al ldquoActivation of phosphatidyl-choline cycle enzymes in human epithelial ovarian cancer cellsrdquoCancer Research vol 70 no 5 pp 2126ndash2135 2010

[12] J-I Tzeng B-C Chen H-M Chang et al ldquoInvolvement ofphosphatidylcholine-phospholipase C and protein kinase Cin peptidoglycan-induced nuclear factor-120581B activation andcyclooxygenase-2 expression in RAW 2647 macrophagesrdquoPharmacological Research vol 61 no 2 pp 162ndash166 2010

[13] L Zhang H Y Li H Li et al ldquoLipopolysaccharide activatedphosphatidylcholine-specific phospholipase C and induced IL-8 and MCP-1 production in vascular endothelial cellsrdquo Journalof Cellular Physiology vol 226 no 6 pp 1694ndash1701 2011

[14] H Li L Zhang D Yin Y Zhang and J Miao ldquoTargeting pho-sphatidylcholine-specific phospholipase C for atherogenesistherapyrdquo Trends in Cardiovascular Medicine vol 20 no 5 pp172ndash176 2010

[15] L Zhang J Zhao L Su et al ldquoD609 inhibits progression ofpreexisting atheroma and promotes lesion stability in apoli-poprotein Eminusminus Mice a role of phosphatidylcholine-specificphospholipase in atherosclerosisrdquo Arteriosclerosis Thrombosisand Vascular Biology vol 30 no 3 pp 411ndash418 2010

[16] H Li S Huang SWang et al ldquoTargeting annexin A7 by a smallmolecule suppressed the activity of phosphatidylcholine-spec-ific phospholipase C in vascular endothelial cells and inhibitedatherosclerosis in apolipoprotein Eminusminus micerdquo Cell Death andDisease vol 4 Article ID e806 2013

[17] F Hu H Xuan R Zhu and Y Li ldquoInhibitory effect of chin-ese propolis on phosphatidylcholine-specific phospholipase Cactivity in vascular endothelial cellsrdquo Evidence-Based Comple-mentary and Alternative Medicine vol 2011 Article ID 9852788 pages 2011

[18] H Xuan J Zhao J Miao Y Li Y Chu and F Hu ldquoEffect ofBrazilian propolis on human umbilical vein endothelial cellapoptosisrdquo Food and Chemical Toxicology vol 49 no 1 pp 78ndash85 2011

[19] X-Z Wu ldquoCorrelation of calcium-independent phosphatidyl-choline-specific phospholipase C with 120574-GT in hepatoma cellsrdquoActa Biochimica et Biophysica Sinica vol 30 no 1 pp 104ndash1061998

[20] W Xingzhong H Lu L Zhou Y Huang and H Chen ldquoCha-nges of phosphatidylcholine-specific phospholipase C in hepa-tocarcinogenesis and in the proliferation and differentiation ofrat liver cancer cellsrdquo Cell Biology International vol 21 no 6pp 375ndash381 1997

[21] J Zhao J Miao B Zhao and S Zhang ldquoUpregulating of Fasintegrin1205734 and P53 and depressing of PC-PLC activity andROSlevel in VEC apoptosis by safrole oxiderdquo FEBS Letters vol 579no 25 pp 5809ndash5813 2005

[22] N SuematsuH Tsutsui JWen et al ldquoOxidative stressmediatestumor necrosis factor-120572-induced mitochondrial DNA damageand dysfunction in cardiac myocytesrdquo Circulation vol 107 no10 pp 1418ndash1423 2003

[23] H Izuta M Shimazawa S Tazawa Y Araki S Mishima andH Hara ldquoProtective effects of Chinese propolis and its com-ponent chrysin against neuronal cell death via inhibition ofmitochondrial apoptosis pathway in SH-SY5Y cellsrdquo Journal ofAgricultural and Food Chemistry vol 56 no 19 pp 8944ndash89532008

[24] L Ma X Liu Y Zhao B Chen X Li and R Qi Lina MaldquoGinkgolide B reduces LOX-1 expression by inhibiting Aktphosphorylation and increasing Sirt1 expression in oxidizedLDL-stimulated human umbilical vein endothelial cellsrdquo PLoSONE vol 8 no 9 Article ID e74769 2013

[25] L Zhang G Z ZhouW Song et al ldquoPterostilbene protects vas-cular endothelial cells against oxidized low-density lipoprotein-induced apoptosis in vitro and in vivordquo Apoptosis vol 17 no 1pp 25ndash36 2012

[26] X Liu J Zhao J Xu et al ldquoProtective effects of a benzoxazinederivative against oxidized LDL-induced apoptosis and theincreases of integrin 1205734 ROS NF-120581B and P53 in humanumbilical vein endothelial cellsrdquo Bioorganic and MedicinalChemistry Letters vol 19 no 10 pp 2896ndash2900 2009

[27] C C Munari J M Alves J K Bastos and D C Tavaresa ldquoEva-luation of the genotoxic and antigenotoxic potential of Bac-charis dracunculifolia extract on V79 cells by the comet assayrdquoJournal of Applied Toxicology vol 30 no 1 pp 22ndash28 2010

[28] M Srivastava L Bubendorf V Srikantan et al ldquoAnx7 a candi-date tumor suppressor gene for prostate cancerrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 98 no 8 pp 4575ndash4580 2001

[29] H Li S Huang S Wang et al ldquoRelationship between annexinA7 and integrin 4 in autophagyrdquo International Journal of Bio-chemistry and Cell Biology vol 45 no 11 pp 2605ndash2611 2013


[30] M Srivastava C Montagna X Leighton et al ldquoHaploinsuffi-ciency of Anx7 tumor suppressor gene and consequent genomicinstability promotes tumorigenesis in the Anx7(+minus) mouserdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 100 no 2 pp 14287ndash14292 2003

[31] W Liu Y Yin Z Zhou M He and Y Dai ldquoOx-LDL-indu-ced IL-1beta secretion promoting foam cells formation wasmainly via CD36 mediated ROS production leading to NLRP3inflammasome activationrdquo Inflammation Research vol 63 no1 2014

[32] D Pastori R Carnevale and P Pignatelli ldquoIs there a clinicalrole for oxidative stress biomarkers in atherosclerotic diseasesrdquoInternal and EmergencyMedicine vol 9 no 2 pp 123ndash131 2014

[33] L Cominacini A Fratta Pasini U Garbin et al ldquoOxidized lowdensity lipoprotein (ox-LDL) binding to ox-LDL receptor-1 inendothelial cells induces the activation of NF-120581B through anincreased production of intracellular reactive oxygen speciesrdquoJournal of Biological Chemistry vol 275 no 17 pp 12633ndash126382000

[34] N H Nam ldquoNaturally occurring NF-kappaB inhibitorsrdquo MiniReviews inMedicinal Chemistry vol 6 no 8 pp 945ndash951 2006

[35] K Wang S Ping S Huang et al ldquoMolecular mechanisms und-erlying the in vitro anti-inflammatory effects of a flavonoid-rich ethanol extract from Chinese propolis (Poplar Type)rdquoEvidence-Based Complementary and Alternative Medicine vol2013 Article ID 127672 11 pages 2013

[36] Z Ding S Liu X Wang M Khaidakov Y Dai and J L MehtaldquoOxidant stress in mitochondrial DNA damage autophagyand inflammation in Atherosclerosisrdquo Scientific Reports vol 3Article ID 1077 2013

[37] C Y Lin P N Chen Y S Hsieh and S C Chu ldquoKoelreuteriaformosana extract impedes in vitro human LDL and preventsoxidised LDL-induced apoptosis in human umbilical veinendothelial cellsrdquo Food Chemistry vol 146 pp 299ndash307 2014

[38] F HuW ZhuM Chen Q Shou and Y Li ldquoBiological activitiesof Chinese propolis and Brazilian propolis on streptozotocin-induced type 1 diabetes mellitus in ratsrdquo Evidence-Based Com-plementary and Alternative Medicine vol 2011 Article ID468529 8 pages 2011

[39] Z LiH Xuan L Zhang C FuQ Sang andY Song ldquoProtectiveeffect of propolis on the injury of rat BRL hepatocytes tnducedby hydrogen peroxide in vitrordquo Food Research andDevelopmentvol 34 no 15 pp 5ndash8 2013

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


study indicated that annexin a7 (ANXA7) a member of theannexin family of calcium-dependent phospholipid bindingproteins was an endogenous regulator of PC-PLC ANXA7also participated in the progression of atherosclerosis andtargeting ANXA7 inhibited atherosclerosis in apoEminusminus miceANXA7PC-PLC signaling pathway may represent a noveltarget for the treatment of atherosclerosis [16]

Chinese propolis affected PC-PLC activity Our previ-ous study indicated that Chinese propolis played an anti-inflammatory role partly through its inhibitory effect on theactivity of PC-PLC [17] Considering the important rolesof ANXA7 and PC-PLC in atherosclerosis and propolismodulated atherosclerosis and affected PC-PLC activity wehypothesized that propolis may also affect ANXA7 theendogenous regulator of PC-PLC In present study we testedthe hypothesis by investigating the effects of Chinese propo-lis and Brazilian green propolis on PC-PLC activity andANXA7 level in ox-LDL-stimulated HUVECs ox-LDL playscrucial role in triggering the development of atherosclerosisFurthermore we investigated the effects of both types ofpropolis on reactive oxygen species (ROS) levels nuclearfactor-KappaB p65 (NF-120581B p65) and mitochondrial mem-brane potential (MMP) which were regulated by PC-PLC inHUVECs

2 Materials and Methods

21 Chemicals and Reagents DMEMwas fromGibco (USA)Fetal bovine serum (FBS) was from Hyclone Lab Inc(USA) L-120572-phosphatidylcholine 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide (MTT) 2101584071015840-dichloro-dihydrofluorescein diacetate (DCFH) and JC-1 were fromSigmaCo (USA) Acridine orangewas fromAmresco (USA)Ox-LDL was from Beijing Union-Biology Co (China) Pri-mary antibodies against ANXA7 NF-120581B p65 GAPDH andsecondary antibody (horseradish peroxidase) were fromSanta Cruz Biotechnology (USA) Secondary antibody forimmunofluorescence donkey anti-rabbit IgG Alexa Fluor-488 was purchased from Life Technologies (USA) All otherreagents were of ultrapure grade

22 Preparation of Propolis Extracts Propolis used in presentstudy was Chinese propolis and Brazilian green propolisBoth types of propolis had been used in previous studies [1718] The extraction method was as before Briefly extractedChinese propolis was obtained from colonies of honeybeesA mellifera L in Shandong Province of North China andthe main plant origin was poplar (Populus sp) ExtractedBrazilian green propolis was collected in Minas Gerais Stateof Brazil where Baccharis dracunculifolia DC is the mainbotanical source Both types of propolis were stored at minus20∘Cuntil used Chinese propolis and Brazilian green propolissamples were extracted with ethanol and then filtered underreduced pressure and the filter liquid was concentratedunder reduced pressure at 40∘C until reaching a constantweight and then redissolved in ethanolThe ethanol-extractedChinese propolis (EECP) and ethanol-extracted Brazilian

green propolis (EEBP) had a brown colorTheprepared EECPand EEBP were stored under a dry condition at 4∘C

23 Cell Culture HUVECs were gifted by AtherosclerosisResearch Institute of Taishan Medical University of Chinapurchased from ATCC HUVECs were cultured in DMEM(high glucose) supplemented with 100UmL of penicillin100 120583gmL streptomycin and 10 heat-inactivated FBS at37∘C under humidified 95ndash5 (vv) air and CO

2

24 Exposure of HUVECs to EECP and EEBP When theHUVEC cultures reached 80 confluence then the cellswere divided for treatment (a) culture in 35 DMEMmedium with ethanol at lt01 (vv) (control) (b) culture inbasal DMEM medium with 45120583gmL ox-LDL with ethanolat lt01 (vv) and (c) culture in basal DMEM mediumwith 45 120583gmL ox-LDL with EECP and EEBP (125 120583gmL)respectively EECP and EEBP were dissolved in ethanol withfinal concentration of ethanol in the culture medium lt01(vv) Ethanol at 01 (vv) did not affect cell viability

25 Cell Viability Assay The viability of HUVECs was deter-mined by MTT assay HUVECs were seeded in 96-well cellculture plates and grown to 80 confluence and then treatedwith ox-LDL or EECP and EEBP for 12 and 24 h respectivelyMTT solution was added to each well and incubated for 4 hThe MTT-formazan product dissolved in DMSO to estimatebymeasuring absorbance at 570 nm in an ELISA plate readerThe viability () was expressed as (OD of treated groupODof ox-LDL group) times 100 The viability of the ox-LDL groupwas set at 100

26 Nuclear Fragmentation Assay The morphological chan-ges of nuclei were detected by acridine orange staining At24 h cells were stained with 5120583gmL acridine orange at roomtemperature for 5min and observed under a laser scanningconfocal microscopy (Olympus FV1200 Japan)

27 PC-PLC Activity Assay PC-PLC activity assay was per-formed as the described methods in [19 20] Briefly we usedL-120572-phosphatidylcholine as the substrate of PC-PLC Theoptical density wasmeasured at 660 nm Enzyme activity wasexpressed as nanomoles per minute per milligram protein

28 Immunofluorescence Microscopy After treatment cellswere fixed with 4 paraformaldehyde and blocked with5 normal donkey serum for 20min at room temperatureCells were incubated with ANXA7 and NF-120581B p65 primaryantibodies (1100) at 4∘C overnight After three rinses in01M phosphate-buffered saline cells were treated with acorresponding FITC-conjugated secondary antibody (1200)in a humid chamber at 37∘C for 1 h Cells were rinsed threetimes with 01M phosphate-buffered saline to eliminate theuncombined secondary antibody A laser scanning confocalmicroscope (Olympus FV1200 Japan) was used for fluores-cence detection Analysis used the Image-Pro Plus software(USA) Images are representative of three independent exper-iments


020406080

100120140

Cel

l via

bilit

y

minus

minus minus

+ + ++ +

Ox-LDLOx-LDL


24h12h

Control



EECP EEBP

(a)

Ox-LDL

24h

Control EECP EEBP

(b)

lowastlowast


minus

minus minus

+ + ++ +

Ox-LDLOx-LDL



101214

The r

atio

of a

popt

otic

cells

()

(c)







002040608

112

of P

C-PL

CTh

e rel

ativ

e lev


minus

minus minus

+ + ++ +

Ox-LDLOx-LDL


Control EECP EEBP



3 Results









4 Discussion





Ox-LDL

24 h

Control EECP EEBP

(a)


ANXA7

GAPDH

24 h

ANXA7

GAPDH

(b)

002040608

1121416

The r

elat

ive l

evel

of

AN

XA

7G

APD

H

lowastlowast

lowastlowast


minus

minus minus

+ + ++ +

Ox-LDLOx-LDL


24h12h

Control EECP EEBP

(c)







Mer

geN

ucle

iN

F-120581

B p65

(a)

NF-120581

B p65

012345

67

89

The r

elat

ive l

evel

of



lowastlowast

minus

minus minus

+ + ++ +


(b)







Ox-LDL

24h

Control EECP EEBP

(a)

012345678

The r

elat

ive l

evel

of R

OS


minus minus

+ + ++ +


lowast

lowast lowast

(b)


Ox-LDL

24h

Control EECP EEBP

(a)


minus minus

+ + ++ +


0

02

04

06

08

1

12

14

16

18

The r

elat

ive l

evel

of

mito

chon

dria

mem

bran

epo

tent

ial


(b)





Acknowledgments



References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















020406080

100120140

Cel

l via

bilit

y

minus

minus minus

+ + ++ +

Ox-LDLOx-LDL


24h12h

Control



EECP EEBP

(a)

Ox-LDL

24h

Control EECP EEBP

(b)

lowastlowast


minus

minus minus

+ + ++ +

Ox-LDLOx-LDL



101214

The r

atio

of a

popt

otic

cells

()

(c)







002040608

112

of P

C-PL

CTh

e rel

ativ

e lev


minus

minus minus

+ + ++ +

Ox-LDLOx-LDL


Control EECP EEBP



3 Results









4 Discussion





Ox-LDL

24 h

Control EECP EEBP

(a)


ANXA7

GAPDH

24 h

ANXA7

GAPDH

(b)

002040608

1121416

The r

elat

ive l

evel

of

AN

XA

7G

APD

H

lowastlowast

lowastlowast


minus

minus minus

+ + ++ +

Ox-LDLOx-LDL


24h12h

Control EECP EEBP

(c)







Mer

geN

ucle

iN

F-120581

B p65

(a)

NF-120581

B p65

012345

67

89

The r

elat

ive l

evel

of



lowastlowast

minus

minus minus

+ + ++ +


(b)







Ox-LDL

24h

Control EECP EEBP

(a)

012345678

The r

elat

ive l

evel

of R

OS


minus minus

+ + ++ +


lowast

lowast lowast

(b)


Ox-LDL

24h

Control EECP EEBP

(a)


minus minus

+ + ++ +


0

02

04

06

08

1

12

14

16

18

The r

elat

ive l

evel

of

mito

chon

dria

mem

bran

epo

tent

ial


(b)





Acknowledgments



References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















002040608

112

of P

C-PL

CTh

e rel

ativ

e lev


minus

minus minus

+ + ++ +

Ox-LDLOx-LDL


Control EECP EEBP



3 Results









4 Discussion





Ox-LDL

24 h

Control EECP EEBP

(a)


ANXA7

GAPDH

24 h

ANXA7

GAPDH

(b)

002040608

1121416

The r

elat

ive l

evel

of

AN

XA

7G

APD

H

lowastlowast

lowastlowast


minus

minus minus

+ + ++ +

Ox-LDLOx-LDL


24h12h

Control EECP EEBP

(c)







Mer

geN

ucle

iN

F-120581

B p65

(a)

NF-120581

B p65

012345

67

89

The r

elat

ive l

evel

of



lowastlowast

minus

minus minus

+ + ++ +


(b)







Ox-LDL

24h

Control EECP EEBP

(a)

012345678

The r

elat

ive l

evel

of R

OS


minus minus

+ + ++ +


lowast

lowast lowast

(b)


Ox-LDL

24h

Control EECP EEBP

(a)


minus minus

+ + ++ +


0

02

04

06

08

1

12

14

16

18

The r

elat

ive l

evel

of

mito

chon

dria

mem

bran

epo

tent

ial


(b)





Acknowledgments



References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Ox-LDL

24 h

Control EECP EEBP

(a)


ANXA7

GAPDH

24 h

ANXA7

GAPDH

(b)

002040608

1121416

The r

elat

ive l

evel

of

AN

XA

7G

APD

H

lowastlowast

lowastlowast


minus

minus minus

+ + ++ +

Ox-LDLOx-LDL


24h12h

Control EECP EEBP

(c)







Mer

geN

ucle

iN

F-120581

B p65

(a)

NF-120581

B p65

012345

67

89

The r

elat

ive l

evel

of



lowastlowast

minus

minus minus

+ + ++ +


(b)







Ox-LDL

24h

Control EECP EEBP

(a)

012345678

The r

elat

ive l

evel

of R

OS


minus minus

+ + ++ +


lowast

lowast lowast

(b)


Ox-LDL

24h

Control EECP EEBP

(a)


minus minus

+ + ++ +


0

02

04

06

08

1

12

14

16

18

The r

elat

ive l

evel

of

mito

chon

dria

mem

bran

epo

tent

ial


(b)





Acknowledgments



References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Mer

geN

ucle

iN

F-120581

B p65

(a)

NF-120581

B p65

012345

67

89

The r

elat

ive l

evel

of



lowastlowast

minus

minus minus

+ + ++ +


(b)







Ox-LDL

24h

Control EECP EEBP

(a)

012345678

The r

elat

ive l

evel

of R

OS


minus minus

+ + ++ +


lowast

lowast lowast

(b)


Ox-LDL

24h

Control EECP EEBP

(a)


minus minus

+ + ++ +


0

02

04

06

08

1

12

14

16

18

The r

elat

ive l

evel

of

mito

chon

dria

mem

bran

epo

tent

ial


(b)





Acknowledgments



References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Ox-LDL

24h

Control EECP EEBP

(a)

012345678

The r

elat

ive l

evel

of R

OS


minus minus

+ + ++ +


lowast

lowast lowast

(b)


Ox-LDL

24h

Control EECP EEBP

(a)


minus minus

+ + ++ +


0

02

04

06

08

1

12

14

16

18

The r

elat

ive l

evel

of

mito

chon

dria

mem

bran

epo

tent

ial


(b)





Acknowledgments



References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















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 propolis reduces phosphatidylcholine...

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