|叫.一ticle 叫[1J!I"li!ti1.. 11lI川:“:235-41

Basic Scicnιe

Agaricus Blazei Murill Ameliorates Myocardial Ischemia-Reperfusion Injury

Chicl1 -j-lu(I /-I11011g. U Hon- Ni TS lI Î , .I Ren-Hu ll' P.的嗨• I }'i- /-Isua l1 Hsie !J, 3 Wοei-.Jer CI1IIong.J../ Cα-Chillg HSlf ， 可I'Velll叫 HlI{lI1g~.4. 1I1ld I'Ven-.Jone Clrel1 l.~ 均

8ackgrolllld: AgariclIs bla~ei MlIri ll (ABM) is a widcly acceptcd heallh food and is known 10 prescnt anli-ll1 morigenic

activities . ABM has also been rcported 10 11l0dll late oxidati ve strcss aner inJl a111111a tion and infec tion. Reactive

oxygen fl閃閃dica l s havc been shown to bc criti ca ll y in vo lved in i schc l1l ia-rcpcr仇， sion injllry cascadcs. We ail1lcd to

stlldy Ihe antioxidant activ圳的 ofABM aga inst tiss lIc injllry causcd by 111yO巳ard i a l ischcmia- repcrfllsion proccsses

MethOlls: A rat card io l1lyocy tc H9c2 ccl l line was sct up and lIsed for thc stlldy. Cc ll viabi li tics afler di仟erent

dosages 0 1' ABM ex tracl trcat l11cnt and hydrogcn peroxidc injuries wc rc c he氾kcd . A card iac ische l1l ia-rcpcrfllsion

injllry modcl was set lIP to elllcidate the cardioprotcctive roles 0 1" ABM cxtrac t. Diffcrcnt doses of 22.5 I11 g/kg of

body wcighl and 45 I1lg/k且 of body wc ight AB M cxlraCI wcrc p悶-Ircatcd al 24 and 4日 hO llrs bctòre indllcing the ischc l11 ia-rcpcrl"lIsion injllry

Re.", lts: ABM cx tract trCatll1enl increased thc slI rviva l o f ca rdiomyocytes witholll cytotoxicily. Pret rcatment with ABM ex tract redllced hydrogen pcrox ide- indllced cc ll da mage and increased ccll slI rviva l (0 日 6 :t 0. 1 5 in ABM 6.0

mg/ l1l l vs. 0.45 :t 0.03 in control, p = 0.0 19). ABM-pretreatcd rats lhat lInderwent myoca rdial ischcmia-rcpcrfllsion had greatl y rcdllced infarcl areas comparcd to those in thc control grollp (14.3 :t 3.3% in contro l grollp . 4.6 士 1.9% in

22.5 111 g/kg grollp, 4.9 士 2. 1 % in 45 mg/kg grollp. p < 0.05) COll cIIlS;O Il: ABM incrcascs anlioxidant ac tivi旬， which grea tl y amc li ora tcs myoca rdial injllries callsed by

l1lyocardi al ischc l11 ia- repcrfllsion injllrics. Us ing ABM as a hea lth food l11ay providc ca rdioprotect ivc cffect aga inst

ischemia-rcperfll sion i 叮 uncs

Key WO l"ds : AgariclIs h1a~ei Murill • An ti ox idant • Ischcmi a• cpcrlùsion . Myocardia l infa rcti on

INTRODUCTION known fo r it s med ic ina l propertics in diabetcs ‘ osteopo-

ros悶、 and ca nce r. 1.1 The ABM mushroom is anti c las-Agaric lIs bla工的 M lI rill (ABM) , an edi bl e Basid i o-ωogeni c in m刊I ce W I川1叫山h t仙u l11 0 r xe釘enog♂ra ft心s

l11ycota r，心1I叫11川ngll s f，ωOll叫l叮II1 d pril川maril抄Y 11川n SOllt仙h America , i的s well ca別te的5 t山h a削t ABM i陌s t仙u叫lmor i cida l. AB M also like ly plays a

ro le in preventing tumor initiation by reducing geno-

Rece ived: JanLHlly 7. 20 10 Accepled: .I unc 2X. 20 10 I Depart mcnt 01' Emc l 且c l1 cy Mcdicinc; 2Dcp i:l rt Il1 Ci11 of Int crn al Mcdicinc司 National Taiwan Un ivers ity Hospita l‘ Taipei: 3Bas ic Med ica l Sc iel時的、 Medica l Laboratory Sc iencc and ßiotechnology: -I Biochcmistl)'. Collegc ofMed icÎ nc可 Nationnl Chcng-Kul1且 Univcrs i ty.Tainan: 雪Simpso l1 B i o l cchno\o且y Il1c.. Taoyu él ll ‘ 4、 Dcpartmcn t 01 Intemal Mcdic ine可 Shin Chu Gcneral Hospilal‘ Shin Chu. Taiwan Addrcss correspondcnce and reprint reqllc心的 1 0: Dr. Wcn- .I ol1e Chcl1 , Depanment of Emcr且cncy Medicinc. Nat Îona l Tai wan University Hospilal , No. 7‘ Zhollgshall S. Rd ‘ Taipci City 100. Ta iwall. Tel : XX6-2-2356-2831 ‘ Fax: 886-2-2322-31 50: E-mai l: wjchcI1 1 955(ç~lllu.cdll. tw The two cO lTcsponding au t l叮叮 contribll tcd eq llally

235

tox ic itics ca used by DNA-damaging agents5 ABM has

a lso becn shown to cnhance innatc as we ll as adapt ivc 6.7 i l11 mune rcsponses in l11 ice.".' It st imulates the prod uc-

tion of inter le ll kin and cyto kin的 ， which inc rcase host

immuniti cs against bacteria l and viral infect ions. S. 'O It

also induccs inter leukin-12- and intcrfero n-y-mcdiated

natura l kill er cell ac tivation ." These find ings inva ri ab ly

suggest that A BM is a promising “hea lth food" thal pro-tects hos ts from infcctions and cancers. O nc recent

study' l reported that ABM prevented di ethylnit ros3m ine,

AC(fI Cflrdiol Sill 2010:2 f1:l35- 41

Chien-Hlla Hllang et al

a strong hepatic cytotoxin and genotoxin，丘。 111 c3 11 sing fied Eagle l11 edill m (DMEM ) sllpp lemented with 10% fe-

liver ce ll injury; thi s indicated thal ABM helps maintain tal bov ine serum (FBS), 2 111M L-g lll ta l11 ine, 1 x non-

tissue in tegrily. The deh ydrated ABM fruct ifica tion body essent ia l amino ac id , and 1 x antibiotic/anti l11 ycotic

is ri ch in proteins and carboh ydrates. The po lysaccharide mixtllre (Gibco BRL, Grand Island , NY). 執le grcw lhe cOl11ponent，但-glllcans ， i 日 key to the l11 edicina l effect s of ce ll s at 37 oC in a 5% CO2 的mosphere.ABM. 1 The ß-glucans exlracted from ABM 1110dll laled T he dried ABM powdcr was offered by Si l11 pson

im l11 une responses by stimu lati ng the re lease of cylo- Biolechnologies, lnc . (Taoyuan, Taiwan). We mixed 2.5 kines , which comprise ABM's il11l11 une regu latory eι grams of ABM powder with 100 mL of phosphate bllι fects U fered sa li ne (PBS) [pH 7.4] and shook it at room temper-

Two parallel but somew hal contradictory immune alure for 1 h. The ABM so luti on was filt ered us in g

reg lllato ry effec ts of ABM have been reported . O ne Whatman No. 1 filter pape r. The filtra惚 ， which com-

stlld/ reported that, in macrophages , ABM stimu lated prised the water-soluble cO l11ponents of ABM、 was ster-nitri c oxide production and increased infla l11 mati on, i1 ized by pass ing il through a 0. 22-~lm-sy rin ge fi lter. It which creates cell ular oxidative stress; a second stlldy,14 was then slored al -20 oC unti l ll se.

in li ver and lung ce ll s, reported that ABM redllced the

prod llction of reacti ve oxygen species (ROS) and main- MTT assay

tained DNA and ce ll ul ar integrity. The mo lecular mecha- A 3-阱， 5-d imethylthiazo l-2-y 1 ] 孔， 5-diphenyl l e tn卜

ni sms to reconcile these two di stinc t pheno l11ena have zo liu l11 bro l11 ide (MTT) ce ll pro liferation assay was l1sed

not yct been identified . However, ABM l11ay affect ox i- to detect ce ll viability by eva lllating the l11itochondrial

da ti ve responses in different ti ss ll e-types in di stinct redllctase ac ti vity after treatments with ABM extract and

ways. hyd rogen pe rox ide. The protoco ls bas ica ll y fo ll owed

In lhe presenl s rudy, we wanted to kno w whelher those previously described 22 Brie fl y, lhe ce ll s that were ABM is an anti oxidant in the card iac sys tem and g ro wn to 5 x 104/we ll in a 24-we ll clllture pl ale we re

whether it protec ts cardiomyocytes [rom permanent trealed with vario l1 s doses of ABM exlrac l for 24 h al

damage callsed by aCllte myoca rdial infarction, which 37 oC and then treated with 100μM hydrogen peroxide

know whether ABM protecls cardiomyocytes [rom the Intracellular reactive oxygen species (ROS)

ox idative stress callsed by [巴perflls i on therapy. measurement

lntracellu la r ROS level was sludi ed by measuring

the changes in flllorescence resulting from intrace ll ulal

MATERIALS AND METHODS probe ox idation. The probe 2' ,7'-d ich loroflllorescin dia-

cetate (DCFH -DA、 5.0 mM 、 S i gma ， St. LOll is , MO ‘

Cell lines and the ABM mushroom U.S.A.) entered the ce ll and th e acetate g ro l1 p on

We used rat cardiomyocy le H9c2 cell s for thi s study. DC FH-DA was c leaved by cell l1 lar es te ra ses. S l1 bsc-

The cell s were 111aintained in reglllar Dulbecco 's mod i- quent oxidation by ROS , particu larly hydroxyl radica l 、

ACfα C(I川Iiο1 Sil/ 2010:26:235-41 236

Antioxidant Activity in AgariclIs B/azei Muri ll

y ic lded thc fluo rescent prodllc t DCF. AB M cx trac t- complele ly liga ting the margi na l coronary a rtery fo r 30

lrcated and contro ll ed 1-1 9c2 ce ll s ( 1 x 10") wc rc loaded min . After 30 min 0 1' isc hem ia, the S UI 且ica l li e was rc-w ith 10 ml DCF I-I -DA at 4 ho urs a fte r treatmenl w ith leased and the ischcmic myocardium was reperfused fo r

1-1 20 2, and ana lyzed by f ACSca n ( Bec to n Di ck in son 2.5 h . Myoca rd ial ischemia was con fÏ rmed visua ll y by H e i de l be l 且， Gerl1lany) . reg iona l cyanos is of the myocard ia l Sllrface.

Th e infa rct s ize was de termined as prev io us ly

Cell damage assays by flow cytometry analysis dcscr ibed 24 us ing 1% tripheny l-tetrazo lium ch lo ride

Damagcd ce ll s were detected by pro pid ill l1l iod ide (S igma-Aldrich, U.S.) in phosphate bll ffer [pH 7.4]. The

(叭 ， 50 I1lg/ l1l l) afte r trcatment with 1-1 20 2 fo r 24 h as de- lell ventri cul ar area at risk and the area 0 1' in fa rcted ti s-sc ribed prcvio us ly. 23 Labe ling was pc rlo r l1led with the s lIe were measured by an independent, blindcd obsc rver

sta ining ana lyzed on a FACScan (Becton Dickinson He i- using CO ll1pllter plani l1letry.

dc l berg、 Germany). When nuclear membrane d isrllpted,

which is a s ign of advanced ce ll dama且ed ， the 1'1 we nt Statistical analysis

into thc nllc lc i and showed thc pos iti ve s ta inin且 Data are presented as l1lea n 士 s tand a rd dev iati on

ANOVA tes t with pos t- hoc ana lys is by Bonfe rro ni

Treating rats with ABM l1lethod was lIsed to eva lllate stati st ica l s ignifi cance of

Eightecn 8-wcck-o ld Wistar rats wc igh ing approx i- di ffe rences alllong di行crent grollps. A va lll e of p < 0.05 mate ly 350 g werc d ivided into three gro llps: a contro l was considered stati stica ll y signifïcant

g ro ll p (11 = 6) and two grollps treated w ith A BM 22 .5

mg/k且 of body we ight (11 = 6) and ABM 45 m g/kg of

body we ight (11 = 6) gro llp . The ABM was admini stercd RESULTS

by lIs ing orogastric tllbes at 241h hOllr and 481h hour be-

fore ischemi a- reper fll sion inj uries we re indll ccd . T he ABM increased cell proliferation and was not

contro l gro llp receivcd the cqll iva lcnt amollnt of water at cytotoxic

thc sa lll e tillle po ints as the treatlllent groups did. In thc H9c2 ce ll li ne treated with ABM ex tract only

without hydrogen p巳rox i dc ， the cc ll num bers were s ig-

Cardiac ischemia-reperfusion animal model nifìcantl y hi g hcr in the ABM-treated ce ll s than in the

After wc had prctreated the rats w ith ABM, we in- controls ( 1.47 fo ld in ABM 6.0 111 g/ l11 1 cOlll pared 10 con-

duced myocard ial ischemi a by completc ly ligat ing lhe t ro l 、 p = 0 .047, Figllre 1), whi

D7 Aι ta Cardiο/ Sill 20/IJ:26:235- 41

Chicn-Hua Hua n且 c t a1

_ 2

E E

m1.5 。O QI cl E 甸

舌。 5

1J

D LL 0

。 2 4 6 ) ABM concentr悶ation (mgωImt)

fïgl仙d“re 1. Th e:“e 1川t川川1/圳圳/1川州/1川叫IIh阿削

A nli ox id叫11 Act lvlty In Agω iC IIS 8icCl!i Muri ll

apy lIsed after aC lI le myocardia l int~1 rct i o n . Wc f"ollnd m句0 1' mec hanisms by wh ich AB M cO ll ld ameliorale thc Ihat rats prelreated Wilh ABM (both 22.5 I11g/kg and 45 l11yocardial ischemi a-reperfll sio l1 injllry in an imal 1110del

mg/kg doses) had s ignificant ly s l11 all cr infarcI arcas Posl-myocardial intàrcl ion injllrics are rclated to the

compal 以.1 10 lhe conlro l grollp (14.3 :!: 3.3% in control IIp-reglllaled local neurohllmoral facto rs. i n c llldin且 pro-

grollp . 4.6 :!: 1.9% in 22.5 m且/kg gro llp. 4.9 :!: 2 .1 'Yo in45 inna l11 matory cytok ines and rcaclive oxygcn specics 29

mg/kg gro llp, p < 0.05 by ANOVA tcst, Figllres 5A and Reac ti ve oxygcn spccies l11 ay conlr iblllc 10 myocardial

B). These tìndings indicated thal the ABM pretrcat ll1ent damagc via va ri o ll s proccsses , in c llldi ng dirccI cyto-

amc li oraled th c myoca rdi al injllry by red uc ing the in- loxic ‘ negali ve inotrop ic , and cy tok ine-slimll lating cf-farct area after myocardia l 川 tà rc ti o n . fec俗. and callse apoptosis J o.J ' Somc antioxidant drllgs

sll ch as probll co l (an antihyper li pide l11 ic drllg). di l1le-

thylthiollrea (DMTU; a hydroxyl radical scavcnger)、 and

DISCUSSION vi ta l1l in E - have been tcsted in ani l11 al models 01" myo-

card ial ische l11 ia-reperfll sion and signifìcant ly attenllated

ABM has cardioprotective elTec ts aga inst oxidat ive ox idativc Slress 司 in h ibited thc activa tion 0 1" pro-intla l1l-

stress InJ lII"I CS ‘ as shown in this stlldy. Prctreatmcnt o f

ABM ca n a l1leliorate hydrogen peroxide injuries‘ which

is a strong donor 01' ox idati ve stress ‘ to cardio ll1yocytes 1'h巴 ce ll damage as shown by Ihc decrease of mitochon

dr ial reductase activity and increasc of nuclear stain ing

by PI was Icss lI ndcr ABM pretreatment in cardio-

myocy tes. ABM prctreatment as a nlltri ent in the food

cOllld dccreasc the myocardial inià rct size in cx pcrimen- A

ta l 3nimals, imp lying lhc ABM cOll ld be helpflll 10 pre-

vcnt thc myoca rdial damagc in aC llle card iac event

Ox idati vc st rcss plays an i mport日 nt role in ca rdi ac

ischc l11 ia-reperflls ion injllri es. T l1 e cxcess ive oxygen

molecu|CSEO Into I|1C Cflt d10lnyocyIES Elu l lng 111C l e-qA

pcrfll sion stage. Thesc oxygcn 1110 lecll lcs will co mpro-

l1l ise thc functions of complex 1 anc\ cO ll1 plex 111 of clec-

tron transport chains in t l1巴 mitoc h ondr i a ， thcn SllpCroX idcs are generatcd exccss ivc ly. Thc sllperoxides bind to

白，焉"、Ihe va rioll s protcin and damage Ihcir fllnctions. "" "" In

another way‘ tl1c s lI pcrox ides are conve rred into hydro- B gcn peroxide and frce rad ica ls whi ch can indllcc 1110rc

potcnt and severe ox idat ivc injurics. Th c 的 ee radi ca ls

and cytochrome C cnter cy tosol after da l11a且 i ng thc l11 ito-chondr ia pcrmcabi lity trans iti on pores leadin g 10 ca r-

diomyocyte necrosis and apoptosis. 27 The anti-ox idant

c lTec ts of ABM l1 ave been stlldi ed in bl ood ce ll s. 1'he

ABM extract shows a signifìcant dose-depcndenl protcc

ti ve cffec t against DN A damage indllced by hydrogcn

pcroxidc in hllman lymphocytes. l• Hot wa ter ex tract of

ABM ca n signi ficant ly dccrease chcmi ca ll y indllced

lipid-perox id at ion in live r ti SS lI C2X In oll r St llct y, we

found ABM could protec t ca rdiomyocytes rro ll1 oxi da-

tive injuries by hydrogen peroxide. It cOllld be onc ofth e

Control 45 mg/kg ABM 22 .5 m g/kg ABM

35 口 Control

30

25

. 22.5 mg/kg ABM

油

5050

，a

呦，

4，

Area at risk ILV

Infarct I area at risk

fïgllre 5. E(1"cc/.\" '!f" A 8/vl υ11 lhe infllru arC!a (~(i er lII.n削川lia/ is-cll elllìu- rf!IJeI:tilsiυ11 ;11 m!s. The rals prem! lI fed 1\ ';111 !l BM ('X/ I'O< fs 111 12.5 IIIg/kg /)m(1' \1'(' /);111 Ofu' .fìlh叫 b叫吋叫 45 mg/kg ho((1' ,, 'cighl (1IIt'

slwc/ed ha/"sJ οf else II/ ock-II ι叫f的I (tlte cleo/' hu/'s) \1 '(.'1'(' SI/N('clecl /0

叫l'( l('ordia! ischeJ/l ill-rt'per/ifsiu lI . (.'1) I\ /~\'(J( 州 'clia/ se('/ifJI/s υ(111 1..' m(s ìïre ar(!{lS lI f risk ('(I/ /sed hy lIIyoca川市0/ isc!t叫nia (l r (J ill fJink, TlIc: iI !fúrct

arC{f f /ll 'lIcd \I'!lif c: ((f/'rOH~ ， Tlle (.υ1/(/'0/ gUJ/lp /wd (/ {mgc:/' ù!f{I/'CI {/I'C:(I

111(/11 凹的的。/阱， 川'() AB/'v/. frearc:d glυups did. (B) Quu川/10/ /(11/ υ11/.丸，

u間的叫 risk 帥/{{ infiJrcl {/I叫s， TII ~ 叫別2月叫 risk ω '(' 1)1的emc:d (1;、 ρ叫一

“ 'JIlage {//,e{Js (?f' I /te leþ 叫作Ilricles 0(' 11丸! /t('(I/,ls fω11:'(1， T/If! i l!I'a /'('1 arc:as (J re fJl'I:senl l!d 川 pel，(，(! lI lage arf!lIS υ(' ,I/e oreas (11 risk ;11 I!W StlllJ(! he.糾制 T!J吋附c的，吋官 刊川.，“圳t方 "仰0 )'1仰lifìcζC(/圳仰'u川F川川(/

Chicn-Hua H uan且 et al

matory cytokin e丸 and preserved ventricular function af- fusio l1 injuries model in the study were healthy ani l11als

ler myocardial intàrction. " The present study showed The responses to the injuries and treatment may be diι

that ABM treatment reduced infarcted areas after cardiac ferent in the clinical setting. Hllman studies are needed

ischemia-reperfusion, and that the antioxidant acti vity of belore c1 inical application ABM like ly plays lhe key protec ti ve ro le. Based on a

prcv ious report l4 that AB此1 contributes to ant卜 infla l11-

l11atorye仟ects ， AB M might also protect l11yoca rdial ce ll s CONCLUSION

fro l1l infl ammati on-induced ox idati ve ti ssue damage.

However、 n U l11e rous studies l.l . 1J have pointed out that 1n lhi s study、 we found that ABM reduced ox idative ß-glucans are the 111句or components of a仙oxid ant and stress in cc ll s and protccted them fro ll1 perll1anent cell

anti-inlla ll1 matory ac ti vities. Whether ß-glucan CO l11pO- da ll1agc. Using ABM as a health food l11ay help decrease

nenls are the key pl aye rs contr ibuting to these eff，巴cts l1l yoca rdi al injury after aC llte myocardia l infarcti on

was not clear ly demonstrated in the present stlld y. ß- Based on the findings in the present study, we conclude

glllcan s are polysaccharid es of D-glllcose 11l0n0 ll1ers that ABM is a promi sing nlltritional sllppl el1lent for indi-

whi ch are li此ed by 戶名Iyco且 idic bonds. They OCC llr vidllals with a high ri sk ofmyocard叫 infa rct i o n

most cO ll1 lllo l1 ly as ce lllll ose in plants , the cell wa ll of

ce rtain fllngi. lllushroo ll1s and bacteria. Glucans exhibits

thei r protec ti ve effect aga inst ox idative damagc as a con-

seqllence of scaven皂 ing both O H radica ls and si ng let

oxygenJ3 Therefore, we speclllate that ß-glllcans cOllld

be the key contriblltors in ABM 's l1lyocardial protcction

It has beco l1le an ill1 portant iss lle whether natura l

in gredients and nlltri ents can lowe r the seve rity and

chances of deve lopi ng heart diseases in the preven ti ve

medi ci ne. Some ingredi ents, such as red yeas t ri ce

(Mollasc /ls 1'/1"1' 111叫s) and tï sh oil are proved for their lipid-Iower ing effec tsJ 4..l5 Mushroom extract , sllch as

Gallodel 川。 IIICidll 1l1 (Reishi l1lushroo l1l) is reported for

1tS a nt卜ox id a nt and card ioprotecti ve effects against etha-

no l and isc hem ia-reperfus ion cardiac injuri es throllgh mitochondria-dependen t path ways J(>.l 7 ABM has il1l

ll1 une- ll1odulating effects. In our stud y. we proved anti -

ox idant and cardiac protect ive effects o fpret reated ABM

aga inst ischemia- reperfl1sion inj l1 ri es . ABM also showed

110 cyto tox icity in the cardiomyocyte c l1 lture st l1dy. This

imp lies ABM may have potential as a nl1 trient ingredient

for cardiac health

The study has limitations. Firstly, the ABM extract 111 ay contain SO l1le c0111pounds rather than a single che-

mical c0111pound. The rea l ac ti ve ingredi ent of ABM

extract for cardi oprotecti on is not c1ear so fa r. However,

as a nutri ent fo r dail y l1se , the 111ixture of S0 111 e C0111-

pOl1 nd is acceptable and convenicnt in l1se. Secondly. the

cO l11pari son of ant卜ox ida nt activ ily a1110ng ABM extract

and other an ti-oxidants was not checked in the study

Third ly, the an imals l1sed for inducing ischem ia-reper-

AC /(f Cardiv! Si月 2010:26:235-41 240

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