Skeletal muscle necrosis in rat induced by
Trimeresurus flavoviridis venom can be
prevented by its serum proteins
著者
KITANO Motoo, HIRANO Masato, ISHIHARA Takashi,
YOSHIDA Aichi, HATTORI Shosaku, UEDA Naoko,
CHIJIWA Takahito, OHNO Motonori
journal or
publication title
南太平洋研究=South Pacific Study
volume
23
number
1
page range
11-18
South paciflc Study Vol.23,No l,2002
Skeletal muscle necrosis in rat induced
by rrmιrFs“
r“
sノ
Zα
ツ
θツ
tridis venom
can be prevented by its serunl proteins
Ⅳlotoo KITANOl*,Ⅳ Iasato HIRANOl,Takashi lsHIHARAl,
Aichi YosHIDA2,shosaku HATTOR13,
Naoko UEDA4,TakahitO CHIJIwA4,Ⅳ
lotonori OHN04
Abstract
財 ″θ″s″″s′αツοツプ″グノsの sertlm proteins wcrc fractionatcd by anmnonium sulfatc precipitation to i、e pOrtiOns dcpcnding on the dittcrcnces ofits saturation percentagcs,that is,0‐20%,20-30%,30-409る ,
4050%,and 50-70%Thc cmccts ofthese proteins on 7venOm_induced rat skeletal musclc damage were in、cstigatcd、/ith cioscr attention to histopath。10gical features ofimpainllcnt,necrosis,and regeneration of musclc flbers The kllowledgcs which portion of y senlln prOteins is effectivc fOr prevention of local lcJOns causcd by 7Venom should shed light On the effccJve medicaltreatmcnt afterbi■cn by γsnakC In
conscquence,it was fOund thatthe necrotic changc ofthe rats inoculated witll ηrcrlldC vcnOm together with
thc senlm prOtein fraction ofanlmOnium sulfate saturation percentagc 40-50%was the smallcst cOmparcd to thOse ofthc rats tested with othcr ttrSertlm protein fractiOns
Key words: r7′ 772′″S′77/Sノ αツοツ′′″お (助 ,envenomation,skelctal muscle nccrOsis(町 OneCrOsis), regcncration,myoblast,7scrum protcins,allllnonium sulf・ atc
Introduction
ln conforlnity with recent improvement of the mcdical treatlncnt of ttt777θ′lθS夕″ZrS
/7αソοッ″た力s cγ:habu snakc)snakC bites,thc mttOrity Ofrcccnt victirns has bccn savcd frOm scvere gencralizcd symptoms and dcath. HOwcvcr, local tissue damagc is
incvitablc,and in some sevcrc cascs this is responsiblc for sequclac such as a se五ous
slough in the mttor extrcmity and eventually leads tO alllputation(HoMMA and TU,1971).
In the AInaFni iSlands of Kagoshilna Prcfccture and the several islands of Okina、va Prcfccturc,this accident accounts for abOut 90%Of γ Snake bitcs.Thcrcfore,it is an irnpOrtant task to prevent the scvcrc and cxtensive damage of skeletal musclc tissues caused by myotoxins containcd in 7venOm.
Five phospholipase A2(PLA2)iSOZymcs have becn is01ated from r/venom
(TANAKA ι′α′,1986:LIu θ′α′,1990;YOsHIzUMI θ′α′,1990;KIHARA`′ α′.,1992):
駆踏 郷 肱鵠 翌堂管
糊 器 鋼 蝠 鳴鍬躍 比
called PLA―N(p110.3,neuroto対 c),and tWO
and 10.2, rcspcctivcly. extremcly low lipolytic to egg―yolk cmulsion but strongly
椰y掘
鰹F職
換都躙I亭
羅A善
覆
鵞鐵 醜五
牌
E n g i n c c H n g , S o J o U n i V C r s i t y *Corresponding Author
South Pacinc study Vo1 23,No l,2002
myolytic)(LIu θ′α′.,1990;IαHARA θ′αノ。,1992;SHIMOHIGAGASHI θ′α′.,1995).BPI and BPII can clcavc arachidonatc at sん…2 position of phospholipids in lniccllar state and in bilaycr rnembranes with siinilar activity as PLA2.Thus,Inost of PLA2 iSOZymcs can be considered to bc rnyotoxins.Thc rnyotoxic action of PLA2S iS nOt neccssarily associated 、vith thcir catalytic activity toward ordinary micenar substrates such as egg¨ yolk emulsion.Thcy are able to disrupt the integrity ofskclctal rnusclc plasma rnembrancs and possibly causc an increased perlncability to C′+and Othcr ions.
Reccntlェ NoBllHISA θ′α′.(1997)reported that thc proteins with binding afflnity to γ VCnOm PLA2S WCre fractionatcd from its scrum on four colums,cach cottugatcd with
onc offour PLA2iSOZymes,PLA2,PLA― B,BPI,and BPH.Five PLA2 inhibitory protcins,
tcrllled PLA2 inhibitors,PLI―I∼PLI―ヽ;were Obtaincd.PLI― IV and PLI―V is mostly bound to PLA2,whereas PLI―I has attnity toward basic PLA2S,PLA―B,BPI,alld BPⅡ .
PurincatiOn of PLA2 inhibitors from γ Seruln by afflnity chromatography is quite laborous and this lnethod is difflcult to obtain thc inhibitors in a large quantity.For thc
reasons,η
rSerum proteins were fractionatcd by ammonium sulfate precipitation to ive
portions dcpcnding on thc diffcrcnccs of its saturation pcrcentagcs, 0-209る
, 20…
309る
,
30-40%,40¨50%,and 50-70%.Thc cffccts ofthcsc protcins on
γvenom―induccd rat
skeletal muscle damage 、vcrc invcstigated with closcr attcntion to histopathological featurcs of impairlncnt,nccrosis,and regeneration of lnuscle fibcrs.Such studics havc s t e m m e d f r o m o u r r e c e n t d e t a i l c d s t u d i c s o f t h e i n 」u 五o u s a c t i o n o f 7 v c n O m a n d i t s componcnts,some PLA2S,On thc rat skeletal lnuscle flbcrs(● TANO θ′α′.,2001).The knowlegdc which portion of 7FSerum protcins is cffcctivc for prcvcntion oflocal lcsions causcd by[∫ VCnOln should contributc to advanccmcnt ofthe effcctivc lncdical treatmcnt aftcr bittcn by γ snake.
ⅣIaterials and PIethods Rαお
Y o u n g a d u l t f c m a l c a n d m a l c r a t s ( 8 0 ∼1 0 0 g i n b o d y w c i g h t ) o f W i S t a r / F u r t h s t r a i n maintaincd in Dcpartrnent of(Dral Pathology Kagoshirna l」 niversity Dcntal School by
sister―brothcr mating(● Iぶ Oθノαノ,1992;KITANO θ′α′.,2001)were dividcd into scvcn groups,cach group consisting ёf 6 rats(tOtal;42)(Table,1).
― ―
Cr“ル 7降 “
θ″
7 VCnOm was collected in Amami Laboratory of ltturiOus Animals,Institutc of Mcdical Science,Univcrsity ofTokyo and lyophilized aftcr dilution with a small volumc of、vater. Thc crudc venoln、 vere、vcighcd and dissolvcd in sterilc physiological saline immediatcly prior to usc.
き
、
崎 α″r i a “
α
“
″F r α
c r i a κ
α″θ
“
θ′
` γ
S ι
r “
″P r a 彪
れs
Blood of ηrsnake was collected at Amami Laboratory of ltturiOus Animals.Its scruln was prcparcd by rcmoving prccipitated cげ hrocytes and fibrous protcins aftcr kccping the blood at 49C overllight and stocked at-8013.At Faculty of Engincc五ng,
S●O UniVersity,ammonium sulfate was added to the serum portionwise at O℃
.At 20%
Prevcntion Of r βαッ。ップ/7dis vcnOm―induced ryonccrosis by its scrllrn protcins 13
supcmatant waS then brought to 30%ammonium sulfate saturation and thc prccipitate 、as conectcd.The samc proccdurc was repeated.Thc protcins cOHcctcd wcrc dissolvcd in water(or O.05 M Tris―HCl,pH 8.0),dialyzcd against water,and lyophilizcd.Thc protcins obtaincd'om 50 mlof 7scrum WCrc O.15g,0.36g,0.35g,1.33g,and O.55g for
O-20%,20… 30%,30-40%,40-50%,and 50-70%ammonium sulfatc saturation,
respcctively. Each serunl protcin iaction was weighed and dissolvcd in sterile p h y s i o l o g i c a l s a l i n c j u s t b c f o r c u s c .
Expι ri″ι″″′Praιι″ “
″s
Thc animal cxperilncnts wcrc conductcd at the Animal Center of Kagoshima
University IDcntal School follo、ving“Guidline ofthc Anilnal Expcrilncnt"ofthis School. All thc rats of seven groups wcrc ancsthctizcd with pentobarbital sodium(Nembutal:
A b b o t L a b . , U S . A . ) . A m i x t u r e o f 7 F C r u d e V e n O m ( 5 0 μg c a c h ) a n d C a c h s c r u m p r o t c i n
iaction(50
μg each)in 100
μl physiological salinc、
vere ittcctCd into thc antcrior aspect
ofthc quadriccps fcmoris muscle(QFM)of caCh rat of irst ttve groups(Groups l-5)in
the ordcr of incrcasing ammonium sulfatc satura●
on pcrccntagc,0∼
20%,20∼30%,
30∼
409る, 40∼
509る, and 50∼
70°0(TablC l). The Crude vcnolll(50
μg)in 100 μl
Physiological saline 、vas illlectcd into the rats of the sixth group (Group 6). Sterile physiological salinc alonc was in」 cctcd into thc rats of thc seventh group(Group 7)as a control Thc anilnals、vere aHowed to suぃ′ive for a period of48 hours and sacrinced under cthyl ether ancsthesia.Both thc right and left hindlimbs and visccral organs including the brain wcrc immcrscd for scveral days in 10%buffcrcd formalin(pH 7.4),and prOccssed lor parafflnic cmbedding Thc scctions of 5∼6 μ rn thickness wcrc cut and staincd with hcmatoxylin and eosin.
Pathological indings
Thc musclc tissuc ofthe rats of Group 7 ill」ectcd only with physiological saline had a typical histology ofskeletal llluscle、vith no abnorlllality in inuscle ibers,nerves,blood ヽCSSClS,and ibro―adiposc layers.
In thc rats inoculated with 2γ Crude venom(Group 6),and With a mixturc of the crudc vcnonl and a scrunl protein fraction of ammoniuln sulfate saturation perccntagcs (Groups l∼ 5),no hiStOpathologicaHy signincant differences、 verc dctcctcd among the
imale and male rats of cach group,so wc prcfcrrcd to dcscribc hcrc all together(TablC l)
In the rats inoculatcd with only ηrcrtldC vcnom(Group 6)and with a mixture oftllc
crude venonl and thc scrulln protcin fraction of allallloniulll sulfatc saturation perccntagc O∼20%(Group l),thcrC Was a widespread musclc nccrosis.Thc myo■brillar matcrials in necrotic ccns wcrc more amorphous and their distribution within the cenular space was dense Clampcd masses showcd liqucfactiOn nccrosis(Fig.1)instcad of being morc
homogeneous looking coagulative necrosis 2へ n abundant innanllnatory inflltratc was prescnt outsidc thc nccrotic ccH arcas, mainly at thc surrOunding tissuc layers of thc llccrotic muscle flbcrs. SiFllultancousl)Ъ rcgcncrativc prolifcration of myoblasts,which 、crc characterized by thc prcscnce of scanty basophilic cytoplasnl,was obscⅣ cd in thc pcriphery of the nccrotic ccH arcas.Thc rcgcncrating cclls wcrc spindlc in shapc、vith a ccntral nucleus,some rcvcaling lnitotic activitics.There、vas little hemorrllagc,although
14 South Paciflc Study Vol.23,No.1,2002
edema was considcrably markcd.Fibrosis was not so conspicuous.
Thc sizes of thc necrotic arcas became smaner and slnallcr in accord with the phenomenon that prolifcration of sman regenerating muscle flbers seemed more and
morc rnarked in theく3roups 2,3,4 in thc ordcr ofincrcasing aIIIlmonium sulfate saturation pcrccntage,20∼30%,30∼40%,and 40∼50%(Figs.2‐4).HoweVet the rats ofGЮup 5 of ammonium sulfatc satllration pcrccntage 50∼70%showed rclativcly greater necrotic changcs than thosc ofGroups 3 or 4.
T a b l c l . H i s t o p a t h o l o g i c i n d i n g s i n Q F M o f t h e r a t s w h c n t r c a t c d w i t h t h e m i x t u r e s o f
TF venorn and its senll■protein fractions indicated.
7VenOm
+ Necrosis lnflanlm atory Rcgcncration Hcmorrhagc Edenla Others Fraction*of of lnflltradon Ofskeletal
7Semm SkCkCtJ protcin musclc (Group of tissuc ratS)** muscle Flbers
0∼
20%*
…
Ⅲ
・
「H
‖
―F
―
(Group l) 2 0 ∼3 0 % I 「 I ‐ F F ‖ ― F F F F (Group 2)30∼
40%
―++
‐丼 I「
¨
―F
―IF
(Group 3)40∼
50%
‐
―「F I‐
IF
‖
―
―l‐
―I
(Group 4) 50∼ 70% ‐「‐ ・FI‐ 4‐ 丼 IF ― l・ 一 IF (Group 5) Crudc venom 一I「 丼 ¨―F IFI thro壺 bosis (Group 6)
Salinc only ¨ (Group 7)
* The values of%represcnt thc anlmcl五
urn sulfatc saturati(m rangcs for fractional prccゎ
量atiOn Ofthe
scrtlm protcins
** Each gЮ up consists of 6 rats(｀ViStar/Furth strain;F+M)
# The necrodc changcs are composed nlainly of liquefaction necrosis but coagulativc necrosis are also present.
Prevention of r/7aッ9ν′″″s venom―induced lnyonecrosis by its serum protcins
Fig■ Fig 2
Fig l(lCft):A photomicrograph of a peHphcral area of a largc focus of liqucfaction nccrosis(N)obscrVCd in thc QFM ofa rat ofGroup 6.Thc necЮ sis was induccd by
ηr CrtldC VCnom, inoculated 48 hours beforc.The focus is surrounded by a
granulation tissuc (Astc五 sks) containing numerous inflammatory ccHs and rcgcncrating lnyoblastic spindlc cclls(HE).
Fig 2(right):A photomicrograph ofa vcry smali necrotic focus(Arrow hcads)inthe QFM
ofa rat of Group 4(HE).
Fig 3 Fig.4
Fig.3(lcft):A larger magnincation ofFig.2.The focusis composcd mainly ofa numerous
inflalnmatory cclls and regenerating myoblastic spindlc cclls.A couplc of nccrotic musclc■ bcrs(Arrows)arc interlninglcd with them at thc ccntral rcgion ofthc focus. Fig.4(五ght):A pc五 phcral arca ofthe granulation tissuc of Fig.2.Notc thc charactcristic
South Paciflc Study Vo1 23,No l,2002
Discuss10m
Many vcnomous snakcs are rcsistantto their o、vn vcnoms.Thcir natural rcsistancc to thcir toxins is duc to neutralizing factors in thcir scra.Thcse factors must protcct against toxicity duc tO accidcntal bitcs by the snake itsclfOr by othcr snakes ofthc same spccics. Inhibitors of snake venonI PLA2S haVe becn is01atcd from the scra ofvaHous snakes and
their prilnary structurcs detcllllined. Two kinds of PLA2 inhibitOrs against ttr vcnom
PLA2,namcd PLI― A and PLI― B,were pu五 icd frOm its serum(INOUE θ′α′.,1991),and an inhibitor that ncutralizcs Иgた,s′Юグθ
“bあ ″力晰傷 S″′′Jθ“
s(mamushi snakc)PLA2 WaS isoslated from its serum(OHKURA θ′α′.,1993)CЮ ′αルS PLA2 neutralizing factor was isolatcd from CЮ rr7ルs″夕′おs“s′θ″′θ夕S SCrum(FoRTES― DIAS θ′α′.,1994;PERALES α α′.,1995).
NOBuHISA θ′α′。(1997)rcported thc is01ation Of flve PLA2 inhibitoぃ /protcins (PLI一I∼V)from πFSCrum in which PLI― IV and PLI― V arc the samc as PLI― A and PLI― B,
rcspect市
ely.HOwevet the contents ofthc inhibitors in lγ
serum scem to bc to a minOr
cxtcnt,so thatthcir scparation from γ scrum is vcry dimcult.Thus,thc cstablishnlcnt of thc cffcctive mcthod ibr prcparation ofthc scrum fractiOns containing thc inhibitors was rcquircd.Our prcscnt study has startcd to fractiOnatc■iseruln protcins to scvcral pOrtions by prccipitation v/ith increasing conccntration of alllmlonium sulfatc.
It is、vcll known that the pathological signs charactcristic of E∫bitcS in humans, rabbits,and micc arc markcd and cxtcnsivc hcmorrhagc and musclc necrosis(OKONOGI θ′α′, 1960).PrCViOusly we rcported that Our cxpe五 mcntal rats,howevet failed to dcmonstrate thc hcmorrhage as a predominant slgn whcn 7 venOm was inJccted.7
venom did not givc rise to any promincnt local bleeding in the rats, but produccd a massivc nccrosis in tlle skcletal muscles accompanied by marked edema(KITぶoθ′α′.,
2001)Additionally wc repOrtcd that in 48 hours aftcr the ittcctiOn Ofヱ
FVCnOm intO the
QFM of the rats,a widcspread myolytic muscle necrosis was fOund,which prObably
resulted fronl a dircct venonl action On thc musclc ibers.Muscle nccrosis by thc 10cal inoculation of Ei VenOm is usuaHy fol10wcd by rapid phagocytosis of dcbris at thc periphcry ofthc nccrotic area.Thcn thc clcaHng Ofnecrotic rllatc五als by phagocytes、vas followcd by a rapid regcncration prOccss. Numcrous myoblasts wcrc obscⅣ ed.「Fhc
exccss and rnarkcd regencration activity ofmyoblasts aftcr rnusclc nccrosis induced by=r myotoxins lnight bc ascribcd to thc cOnditions that ncithcr neⅣcs nor blood vessels are
markedly damagcd by thcsc tOxins,since adcquatc b10od supply and innervaton are thc
essential rcquircmcnts for muscic rcgencration(QuEIROZ θ′αム,1984;GuITIERREZ θ′α′,
1989).ThCrcfOrc,for analysis ofthc cffcct市
c functions of7sCrum inhibitors againstthc
myolytic itturicS by
η
rVCnOm,thc ratis considcred to bc onc ofthc most appropriatc alld
uSeflll CXpcrilnental anilnals.
In thc present studンЪthe rats ofGroups 3 and 4 inoculated byごfvenOm togethcr、 vith
t h c s e r u m p r o t e i n f r a c t i O n s o f a m m o n i u m s u l f a t c s a t u r a t i o n p e r c c n t a g e s 3 0 - 4 0 % a n d 40-509る demonstrated the most dOminant changcs in thc foci Of musclc necrosis. Thc
nccrotic foci were lilnited in a narro、v arca and、verc su■ounded mainly by granulation tissucs cOntaining numcrous lnacrophages and rcgcncrativc myoblastic ccns.Edcma was not so marked, cither. These facts suggestcd that thc effcctivc myotOxin inhibitory
protcins wcrc cOntaincd in thc scrum fractiOns prccipitated in thc particular conccntration rangcs ofalllnlonium sulfate
Prevention of rノαッ。ッJガ
`″s venom―induced myonecrosis by its sertlm protcins 17
ConclusivelシЪ the results obtained in thc present study suggest that thc myotoxin inhibitors in ηrserum are effect市e in healing the myotoxic ittuHeS induced by ηrcrude 、enom Since application of excess市 c doscs an″ or repetitive application of anti-7 ヽenom antisera are known to bring about se五ous lncdical problems,we certainly expect
that applicati9n OfrFScrum inhibitors in appropriate ways could providc a uscfulthcrapy
ぉrr/snake bites in near futurc.
Acknowledgements
This work、 vas supportcd,in parts,by a Fund for Promotion and Dcvclopment of Arnami lslands sponsored by Kagoshilna Prefecturc and Ministry ofLand,Infrastructurc and Transport of Japan and by a KagoshiFna University Fund for lmprovcment of Educatlon and Research Activltles.
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