532 damazolgroupshrinked,glandularepitheliumbecamethinner,andthenumberoftheglandsdecreased.ectopicendome trialcelswereshot cylindricaleventabularwithi

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1 )** ChineseJournalofPathophysiology 2013,29(3): [] (2013) !"#$%&' caspase 3 Bcl 2()*+,-. 1, 2, 3, 2, 2, 2,! 2, "#$ 2 ( 1 "#$%$%,& "# ; 2 $%' $%, ; 3 & (%)*,& "# ) [/ 0],:+,-./01. 2(EMT) :$; caspase 3 Bcl 2 <=>?,@A-./BC EMT= DE0 1 :D. -0 EMT3 4 F,G EMT34HEIJ FK <LMBCK -./BC 1K -./BC 2K,NO 4, K FK ) P3Q,RD HEST UVWX Y K FK 569: = D K Z K caspase 3 Bcl 2 = <[\ :-./BCK, P] ^_Q(P<0.01),) `a]bc0 a], ;56, 78, a]; -./K <LMK6 _,,6 _, ST, ;56 78Q 9 ;, K ], FK Bcl 2 < ;-./K 56 caspase 3 < (P<0.01), Bcl 2 < (P<0.05) :-./ 34 EMT a]= c0 D, DE / 56= caspase Bcl 2 EMT= <, 56=78, E0 [ 2]. 2;-./;5678 [3456] R711 [789:] A doi: /j.isn EfectofFugongningonmorphologyandproteinexpresionofcaspase 3 andbcl 2inratendometrioticcels HUANGYue yan 1,XIAOChun 2,YINMan xiang 3,LIUChun hua 2,QIAOYu dan 2,SHI Min 2,WANGXiao min 2,XUYou hui 2 ( 1 JiaxingUniversityColegeofMedicine,Jiaxing314001,China; 2 SchoolofBasicMedicalSciences,JiangxiUniversityof TraditionalChineseMedicine,Nanchang330004,China; 3 DepartmentofPathology,ZhejiangProvincialCorpsHospital, ChinesePeople sarmedpoliceforce,jiaxing314000,china.e mail:hyylinda@163.com) [ABSTRACT] AIM:ToinvestigatetheefectofFugongning(FGN)ontreatingendometriosis(EMT)byobserv ingthechangesofhistopathologicalmorphologyandultrastructure,andtheexpresionofcaspase 3andBcl 2inanendom etrioticratmodel.methods:theratmodelofemtwasestablishedbyautotransplantationofendometrium,andthe modelratswererandomlydividedinto4groups:emtgroup,danazolgroup,fgn1groupandfgn2group.theratswere treatedwithnormalsalineinemtgroup,danazolindanazolgroupanddiferentdosesoffgninfgn1groupandfgn2 groupbyintragastricadministrationfor4weeks.after4weeksoftreatment,thesizeofthelesionandhistopathological morphologyofectopicendometrialtisueswereobservedundertransmisionelectronmicroscope.usingimmunohistochemis trymethod,theproteinexpresionofcaspase 3andBcl 2inendometriumandendometriotictisueswasdetermined.RE SULTS:ThesizeoftransplantedobjectinFGNtreatmentgroupswasmuchsmalerthanthatincontrolgroup,andthe growthoffocuswasinhibited.undertheelectronmicroscope,theectopicendometrialtisuesinfgntreatmentgroupsand [] [ ] [ ] (No )! Tel: ;E mail:hyylinda@163.com

2 532 damazolgroupshrinked,glandularepitheliumbecamethinner,andthenumberoftheglandsdecreased.ectopicendome trialcelswereshot cylindricaleventabularwithincompactarangement,andapoptosisandinterstitialfibrosiswere observed.eutopicendometriadidn tshowanypathologicalchanges.besides,mitochondrionswelingandlow,disappearedor vacuolatedmitochondrialcristainectopicendometrialcelswereobserved.immunohistochemistryresultsshowedthathigher expresionofbcl 2proteininectopicendometrialtisueinEMTgroupwasobserved.Theproteinlevelofcaspase 3intheec topicendometrialcelsinfgntreatmentgroupsincreased,whiletheproteinexpresionofbcl 2decreased,thuspromoting theapoptosisoftheectopicendometrialcels.conclusion:fgninhibitstheproliferationofectopicendometriumby inducingtheapoptosisofendometrioticcelsthroughactivatingcaspaseactivityandreducingbcl 2expresioninthecels. [KEYWORDS] Endometriosis;Fugongning;Apoptosis. 2(endometriosis,EMT) ), ) [1] EMT= ) )E0, BC = $BC) YJ, Da],-, J! b BC. 2P =!"# [2 4] -./ %!BC 2="#Z, $ % & ', # R D HEST UVWX Y K Z -. / #K 569: 78 <=, FK <LMBCK,(78= ),@A-./01= D E0 ; 1 1 < -./01*+, -Y./ " ml8 2g, g/kg 2.0g/kg <LM (danazol):;: 0<,=>?@J ,DAB5C 7 ml8:; 25mg Caspase 3 Bcl 2D E Y 2 =>?@ A FGH SD34,50I, (200±50)g,* 3$ #JK6L, $%)* #MN # #JO 15d,73 PQ, 34J[ R S 3T J[ R=J[R,RD. [5] U V EMT34 F 1%W XY,.,Z[\].)^`_ 1cm,`a ab cy G =. K 0.5cm 0.5cm 0.5cm3Q=, c \]., c Z Y JI, 3 5B C 4 " =34 40 I,HEIJ 4K, :-./BC 1K -./BC 2K,<LMBCK FK,7K 10I -. / 1K 2KI 1.0g kg -1 d g kg -1 d -1,<LMK 100mg kg -1 d -1, FK 4 9* NO 4 4 DE SZKFD,PH140AF /,LXJ 64 01F KE,SZ 93F J 5A B,BX 41F $] X,Q550CW R I 4 5 8, D 24h,, [ ),I K ].^( )1cm J, 10% 2 5%W D ( ) ) : 3, =Q, 9 =;, 1T, K % 9, I 1T TQ 9 P(` ), mm 3 2.5%W =K D 0 ZQ,UV WX. 56=78[\ 6 #$ F$&' HEGH IJ 48h," 5 Ua ZQ(Q 3~4μm) 5,HEST 7 KLBM 5NOM caspase 3 Bcl 2(),*+ G ZQ, ` K ST,RD SP ST,DAB]T :RD H score [6],* )* STE = ZQ,7 ZQHE 5T ( 400) K = 56, ST ( 6 / 6 1T)=56

533 I I <5%= 56, 0I;5% ~ 10%=56, 1I;11% ~50%=56, 2I;>50%=56, 3I 56 T 56 =ST(1T)J T, 3I; 1TJ, 1I;! =1 TJ4 T, 2I G 56I I ST I,J K PI(,! I = E ) 8 D SPSS13.0 `* E ±!(mean±sd),k E RD "Z!

A:endometrioticlesion,withhighcolumnar epitheliumandvascularhyperemia;b:cysticendom etrioticlesion, withcolumnarglandularepithelium andinflammatorycelsinthecavity;c:eutopicen

05) <LMK FK ) P_Q (P<0.01), D' ) P_Q(P<0.01) FK ', P _Q, `(P<0.05), a) (, 1 * 1 #$ P,-. Table1.EfectofFugongningonvolumeofectopiccystsinrats withendometriosis(mm 3.Mean±SD.

3 533 I I <5%= 56, 0I;5% ~ 10%=56, 1I;11% ~50%=56, 2I;>50%=56, 3I 56 T 56 =ST(1T)J T, 3I; 1TJ, 1I;! =1 TJ4 T, 2I G 56I I ST I,J K PI(,! I = E ) 8 D SPSS13.0 `* E ±!(mean±sd),k E RD "Z!I SNK q##,rd Spearman4$ I ## caspase 3 Bcl 2 <=, P <0.05J! $%& T ) 9 =. / a], 56 56E 0_, 3 Figure1. Endometrialtisuesinmodelgroup(HE staining, 200).A:endometrioticlesion,withhighcolumnar epitheliumandvascularhyperemia;b:cysticendom etrioticlesion, withcolumnarglandularepithelium andinflammatorycelsinthecavity;c:eutopicen dometrium,withafewinflammatorycelsinfiltrating inglandularepithelium,andnondecreasingglands. 4 1?@B%&'BQ 1 #$ P,-. D' K) P! ;D,-./ <LM 34 E$ a]=c0 D -./K) a]_q, FK D' E ]^! (P<0.01); <LMK ]^! (P>0.05) <LMK FK ) P_Q (P<0.01), D' ) P_Q(P<0.01) FK ', P _Q, `(P<0.05), a) (, 1 * 1 #$ P,-. Table1.EfectofFugongningonvolumeofectopiccystsinrats withendometriosis(mm 3.Mean±SD.n=10) Group Beforetreatment Aftertreatment Model ± ± Danazoltreatment ± ± ## Fugongningtreatment ± ± ## Fugongningtreatment ± ± ## P<0.01vsmodel; ## P<0.01vsbeforetreatment. 2 BQ RM X[ FK 9 )*=+9,, J VZ,3 I 56 I, ( [ ), =%, = 9 98-% 1" 56., 1 <LMK a],56,56 /,, 2 -./K,, I) 0_! 1 56a],, % Figure2. Endometrialtisuesindanazoltreatmentgroup(HE staining, 200).A:high columnarglandularepi thelialhyperplasiainendometrioticlesionaroundovar ian;b:glandularepithelialhyperplasiaandinflamma tionbeinginhibitedinendometrioticlesion;c:vari ousdegreesofatrophyineutopicendometrialepitheli umandglands STUVB%&'BQ Figure3. EndometrialtisuesinFugongningtreatmentgroup (HEstaining, 100).A:significantly reduceden dometrioticlesion,withlitleintracavitarysecretion, andcystwalfibrosis;b:atrophyandloweringin partofendometrioticepithelium,andcystwalhemo siderosis;c:abundantglandsineutopicendometri um,withsecretionbutnoatrophy. 4 3 UVB%&'BQ 3 WX Y R UVWX] : FK N, 56, 5, 2' 456 <LMK, 456, I /K 6 _,, 6 _, ST, ;56 78Q 9, 4

534 Figure4.Ultrastructualchangesofendometrialepithilialcels.A:modelgroup( 4000);B:danazoltreatmentgroup( 4000); C:Fugongningtreatment1group( 4000);D:Fugongningtreatment2group( 8000).

01), FK <LMK caspase 3 < ]^! (P> 0.05), 2 1T=4,7 56. 8956 <,. <, 6 K Bcl 2 <5 6(P>0.05); K Bcl 2 <5,-./ 1K 2K Bcl 2 <a] FK(P<0.01) -./ 1 K 2K Bcl 2 <5 a] (P<0.01) FK::, Bcl 2 <5 a] (P<0.

$treatmentgroup( 200). 4 5 %&'BQOM caspase 3(),*+ * 2 ZBF$ #$&'OM caspase 3()*+[\,]^ Table2.Expresionofactivatedcaspase 3proteinineutopicand ectopicendometrialtisuesfromdiferentgroups(mean ±SD.$

4 534 Figure4.Ultrastructualchangesofendometrialepithilialcels.A:modelgroup( 4000);B:danazoltreatmentgroup( 4000); C:Fugongningtreatment1group( 4000);D:Fugongningtreatment2group( 8000). 4 4 %&',WX R 4 OM caspase 3()*+ caspase 3 K = 56 56=6 <, 3 1 T 1T=4, 5 5Q 56 E T K caspase 3 <5 6(P>0.05); K caspase 3 <5,-./ 1K 2K caspase 3 <a] FK(P<0.01) -./ 1K 2K caspase 3 <5 a] (P<0.01), FK <LMK caspase 3 < ]^! (P> 0.05), 2 1T=4, <,. <, 6 K Bcl 2 <5 6(P>0.05); K Bcl 2 <5,-./ 1K 2K Bcl 2 <a] FK(P<0.01) -./ 1 K 2K Bcl 2 <5 a] (P<0.01) FK::, Bcl 2 <5 a] (P<0.05) <L MK Bcl 2 <5 ] ^! (P>0.05), 3 Figure5.Activatedcaspase 3proteinexpresioninendometrial tisues(immunohistochemicalstaining).a:lowex presionin ectopicendometrium from modelgroup ( 400);B:highexpresioninectopicendometrium fromfugongningtreatmentgroup( 400);C:lowex presion in eutopic endometrium from Fugongning treatmentgroup( 200). 4 5 %&'BQOM caspase 3(),*+ * 2 ZBF$ #$&'OM caspase 3()*+[\,]^ Table2.Expresionofactivatedcaspase 3proteinineutopicand ectopicendometrialtisuesfromdiferentgroups(mean ±SD.n=10) Group Eutopicendometrium Ectopicendometrium Model 2.550± ±0.471 Danazoltreatment 2.450± ±0.412 Fugongningtreatment ± ±0.699 ## Fugongningtreatment ± ±0.675 ## P<0.01vsmodel; ## P<0.01vseutopicendometrium. 5 Bcl 2()*+ Bcl 2 < K = 56, 6 6 <, J 3 Figure6.Bcl 2proteinexpresioninendometrialtisues(immu nohistochemicalstaining, 200).A:highexpresion inectopicendometriumfrommodelgroup;b:lowex presion in ectopic endometrium from Fugongning treatmentgroup;c:highexpresionineutopicendo metriumfromfugongningtreatmentgroup. 4 6 %&'BQ Bcl 2(),*+ * 3 ZBF$ #$&' Bcl 2()*+[\,]^ Table3.ExpresionofBcl 2proteinineutopicandectopicendo metrialtisuesfrom diferentgroups(mean±sd.n= 10) Group Eutopicendometrium Ectopicendometrium Model 2.450± ±0.412 # Danazoltreatment 2.500± ±0.459 Fugongningtreatment ± ±0.738 # Fugongningtreatment ± ±0.978 # P<0.01vsmodel; # P<0.05vseutopicendometrium. 6 ZB#$&' Bcl 2 caspase 3*+,_ ` " Spearman4$ I, K bcl 2 = <5 caspase 3 = <5 ; (r=-0.377,p<0.05); K!= < ]

5 535 a EMT $ ` =. K. = =,! J " <" = >? 4 ) [ -,!@B EMT )E0 NA, ' $!BJ, C ' " % D E 6 = c [7], R = ", 56. C, 78=FDG [8]. H?, IJE = K C,J EMT= )L MN 2() )E B B/ 9 NO 3 < :BJ P, QR[. YLQ4 6, &%ST6. UV ')E,BC 3 % &J3 W 2=) )E, c%=!"#,xv % & JB C /=-./01, Y 6 % S= ",* `% " Z[ +, -Y \,?, ; 4. ) ;./ 014 %" &] ;^ 23 _`a Z[ b,c% /`,&% ; H ` ],c `/%` AZ,KZc*,$ = 78=, _Q ), ",;O<" =, 6 2 >!=b> #RD" = Y.,U V EMT F, # a FK, =,6 ;-./ 2T19KE 3 =c0 =,c 0_ 78 # K. E 7 8, F K , [9 12] 6 D-./, 5678 a], 4 [13] +, 2. 2 F = 6 -./K 5678 a]:wx[ ST <, =, 9 78Q =566 = = 6 -./=C E0, Z 56=78; Z, = 56= ;-./6 = Ia], 56= 9, c 56=0_, ) _Q +, K, -./K,EMT Bcl 2 <5 a], F K -./K34 56 caspase 3 = <5 /a], <LMK!! a] G K Bcl 2 caspase 3 <= " Spearman4$ I, K Bcl 2 = <5 caspase 3 = <5 ; (r=-0.377,p <0 05); K!= < ] *,-./ 56= caspase Bcl 2 EMT= <, 56=78, =c0 D Harada4 [14] ` EMT. 78 = #, EMT! 78= Ea][,!![ a], R, K 78 78= EMT = #,-./BCK a]78, a]78, >? 56 56=78, +,@A 'R #,,-./01 ER PR= < c0 `= D [15],<LM EMT34% E 2 P5,-./01 EMT3 4% E 2 P= D, FK% E 2 P89a ] K, a-./ c034 E 2 P= I,< BC EMT= = -./01Y % &, ) ( `=, DQ, `R D, - -./. 5678, <LMC, = E0 E 2 5 [ Z -./ E2= D <LM, $. = D [b c 7] [1].. 2='!+,(S ) [M].S 1. : 3$,2003: [2] P,,,4.STAT3 SOCS3. 2= +,[J]. )**,2012, 28(1): ,158. [3], M,. & =>?[J].,2008,24(10):54

6 [4],,,4. :; <LM 34. 2BC = [J]. c,2012,32(8): [5],1,,4.34 #. 2 F-0[J]. )**,1995,11(3): [6] OtaH,IgarashiS,SasakiM,etal.Distributionofcy clooxygenase 2ineutopicandectopicendometriuminen dometriosisadenomyosis[j].hum Reprod,2001,16 (3): [7] HastingsJM,FazleabasAT.Futuredirectionsinendome triosisresearch[j].seminreprodmed,2003,21(2): [8] Garcia VelascoJA,AriciA.Apoptosisandthepathogene sisofendometriosis[j].seminreprodmed,2003,21 (2): [9],,.. 2 % c >2= c C [J]. c,1991,11(6): [10]DmowskiWP,DingJ,ShenJ,etal.Apoptosisinendom etrialglandularandstromalcelsinwomenwithandwith outendometriosis[j].hum Reprod,2001,16(9): [11] BraunDP,DingJ,ShenJ,etal.Relationshipbetween apoptosisandthenumberofmacrophagesineutopicendo metriumfrom womenwithandwithoutendometriosis[j]. FertilSteril,2002,78(4): [12],,.. 56=78 =+,[J]. =,1999,34(9): [13],U,,4. 2. F34 K 56 78=>?[J]. K $ 56 $,2003,12(3): [14] HaradaT,KaponisA,IwabeT,etal.Apoptosisinhuman endometriumandendometriosis[j].humreprodupdate, 2004,10(1): [15],,,4.-./ H > "b < J[ R=>? [J].,2010,21(11): 櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆 O= J ( ) W $Y J! " 1 S +#A #% $$% $Y&E 10< 1000 ' )**$ $+A$ $+A 11< 9~ ' Y3$ $Y&E 2< 25~ () ' a J*+,-./0$$Y $ $Y&E 10<[1 ) S # )**$Y$% )**+, 10<[ ( `&E $ ( 3 ) V $% $03$ 8< ) J)**$./0$S 18#$ Y+A$ 8 )**$$ :+./0$S ;#$Y3$ $$K $Y$% $Y&E 7< $Y&E 8< 3~ S # <K% $+A$ $Y&E 9< 50 =5> K?)+,$S #@ 3$ $Y&E 6< 30 ~7< 4 50 A BC =5> J*-./0$ CSATVB cd $Y&E 7<[1 C J*+,- E ) $Y$% K J 1 S # )**$Y$% # F 10<[1 100 ( 3 ) 2 V

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Segments Proofs Reference Properties of Equality Addition Property Subtraction Property Multiplication Property Division Property Distributive Property Reflexive Property The properties above may only