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Regulatory effect of Tripterygium wilfordii polycoride (TWP) towards TLR4/ MyD88 independent pathway in TNBS/ethanol ulcerative colitis (UC) rat model

雷公藤多苷对溃疡性结肠炎大鼠TLR4/MyD88非依赖信号通路的作用研究



全 文 :2016
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Vol41,No.6 March,2016
D‚>­ƒ„–<…†‡ˆ
TLR4/MyD88
‰Š‹v<@Nƒhi„…
àák
1,2,
\âã
2,
°äå
2,
æçè
3,
+±
4,
éM

(1
CÃVWz?Z ŒDEWY F…GX

CÃ H5
310006;
2
CÃVWz?Z DEIJWZY

CÃ H5
310053;
3
CÃVWz?Z ŒDEWY KL]^¼—†

CÃ H5
310006;
4
CÃVWz?Z ŒDEWY Ð{X

CÃ H5
310006)

lm
] 
òú„…D‚>
(TWP)
­LðÔ¥ý]
(TNBS)/
²Êƒ„–<…†
(UC)
‡ˆ
TLR4/MyD88
‰Š‹v<@N
ƒêøhi

µiú
TNBS/
²Ê›=[…ƒ€ú¿R
TNBS/
²Ê
UC
‡ˆ5­

5­¿R~

/
90
÷7–
Wistar
‡ˆF
f‘òʸ­\•

5­­\•
,TWP
H

$

1ö•
(3,6,12mg·kg-1),
«Ïþÿ
(AZA)
•
(6g·kg-1),

15
÷


‘_¾.²ó,‹ðÌ[Š
14d。

3d
T=
UC
‡ˆ»¢•/A=
(DAI)。14d
~×}³B‡ˆ

kq²ó<…•i,
&x•‡ˆ<…•i‡’UÛ¢”Üë
,`
­“—nT‘

µi
Westernblot
ú¨
RTPCR
úqu
UC
‡ˆ<…•i$
TLR4/MyD88
‰Š‹v<@N²ô‘3
(TLR4,TRAM,TRIF,NFκB,IFNγ)²mRNA’Ñ ªÝƒÜÝÔg。<Äpÿ,DAI


‡’UÛÜë¨T‘:pÿ
TNBS/
²Ê
UC
‡ˆ5­5
,TWP
­
UC
‡ˆåæÇ®ƒÐђ_`q=DB[
vhi

ñhi†
AZA
²Â²MeŠ°
AZA。RTPCR
’
Westernblot
(:pÿ

†Ê¸­\•²Â

5­­\•$
TLR4/
MyD88
‰Š‹v<@N²ôƒ‘3xã²
mRNA
êéÑ ªÝÜÝ:ãäø
(P<001)。
†5­­\•²Â
,TWP
Á1
öŠ‹–F±ùñv<@Nbxëè‘3
mRNA
’Ñ ªÝƒÜÝ

“$
TWP
1ö•$xëè‘3
mRNA
’Ñ Üݪ
ÝãäH°5­­\•
(P<005)。
†
AZA
•²Â
,TWP
1ö•­ñv<@NbOÕ3
(TLR4,TRAM,TRIF,NFκB)mR
NA
’Ñ Üݪ݃±ùhiÔ$°
AZA
•

ê­ñv<@Nƒwu†ÇÕ3
IFNγmRNA’Ñ ªÝƒ±ùhiÔ!
°
AZA
•

6b$

?±:xâ;d

Õ1

²
TNBS/
²Ê
UC
‡ˆ5­$
,MyD88
‰Š‹v<@N*†ú†Ç•/
ƒêø
,TWP
œì@ò±ù
TLR4/MyD88
‰Š‹v<@N±ù
IFNγƒ+-,mÚe†hi,“hiŠš†1öÁʲô。

nop
] 
D‚>
(TWP);
ƒ„–<…†
(UC);TLR4/MyD88
‰Š‹v<@N

qr)(
] 20151009

stuT
] 
£bø~XZEfij
(81273903)

vwAx
] 
àák

o$WŠ

€

]^*ùúF…MNZ
,Email:qindp19841@sinacom
RegulatoryefectofTripterygiumwilfordipolycoride(TWP)towardsTLR4/
MyD88independentpathwayinTNBS/ethanolulcerativecolitis(UC)ratmodel
QINDanping1,2,ZHOUYijun2,SUNPeina2,CAOJunmin3,ZHANGChunli4,DAIQun3
(1DepartmentofDigestion,FirstAfiliatedHospitalofZhejiangChineseMedicalUniversity,Hangzhou310006,China;
2FirstClinicalMedicalColegeofZhejiangChineseMedicalUniversity,Hangzhou310053,China;
3CentralLaboratory,FirstAfiliatedHospitalofZhejiangChineseMedicalUniversity,Hangzhou310006,China;
4DepartmentofPathology,FirstAfiliatedHospitalofZhejiangChineseMedicalUniversity,Hangzhou310006,China)
[Abstract] InordertostudytheregulatoryefectofTripterygiumwilfordipolycoride(TWP)towardsTLR4/MyD88independent
pathwayinTNBS/ethanolulcerativecolitis(UC)ratmodel,TNBS/ethanolenemawasadoptedtobuildTNBS/ethanolUCratmodel
Afterthesuccessfulmodelingprocedure,90maleWistarratsareweredividedinto6groups,includingnamelynormalgroup,model
·3901·
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group,TWPlow,middle,highdosegroups(3,6,12mg·kg-1)andazathioprine(AZA)group(6g·kg-1),with15ratsineach
groupAlratsineachgroupwereadministratedwithcorespondingmedicinesfor14daysAfter14daysofadministration,core
spondingcolontissuesweretakenforgeneralandmicroscopicevaluationWesternblotinganalysisandRTPCRwereadoptedtotest
themRNAandproteinexpressionsofTLR4/MyD88independentpathwayrelatedmolecules,namelyTLR4,TRAM,TRIF,NFκBand
IFNγTheresultsshowedthatDAI,generalandmicroscopicevaluationsalindicatedthatTNBS/ethanolUCratmodelwassuccess
fulTWPcanimproveUCrelatedclinicalmanifestationandhealcolonicmucosa,whichwasequaltoAZARTPCRandWBresults
showedthattheexpressionofTLR4/MyD88independentpathwayrelatedmoleculesinmodelgroupweresignificantlysuperiortothatin
normalgroupateithermRNAorproteinlevel(P<001)Comparedwithmodelgroup,TWPcaninhibittheexpressionofeachnode
inTLR4/MyD88independentpathwayinadosedependentmannerTheinhibitoryefectofTWPwithhighdosetowardstheabovemol
eculeswasinferiortothatinmodelgroupateithermRNAorproteinlevel(P<005)TheinhibitoryefectofTWPwithhighdoseto
wardsupstreammoleculesofTLR4/MyD88independentpathway(TLR4,TRAM,TRIF,NFκB)wasslightlysuperiortoAZAgroup
ateithermRNAorproteinlevelHowever,suchinhibitoryefecttowardsterminalinflammatorycytokines(IFNγ)wasinferiortoAZA
groupateithermRNAorproteinlevelAltheabovediferenceshadnostatisticalsignificanceTherefore,inTNBS/ethanolUCrat
model,TLR4/MyD88independentpathwaytookpartinregulatinginflammationTWPexerteditsantiinflammationefectbyinhibiting
theexpressionofTLR4/MyD88independentpathwayinadosedependentmanner
[Keywords] Tripterygiumwilfordipolycoride(TWP);ulcerativecolitis(UC);TLR4/MyD88independentpathway
doi:10.4268/cjcmm20160620
  
ƒ„–<…†
(ulcerativecolitis,UC)
†Õ²4
5±¸²ô
[1],
åæi,ª’Ôª]ù1

ÅK
´ÒO,

A9bê{Xi45±ù1
[2],
”«
Ïþÿ
(AZA)
ö

6
AZA
ÖüLhiò蒱ù
ö

†’Ôª]ù1›iO

ýtm°è’±ù

Cùú“²åæ$ƒói

ê$,D‚>
(TWP)
i°Õ²45–»¢œwaQ
[3],
ê
TWP
œ
UC
ƒk.Àu

åæ2ãÿ“DBÃ$
‚

6“hifù£{

Tol
‚“
(TLRs)
é`Ñ üÓ$ƒ[

ґQ_¢Ð²ô‘3
,`
æç»B45óä

†
UC
ƒm¢§%²ô
[4]。
²
UC
«˜8

…
bK»BòÄÅ$
Tol
‚“
4(TLR4)
Ü݇
#š‰
[5],
ê
TLR4
q$é
TLRs
$ƒ[?`Ñ
 “

71ù{
TLR4
v<@N²
UC
m¢$
$%Öü™K

,ì€ÒÕ3†
TLR4
“&véҕ»B4
5õâƒà[H
,TLR4
v<@N[éçÿüèVÑ
 ’‚‘_Õ3
88(MyD88)
æç

Öüò
MyD88
Š‹¨‰Š‹ƒv<@N
[6]。
íî
TLR4/MyD88
Š‹v<@N²
UC
m¢$ƒfù„…wã
[7],
ê
TLR4/MyD88
‰Š‹v<@N

ÑW
TLR4/TRIF
v<@N

7°7¦œÆ°‡öƒ
IFNγ,ê IFNγ
ˆ†<…†Ç•/DB²ô–
[8],
Õ1

N^Ç¥
@òLðÔ¥ý]
(TNBS)/
²Ê
UC
‡ˆ5­hò
„…e

„…
TWP
ƒe†hi’e†fù


„…
TWP
9:
TLR4
ƒhi

ˆœ—[H{‚
TLR4/MyD88
‰Š‹v<@N²†Çêø$ƒh
i
,`
#$
TWP
éç­1êøDB9:hi

˜ê
òåæói’~̃„…p)ÔäJ¸

1 
ʸ
SPF^

Wistar
‡ˆ
90
÷

Ö
(200±20)
g,7
±8

72$A,‡/‹$Sp)

/‹=
þÌ<
2007000556038,¸
ý·¥

D‚>ü

2¸„ù,BCDE

°Æ
o<
1311108B);
«Ïþÿü
(aspen,
°Æo<
008953)。
5% 2,4,6
LðÔ¥ý]
(5% TNBS,Sigma
D
E
);10%
ª=»¨

2˜$A%
);
xª²Ê

R
xLÙÀÄ_gBCDE
);
$S›œç

bHÝ
yAjïBCDE

B|/<
YZB/USA3966
2011);TLR4,TRAM,TRIF,NFκB,IFNγPCR2‹
(Takara
DE=
);βactin(cst,4970S);TLR4e
(abcam,ab22048),TRAM

(R&D,AF4348),
TRIF

(abcam,ab13810),NFκBe (cst,
8242S),IFNγe(R&D,AF585NA);p8e,œ
8e

UœÁ’Uœ¢
(Biorad);RNA
+p¥1µ
(lot
<
9767,Takara,Shiga,Japan);
Òá\¥1µ
(lot
<
RR036A,Takara,Shiga,Japan);PCR
¾Á
(AppliedBiosystems,FosterCity,CA,USA)。
·4901·
m@ö

D‚>­ƒ„–<…†‡ˆ
TLR4/MyD88
‰Š‹v<@Nƒhi„…
2 
€ú
21 
/‹‘•
 
‘_/
90
÷úÎ
Wistar
‡ˆ’w
Ff‘ò

•

‘_òʸ­\•

5­­\•

TWP
H1ö•
、TWP
$1ö•
、TWP
1ö•
、AZA
•


15
÷

22 
5­¿R
 
‡ˆ¬ó–Û¥

±¢s

XÖ
Ýr
(200±20)g,
ŠÍ
24h。10%
ª=»¨
3mL·
kg-1
5y•–~

/Ù(ö)îòƒ!›œØ7
v7*>*<…+
8cm
I

5­­\•¨,‹•
[|÷‡ˆ
0002mL·g-15%TNBS+025mL
50%
²Ê—n[…

ʸ­\•[
0002mL·g-1
°”ªª
+025mL
°”ªª—n[…

‰{
03mL
öÛ

i77+⇈v7

¶.‡ˆ5
1
1min`
ÞØ
5min,
5
՟
Ȫ~¸ýûÍ

5
3d
~¿R‡ˆ
UC


23 
¾,
 ①ʸ­\•、5­­\•:¾.001
mL·g-1
°”ªª[Š
;②AZA•:/ AZA„Ä
w

²Äw$‰°”ªªù9d¢

[‡ˆ
AZA
60mg·kg-1
ƒ1öù|
1mL
sB
06mgAZA
9d¢

ì
001mL·g-1
ƒ[Šö[Š
;③TWPH、
$

1ö•

/
TWP
„Äw

²Äw$‰°”ª
ªù9d¢

[

•‡ˆì
TWP30,60,120mg·
kg-1
ƒ1ö‘_ù|
1mL
sB
03,06,12mg
TWP
9d¢

ì
001mL·g-1
ƒ[Šö[Š

ìb
,‹|N

]

ðÌ
14d。
24 
,&óí
 
|{,&‡ˆÖ

ßm

ûÍ

‡
Íö[UÔg
,`
—n,¡¥(’
DAI


25 
BNµÀ
 
w]¾,
24h
~

Iɇˆ

ªÊ
¸­\•,

:kq¢¯7{ãI<…•iBN—
n²ôqu

26 
<…†ÇTU
 ①»¢•/A=(DAI)T‘:
[Maines
ö
[9]
p$ƒB|

@ò<=5
14d
~
‡ˆÖÛî¬

‡Í–®’‡Í,¡Ôg—nT
‘
;②‡0ؗ˜T‘:*\Walaceö[10]p$ƒ
B|—nT‘
;③UÛ<…—˜T‘:q¢¯•i—
n
HE
hK

[Geboes
ö
[11]
ƒT‘B|—nT‘

27 RTPCR
úuv
 TLR4/TRIF
v<@N²ô‘
3
mRNA
ªÝÜÝÔg³qBN[
RNA
+p¥1µ
ù{rpqu
RNA,¸
,‘¢¢š;uvu
RNA
Ë
š

Öuv

]

q
500ngRNA
[Òá\¥1µù
{r—nÞá\Òó=
cDNA,
i
ABI7500PCR
Á
[
RTPCR
ù{r/
cDNA
—n
PCR
¾

ìβactin
ò8*

ì
95℃
:¯–
30s,
Ÿ~—nNM¾
,95℃
¯–
5s,60℃
áé‰<ê
30s,
a
40
NM

Òó~—n#×Ðȑ›

µi
2-ΔΔCt
ú‘›íƒÔՃ
²­ÜݪÝ

xv<Ñ ‘32‹ÔgÜ
1。
Ü
1 
xv<‘32‹Ôg
Table1 Primersforsignalingmolecules
v<Ñ  bO2‹
(5′3′)
ÛO2‹
(5′3′)
βactin GGAGATTACTGCCCTGGCTCCTA GACTCATCGTACTCCTGCTTGCTG
TLR4 CTCACAACTTCAGTGGCTGGATTTA GTCTCCACAGCCACCAGATTCTC
TRAM ACACTGCCTTTGGGACACGA GCCTAAGTCAGCAGTGGGTAGGAG
TRIF GCCAGCAACTTGGAAATCAGC GGGGTCGTCACAGAGCTTG
NFκB CATGCGTTTCCGTTACAAGTG GTGCGTCTTAGTGGTATCTGTGCT
IFNγ CATCAGCAACAACATAAGTGTCATC CATTGACAGCTTTGTGCTGGA
28 Westernblot
úuv
TLR4/TRIF
v<@N²ô
‘3Ñ ªÝÜÝÔg
 
/³q…•iBN„è§
Ç~

‰

¦1öƒÞÑ Ï×¢
[20mmol·L-1
Tris/HCl(pH75),10mmol·L-1APMSF,1mmol
·L-1EDTA,1mmol·L-1DTT]
ù**¢

q[v
öÑ b‚

°
10%
š®à¯3EFUœ‘•

‘
•ƒÑ ´áâl
PVDF
`
。5%
±W-ä.
2h,
‰
TLR4(1∶500),TRAM(1∶1000),TRIF(1∶
500),NFκB(1∶1000),IFNγ(1∶5000)—ãäe

,4℃
f¾ò
。TBST
ß`
5min×3
]

‘_‰
²óæ¢8e
(1∶15000),¸
-*>ħ
2h,
TBST
ß`
5min×3
]

µi
Odyssey
¿¢f—n
q6ãµ
,Odyssey
錗nq6=.‘›

íƒÑ
 ƒÜÝ2òíƒÑ ƒ­§š2Âb βactinƒ
­§š2

29 
â;€ú
 
µi
SPSS170
â;éŒI”
=.

=.ì 珋x±s0£Üÿ,x•=.sµi—Õ
O€‘›q(
,P<005
ò±Bâ;d

·5901·
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Vol41,No.6 March,2016
3 

31 UC
‡ˆ<…†ÇT‘
 
œ
14d
~

†Ê¸
­\•²Â

5­­\•‡ˆ
DAI,
‡0ؗ˜’
•iT‘{㏉
(P<001);
†5­­\•²
Â
,TWP
1ö•’
AZA
•<…†ÇT‘:B{
㏉
(P<001);
†
TWP
1ö•²Â
,AZA
•
<…†ÇT‘xã䖏±

Ü
2。
Ü
2 TWP
­
TNBS/
²Ê‡ˆ<…†ÇT‘ƒµ¶
(珋x±s,
n=11)
Table2 EfectofTWPtowardsinflammationscoreinTNBS/
ethanolUCrats(珋x±s,n=11)
•_
DAI

‡0Ø
—˜T‘
<…_`•i
—˜T‘
ʸ­0 0 0
5­­160±0761) 540±2211) 1327±3501)
TWP
H1ö
101±039 400±204 1000±392
TWP
$1ö
113±030 580±218 782±3253)
TWP
1ö
087±0252) 393±1942) 545±2732)
AZA 07±0282) 364±1742) 700±2572)
  
 

†Ê¸­\•ÂÃ
1)P<0001;
†5­­\•ÂÃ
2)P<
0001,3)P<005。
ʸ­\•…Ýx/ð

}Ø_`ô0€”È
†

x12¨ƒ„öÜë

5­­\•…݆±P•
i/ð

…$

}؜ƒ„3

ƒ„¢¯I…
ݏJ

±ˆ_`Ù¡ª/
;TWP
H1ö•…݆
±P•i/ð

…$

…ݯJ

1‘}؜
ƒ„

123

o1Ù¡

ª/
;TWP
$1ö•…Ý
†±P•i¶š/ð

…ݏJ

}Øܝ­:ƒ
„

¼_`Ù¡ª/
;TWP
1ö•
、AZA
•…݆
±P•iuB/ð

…b{ã$

…ݶš
J

}Øb{ツ

B¶š_`Ù¡ª/

q
1。
A
ʸ­\•
;B
5­­\•
;CTWP
H1ö•
;DTWP
$1ö
•
;ETWP
1ö•
;FAZA
•

q
2,3
¤
)。
q
1 
bu<…•i‡q
Fig1 Generalpicturesofdistalcolontissue
ʸ­\•

5­­\•œ<…_`ª/

_
`ò’¥Žòœ>öZ|ÄÅ

¥ÄŒ$–½
ÄÅa)

ƒ„0

ƒ„Ü÷œÌÉò

zL•
i0
;TWP
H1ö•Üœ_`Ûòƒ„


½ÄÅ

Z|ÄÅö†–ÄÅa)C’ŽòÀl¥


1‘œ,4²Ì
;TWP
$1ö•_`Öü
Üëò֚>–†Ç

$–½ö†–ÄÅa)C’
Žò

,4²Ìu
;TWP
1ö•†
AZA
•_`
ÖüÜëò$š>–†Ç

1‘_`’_`ÛòB
uö†–ÄÅa)

q
2。
q
2 
bu<…•i¢”q
(×200)
Fig2 Pathologicalpicturesofdistalcolontissue(×200)
32 Westernblot
úqu
TLR4/MyD88
‰Š‹v<
@N²ôÑ ƒÜÝ
 
­
TLR4/MyD88
‰Š‹v
<@Nƒxëè‘3Ñ ÜݪÝ
(TLR4,TRAM,
TRIF,NFκB,IFNγ)êH,†Ê¸­\•²Â,5­
­\•$þÖüëè‘3ƒÑ ÜݪÝãäø

(P<001);
†5­­\•²Â
,TWP
Á1öŠ‹
–F±ùñv<@Nbxëè‘3Ñ ªÝƒÜ
Ý

“$
TWP
1ö•$xëè‘3
mRNA
ÜÝ
ªÝãäH°5­­\•
(P<005);
²†(–­

AZA
•ÂÃ
,TWP
1ö•­ñv<@Nb
OÕ3
(TLR4,TRAM,TRIF,NFκB)Ñ Ü݃±
ùhiÔ$°
AZA
•

ê­ñv<@Nƒwu†Ç
Õ3
IFNγÑ ªÝƒ±ùhiÔ!° AZA•,
6b$

?±:xâ;d

q
3,4。
33 RTPCR
úqu
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yg?z

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