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Effect and mechanism of danshensu on hepatitis B virus reverse transcriptase and antigen expression

丹参素靶向乙肝病毒逆转录酶抗乙肝及抗原表达影响机制研究



全 文 :2016
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Vol41,No.7 April,2016

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EfectandmechanismofdanshensuonhepatitisBvirusreverse
transcriptaseandantigenexpression
DUANShupeng1,ZHULihong2,LIPeng3,SONGXinwen1,WANGHongwei1,SHENBaosheng1
(1.DepartmentofInfectiousDiseases,theFirstAfiliatedHospitalofXinxiangMedicalUniversity,Xinxiang453100,China;
2.DepartmentofObstetricsandGynecology,theFirstAfiliatedHospitalofXinxiangMedicalUniversity,Xinxiang453100,China;
3.ColegeofPharmacy,XinxiangMedicalUniversity,Xinxiang453003,China)
[Abstract] MTTassaywasusedinthisstudytoinvestigatetheinhibitoryefectofdanshensuontheactivityof2215celsamong
humanhepatomacelline(HepG2);indirectfluorescencelabelingmethodwasusedtomeasurethechangesofreactiveoxygenlevels
inthecels;ELISAmethodwasusedtodeterminehepatitisBsurfaceantigen(HBsAg)andhepatitisBeantigen(HBeAg)levelsin
celularsupernatants;HBVDNAlevelwasmeasuredwithfluorogenicquantitativePCRmethodTheinhibitoryefectofdanshensuon
HBVRT(hepatitisBvirusreversetranscriptase)wasstudiedbyusingenzymeinhibitiondynamics,andtheefectofdanshensuonsec
ondarystructureofHBVreversetranscriptasewasmonitoredbyusingcirculardichroismTheresultsshowedthatdanshensuhadagood
inhibitoryefectonthegrowthofHepG2215cels,withahalfinhibitoryconcentration(IC50)of(1535±243)μmol·L
-1;dan
shensucouldsignificantlyinhibitHBsAgandHBeAgexpressions,andshowedaninhibitoryefectonHBVDNAreplicationInaddi
tion,danshensuwasanefectiveinhibitorforHBVreversetranscriptase[IC50(2132±243)μmol·L
-1]Thefluorescencelabeling
resultsshowedthatthereactiveoxygenlevelsinthecelswereincreasedwiththeincreaseofdanshensuconcentrationCirculardichro
·7921·
2016
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Vol41,No.7 April,2016
ismanalysisshowedthatdanshensucouldinducepartialchangeofconformationofHBVreversetranscriptaseandgradualyincreased
αhelicalcontentTheseresultsindicatedthatdanshensucouldmakethestructureoftheenzymebecomecloserbybindingtoHBVre
versetranscriptase,whichwasnotconducivetotheformationoftheactivecenter,soitcouldfinalydecreasetheactivityofHBVre
versetranscriptaseSuchdecreaseinenzymeactivitywoulddirectlyafecttheHBVDNAreplication,andcombinedwiththedecrease
oftheantigenlevels,theefectofdanshensuonHBVwasincreased
[Keywords] danshensu;HepG2215cels;reversetranscripataseofHBV;reactiveoxygenspecies;secondarystructure;inhibi
toryefect
doi:10.4268/cjcmm20160722
  
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(珋x±s,n=3)
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Ãß
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Ž}ìH
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ÜÝÛ
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10 1144±1312)
25 2454±2452)
50 4239±3612)
100 6518±6112)
­`â
10 1187±1662)
  
å

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1)P<005,2)P<001(
*

¦
)。
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HBV

 
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,Y

¿[⾌C8

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HBsAg
¬
HBeAg
*s

ÓQÝ9ˇ—`•
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6234%;
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10,25,50,100μmol·L-1ž@ì
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2。
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2 
ÜÝÛì
HepG2215
Za
HbeAg,HBsAg
¬
HBV
DNA
¢%±òó
(珋x±s,n=3)
Table2 EfectofdanshensuonHbeAg,HbsAgandHBVDNA
levelsofHepG2215cels(珋x±s,n=3)
`
Ãß
/μmol·L-1
9ˇ
/%
HBsAg HBeAg
Log
(HBVDNA)
Ž}ìH
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10 1732±1432) 1956±2052) 422±0561)
25 2758±2552) 3012±2782) 405±0551)
50 4843±4762) 5447±4892) 387±0351)
100 5718±6512) 6234±6682) 365±0451)
­`â
10 1232±2072) 1544±2132) 425±0581)
24 
ÜÝÛì
HBV
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Ü
ÝÛì
HBV
nopq±9Ë0ž]q
2,
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Ûž±øÅ
,HBV
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Þß

6äÜÝ۱žsä
100μmol·L-1Y,
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nopq±€ìðµÞŸ%y

7àÓ1=9
Ëž
(IC50)•(2132±243)μmol·L
-1,
Fݟ
·9921·
2016
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Vol41,No.7 April,2016
™ µ ì H ­ ` â ±
IC50(1644±189)
μmol·L-1,hidÜÝÛì HBVnopqBÕ÷
±9ËÉæ

q
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HBVDNAP
±9Ë0ž
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Dichroweb
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ÝۜVíY±€^œ÷ëß

Ͼ]*
3。
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HBV
nopq± α7ÓÞߗ=
•
356%,β§hÞߗ=• 200%,ɘQ±
α7ÓQ|}Ä,”lHBVnopq$ÅOÜÝÛ
±Å¾sä
100μmol·L-1O,α7ÓÞߗ=øÅ
ä
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nopq±€^œ÷±òó
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RTinthepresenceofdanshensu
ÜÝÛ
/μmol·L-1
α7Ó
/%
β§h
/%
βo¾
/%
r‹{Å.
/%
0 356 200 206 238
10 406 179 215 200
50 444 128 224 199
100 485 89 232 194
3 
Ús
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p¸¹±mÂ

¹;_üt£ê¼a
RNA
•P
TV–
DNA,
ýY_–[PZ[P±
DNA,
ýY
HBV
üÕ
DNA
ÖnM_

Q¸Ø–
HBVDNA
±æ
Ë
[9],
ba1Ÿ
HBV
nopq±0žfË

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HBV
nopq•0žkë

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m-B./d0

HepG2215
ZaɶëB
HBV
1à±.
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HepG2
Za
[10],

G418
:;<
ä±µÖ

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ÉÀí
ôm‚,˜°ð~Qwž±PQ
[11];ROS
ÉÊZ
a8tP›¸¹$ã±-B„ݘðµ±t—
ï
[12]。
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t
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ïð

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[1] 
D<

IA=

!>Š

ÜÝÛ±,SðµÑ,˜¶ñ«¬
Ö×
[J].
K2,-.
,2011,26(4):310.
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I,v

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?xO

ÜÝÛdeS¢3ÅƱ,S0ž
fË«¬Ö×
[J].
$,Š
,2014,45(17):2571.
[3] 
¦ü

DñÆ

@f 

è

ÜÛì8hÛµ‚ƒ„±2e0
žÊÓfË
[J].
KAœV‚O-.
,1999,9(2):30.
[4] 
E~0

dí

I^^

è

ÜÝÛ$ˆ‚hi_ÊÓ0ž
fË«¬
[J].
$Ƃ¾O-.
,2003,11(5):288.
[5] 
À?ú

P¥V

?^Û

è
.5
$,#֘ìAmQ‚0
Ohnopq¬
DNA
KVq±9Ë0ž
[J].
$:Cl
,1997,13(2):71.
[6] FabrizioA,AliceF.LiposomesandMTTcelviabilityassay:an
incompatibleafair[J].ToxicolInVitro,2015,29(2):314.
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