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Protective effects of oxymatrine against H2O2-induced damage in L02 cells

氧化苦参碱对H2O2诱导L02细胞损伤的抑制作用及其机制的研究



全 文 :2016
y

z ³
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Vol41,No.7 April,2016

”•)(
] 20151218

–—˜R
] 
ì(813300486)

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] 
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t_Tݾì
H2O2\] L02Zaƒ„±
9Ë0žÊÓf˱«¬
÷øù
1,2,
2ú¦
2,
ûJJ
1,2,
8Mä
1,2,
küý
2,
þÿ!
2,
S.
2,
#0$
2,
I1
2,
UÓÔ
2,
ÑÒ®
2,
ÕÖ×
2
(1.
8}#%q

×} 8}
067000;2.
þÿ±
302
#q ±!#$€’

}~
100039)

›
] 
|^«¬t_Tݾ
(oxymatrine,OMT)

H2O2\]±L02Zaƒ„±0žÊÓfË。a$?w‚çÞZaL02
•«¬ì-


H2O2\]t_‚ˆ€‚ƒ„PQÖnçƒ,@¸CCK8‚áOMTìL02Zaðµ±òó;CSFE23ç
ƒ‚á
OMT
ì
L02
Zaø™±òó

GóZa)‚á
OMT
ì
H2O2\]± L02Zaë/‡±òó;DCFHDA2345‚á
OMT
ì
H2O2\]±L02Za8ROSëß;ÓTñ(‚áOMTìGSHPX¬SODðµ±òó。œæhi,OMT¿625~
100mg·L-1
@¸\]
NADPH
±ãÂ

øe
L02
Za8
GSHPX

SOD
q±ðµ

Ö§Ö
GSH
×]±ðµt
ROS
±ª
†

¦§9Ë
H2O2\]±L02Zaë/±mÂ,sä9ËH2O2\]L02Zaƒ„±0ž。

œž
] 
Ò­Ø

t_Tݾ
;H2O2;t_‚ˆ;¸ t_˜q
ProtectiveefectsofoxymatrineagainstH2O2induceddamageinL02cels
HANYanzhong1,2,ZHOUYongfeng2,SANGXiuxiu1,2,LIUHuimin1,2,CUIHerong2,
MENGYakun2,LIGuangquan2,HELanzhi2,YINPing2,WANGJiabo2,BAIZhaofang2,XIAOXiaohe2
(1.ChengdeMedicalcolege,Chengde067000,China;
2.ChinaMilitaryInstituteofChineseMedicine,302MilitaryHospital,Beijing100039,China)
[Abstract] Toinvestigatetheprotectiveefectsofoxymatrine(OMT)againstH2O2induceddamageinL02celsandresearchthe
mechanism,L02celswereusedastheresearchobject.TheoxidativestressmodelofL02wasestablishedbyhydrogenperoxide
(H2O2)CCK8wasusedtodetectthecelactivationofL02celstreatedbydiferentOMTFCM(flowcytometry)assaywasusedto
evaluatethecelproliferationofL02celstreatedbyOMTTheapoptosisofL02celswasdetectedusingAnnexinV/7AADapoptosis
detectionkitThelevelofROSwasdetectedbyDCFHDAfluorescenceprobeTheGSHPXandSODweredetectedbymicroplate
andcolorimetricmethodResultsshowedthatwhentheconcentrationofOMTisbetween625and100mg·L-1,itcouldpromotethe
productionofNADPHandstrengthentheactivityofGSHPXandSODtogetridoftheROStoprotecttheL02celfromtheapoptosis
ofL02celinducedbyH2O2
[Keywords] Subprostratesophora;oxymatrine;H2O2;oxydatvestress;peroxydase
doi:10.4268/cjcmm20160723
  
ÎyςOm‡±Ñy÷ÜîÍá«+±Ó
ԁå

É»%Àíò•Ô.±D”&’“ý[

‚O±mÂLÌ+Íá‚çÞZa±ƒ„

§ðµ
tÍm±t_‚ˆZɕ‚Om±OS1
i
[1],
›%4‚

}~‚

Ohµ‚0

‚hi_
è
[25],
àït_‚ˆ¿‚OmÂm×$±0ž¥ê
·2031·
ßxéè

t_Tݾì
H2O2\]L02Zaƒ„±9Ë0žÊÓf˱«¬
:G

ҭؕ­&Ž˜â9Ê
Sophoratonkinensis
Gapnep
±j©Ø¬Ø@

É[ª«7hÄ$,

TݾĘÞÉҭرL/_–—ý[
[6]。
«¬m±

TݾĖ—-BÕ÷±d‚äq0ž

{20•ràÍ᱂ƒ„
,¨
›t_TݾM
a9ËAû_!Íá±_µ‚ƒ„
[7]、
Hmµ‚
ƒ„
[8]、
é¢WÆ傃„
[9]、
Ohµ‚ƒ„
[10]。
§÷gxÄQ±‚ƒ„xfËþ€ßòó

I
‰æ-

¨›%p÷Dô±%4‚mOfË

ۼ
аé
”:
ɉ¿t_‚ˆ¬®|Zaàï


œ‘”¦0ž

€ßòó@

Íá%4µ‚0

Ö§
mז‚hi_¬‚1_±¸¹
[1]。
ÎyÏt_
Tݾd‚0ž±«¬L/¦.ŸÓ®|˜Ï


ôt_d‚0ž±kÚòê

9«¬@¸
€
H2O2\]± L02Zat_‚ˆ‚Zaƒ„
PQ

«¬t_Tݾ±ôt_0žÊÓdef
Ë

•ÇO«¬Ó¿•‚O$ˆ÷±‚ž#G
·>

1 
ŠÌ
?w$‚ZaÔ
L02
Zaߕìa*]*:Â
˜A,()BCDE

žë
10%
†í¢ª±
DMEM
Øw4½1¿
37℃,5% CO2åm¬\¾Ò
J@Önw‹4½

DMEM
Øw4½1ߕ
Hyclone
DE

†í¢
ª

«|}q

öŽÛ¬ÕŽÛuߕ
GIBCO
DE

¸t_ý
(H2O2)ߕSigmaDE;CelCountingKit
8(CCK8)
ߕ5£_&(

÷H

BCDE
;AN
NEXINⅤFITC/7AADapoptosisdetectionkitߕ‘
%¢µ(DE
;CFSECelDivisionTrackerKit
ߕ
Biolegend
DE

t_Tݾ
(OMT)
ߕ$%ò¤,
¤‚⫬>

Õt_˜¨_q
(superoxidedis
mutase,SOD)
ðµ‚áé

ðµt‚áé
(reactiveoxygenspecieskit)、BCA
|}ž‚á
é
(BCAproteinassaykit)、
ýþùY¸t_˜q
‚áé
(celularglutathioneperoxidaseassaykit)
uߕbH˘()DE

q“
(BioTekSynergyH1H1MFD,
‘%
);
€
t_!4½Z
(NAPCOModel5410,
‘%
);
Ãót
öžSf
(TD5A,
@9[K“w½)BCDE
);
G
óZa“
(BDFACSCantoⅡ)。
2 
œ
21 L02
Za±4½
 L02
Za4½©Hw‹4
½±œ

ë
10%
†í¢ª
、100kU·L-1
öŽÛ¬
100g·L-1
Վ۱ݣ4½1
DMEM,37℃,5%
CO24½Z4½。
22 CCK8
‚á
 OMT
ì
L02
Zaðµ±òó•
D…†
OMT
ì
L02
Zahµ±ÃߌC


L02
Z
aa
70×104

/mL
zä
96
&T

|&
100μL,

12h
Zaw15ºY

—`~Þßž•
3200,1600,800,400,200,100,50,25,125,625,
312,156mg·L-1
±
OMT,
烻aµìH&¬
Ž}ìH&

å|Å¾@»

æ&
,24h
Y
CCK8
‚á
L02
Za±øy‡

Zaøy‡

(As-Ac)/(Ac-Ab)×100%,ó$ Ac•ìH&©3
¾
;As•çƒ&©3¾;Ab•Ž}&©3¾。
23 CSFE
Ê(‚á
OMT
ì
L02
Zaø™±ò
ó
 

CSFE
Ê(ì
L02
ZaÖnÊ(

Ê(
¸¹Ôè©HéúrÖn


L02
Zaa
10×
105

/mL
zä
12
&T$

|&
1mL,

12h
Y—`~Þßž•
50,25,125,625mg·L-1
±
OMT
ì
L02
Za\ˆ

|Å¾@»

æ&
。24

6_Za

¶6_@ϱZaÖnGó‚á

24  L02
Zat_‚ˆPQ±€
 
¶Å¾•
70×104

/mL
±
L02
Zazä
96
&T

|&
100μL,z 12hZaw15ºY,» 08,07,
06,05,04,03,02,01mmol·L-1
ž־±
H2O2ÖnìP,烻aµìH&¬Ž}ìH&,
å|Å¾@»

æ&

—`¿ìP
05,1,2,3,
4h

PBS
ªœ

’


CCK8
‚á
L02
Za
±ð‡

çƒ.æ

’

Za±ð‡
=(As-
Ab)/(Ac-Ab)×100%。
25 ANNEXINFITC/7AAD
‚áë/Za
 
ü
t¢€t_ƒ„PQ

MaøÅa8
ROS
¢%

Ö§mÂa8t_‚ˆÖ‚

Q¸]cZa±¹™
µ./

¹ë/
[15],
àï9çƒ6zpžGóœ
‚áZaë/ϊú
OMT
±ôt_0ž


L02
Za©H
10×105

/mL
zä
12
&T$

»Ž}


PQ¬~,

|&
2mL,

12h
Y

PQ
¬~,—`©H
24
¿@ìPÒJìP
,05h
Y

~,—`~Þßž•
50,25,125,625mg
·L-1
±
OMT
ì
L02
Za\ˆ

~,
24h
Y


L02
Za6_@Ï

©H
ANNEXINⅤFITC/7AADéú
rœÖnGó‚á
L02
Zaë/žŸ

26 L02
Za8
SOD,GSHPX
ðñáâ
 
ž

&
·3031·
2016
y

z ³
41
ų

}
Vol41,No.7 April,2016
T4½
L02
Za

—

ìP

~,ݒ

QY|&Å
200mLPBS,
ÖæøjÝ7Za

ÌY

Ô
r·
min-1
žS
10min,
Ö÷ª

©Hééúráâ
©3¾
(A),
¬x-
SOD,GSHPX
ðñ

27 L02
Za8
ROS
ëßáâ
 
ž
12
&T4½
L02
Za

—

ìP

~,ݒ

QYªœZa

©
Hééúr͞rí½|45±œÖna8
Ê(

Ê(
20min,
ž
PBS
ªœ

º

Øðž«q6
_

ÅØw4½1¸06_
,1500r·min-1
žS

t
ֻ

Q¸ž
200μL± PBS.4。¿ˆmVË488
nm,
mÆVË
525nm
@ÖnGó‚áa823e¾

28 
Æx—à
 

SPSS200
Æx[JÖn
S

þÔÕpž

‚ƒÖnS
,P<005
*iÔ
Õ-BÆxd0

3 
Ͼ
31 CCK8
‚á
OMT
ì
L02
Zaðµ±òó
 
OMT
0žŸ
L02
Za
24h
Y
,OMT
Þßž¿
32~100mg·L-1
Oøe
L02
Zaðµ

Œ125mg·L-1

L02
ZaðµQe
;OMT
ÞßÅ
¾¿
400~3200mg·L-1

L02
ZaBhµ0
ž

¬åÐÑ
OMT
ž±øÅhµ0žøe

q
1)。
1Ÿ÷gœæ

9çƒpž
625,125,25,50
mg·L-1
±Þßž־ÖnY,ƒ

q
1 
t_Tݾì
L02
Zaø™ðµ±òó
Fig1 EfectofOMToncelproliferationrateofL02cel
32 CSFE
Ê(‚á
OMT
ì
L02
Zaø™±ò
ó
 
Ø>
CSFE
23±º&¹¾hi

Ž}ìH
¿
24h
8•Ìø™

P
,L02
ZaG¥¦Å¾
OMT

24h
Y


OMT
ž@±
L02
Zaç
ø™

P

ю}€ñ

bd±ø™:


xÔÕ

éú
OMT
¥{Ö
L02
Za±ø™

q
2,
*
1)。

CCK8
‚áœæ¬rS


CSFE
‚áœæ


OMT
MaÖ
L02
Za8
NAD(P)H
±øÅ

1
Ž}
;2
Ž}
24h;3~6
—`•
24h
O
,625,125,25,50
mg·L-1
t_Tݾ

q
2 
t_Tݾì
L02
Zaø™‡±òó
Fig2 EfectofOMToncelproliferationofL02
*
1 
t_Tݾì
L02
Za23e¾ _±òó
(珋x±s,n=3)
Table1 ThefluorescenceintensityofL02celsstimulatedby
OMT(珋x±s,n=3)
`
Ãß
/mg·L-1
23e¾
0h 24h
Ž}
    - 1547 1865±15
t_Tݾ
625 1547 1840±20
125 1547 1835±15
25 1547 1840±20
50 1547 1860±15
33 L02
Zat_‚ˆPQ±€
 
9«¬HQ
¦¤
H2O2ž¬\ˆOþìL02Zaƒ„±òó,
œæhiÐÑ
H2O2ž±øÅ,L02Za±ð‡
ÑUät

åÞßÑUޟ%y

ÐÑìPOþ±x
Ë

€¦
H2O2ž@±L02Za±ð‡ÑUät

q
3)。
‰V¦ÙìPOþ¬ìPž

a1=c
.ߕr{

9«¬;qìPOþ•
05h
“P
ž•
03mmol·L-1
±
H2O2ÖnY,ƒ。
34 ANNEXINⅤFITC/7AAD‚áë/Za 
H2O2ìPY L02Za±ë/‡•256% ±36%,
òŽ}úhøÅ
(P<005);
—`~
625,125,
25,50mg·L-1
±
OMT
jkY

ë/‡úh@ä

—
` •
112% ±25%,998% ±39%,138% ±
31%,153% ±24%(P<005);
ë/‡Ñ~,
·4031·
ßxéè

t_Tݾì
H2O2\]L02Zaƒ„±9Ë0žÊÓf˱«¬
  
q
3 
¥¦üt¢Å¾Ê¥¦0žOþì
L02
Za±ð‡
Fig3 ViabilityofL02celsinducedbyH2O2underdiferent
concentrationandtime
ž۱[â±Ãß·=Å

Œ
OMT
¿
125
mg·L-1
O9Ëë/0žQe

q
4)。
35 L02
Za8
SOD,GSHPX
ðñ
、ROS
ëßá
â
 OMT
9Ëüt¢ì
L02
Za±ƒ„$
SOD,
GSHPX
ðñ _

ю}€ñ

PQ$
SOD
ð
µh·ät
(P<005),GSHPX
ðµh·ät
(P<001);
ÑPQñò

~,$
SOD
ðµu
h·ÜÝ
(P<005,P<001)、GSHPX
ðµuh
·ÜÝ
(P<001),
å
OMT
¿
125mg·L-1
O۟
ðµÜÝQ•

Ñ?wìH€ñ

PQ$
ROS
ëßh·ÜÝ
(P<001);
ÑPQ€ñ

~,
A
Ž}
;B
PQ
;C625mg·L-1

;D125mg·L-1

;E25mg·L-1

;F50mg·L-1


ю}€ñ
1)P<005;
ÑPQ€ñ
2)P<005。
q
4 
t_Tݾìüt¢\]±
L02
Zaë/‡±òó
Fig4 OMTprotectL02celsfrominjuryinducedbyH2O2
·5031·
2016
y

z ³
41
ų

}
Vol41,No.7 April,2016
ROS
ëßh·ät
(P<001,P<005),
å¿
125
mg·L-1
O
ROS
ätQ•

*
2)。
Ͼhi
,OMT
¿
625~50mg·L-1
uMøe
SOD,GSHPX
ðñ

åät
ROS
ëß

*
2 
t_Tݾìüt¢\]±
L02
Zaƒ„$
SOD,ROS,GSHPX
±òó
(珋x±s,n=3)
Table2 EfectofOMTonSOD,ROSandGSHPXinL02celsinducedbyH2O2(珋x±s,n=3)
`
Ãß
/mg·L-1 SOD/kU·L-1 ROS/MFI GSHPX/kU·L-1
Ž}
    - 11562±1665 32538±9195 20098±3234
PQ
    - 7726±04132) 102778±35191) 4903±17551)
t_Tݾ
625 10132±04554) 75685±99874) 72762±40763)
125 15158±19473) 55899±66233) 79086±70463)
25 11398±05354) 51053±102933) 56660±64023)
50 12058±03814) 87395±104464) 55339±62303)
  
å

ю}€ñ
1)P<001,2)P<005;
ÑPQ€ñ
3)P<001,4)P<005。
4 
Ús
TÝÛ

¹t_Tݾ

ɦ­&Êǎ˜T
Ý

Ó­Ø

3JÒ­Ø莘$#ֱ˜¾

…
†÷L/žŸ$ˆñµmQOhµ‚0

…†Éæ
îd

É$ÆA+.ë[Ó$ˆmQ‚0±,˜
ý[

¦O앍‚ƒ„çBò÷±deÉæ
[16]。
‚O±mÂLÌ+Íá‚çÞZa±ƒ„

ðµt
Ím±t_‚ˆZɕ‚Om±OS1i
[1],
ÎyÏt_Tݾ±d‚0žfË«¬L/È$¿
®|˜Ïœ‘

Ó¿¯°¶˜¢%±ôt_d‚0
žkÚòê

¬å€ñ¶˜çƒ±¯°µ¬0žf
˱æ-µ

ZaçƒòóàÛñòU[

-B¶˜
¯°çƒb¥-)±½ßі(

{Ž•6z±é
úÓôt_0ž

àï9çƒpž
H2O2c L02Z
a

‚çÞZa

t_‚ˆƒ„PQ

U‹¦ôt_
0žœ‘Ï4Út_Tݾ
(OMT)
±d‚0ž

CCK8
‚áÉ[‚áZaøyðµ±wž
œ

Ͼhi
,L02
Za¿zã
OMT
\ˆY

ø™
ðµøe

§pž
CSFE
Za—ÝKQ‚á

Ͼ
hi
L02
Zabd±øy±-

1Ÿ÷g

÷ø
±œæ—à
:CSFE
Za—ÝKQÉ[Ž6z‚
áZaø™±œ

ӜæŽÈî
,OMT
¥{Ö
L02
Za±ø™
;CCK8
‚áÉ[þz‚áZ
aø™±œ

ӂárSÉ@¸‚á
96
&ZaT
$
NAD(P)H
øű•êÏþzéúZa±ø™

ba
CCK8
œæ*ú
,L02
Zazã
OMT
\ˆY

çƒ&$
NAD(P)H
uëßøÅ

‰V

œ±
Ͼ

9“ÿ
,OMT
MaÖU
L02
Za
8
NAD(P)H
ëßøÅ

§
NADPH
ÉZa8ôt
_2iÅƱ./––—

ìŸi+
GSH
±Ár
²ß

dŠZaª†]cZat_ƒ„±hµ˜Þ
ROS
-B./d0
[18]。
àïMa[á
OMT
M{B
ôt_d‚0ž

•ï

9“pžüt¢€t_‚ˆƒ„
PQϊú÷g[á

}Éʟ
H2O2±¥Kâµ、
ZaÄaÊ$•èàÛ±¿

¥¦^²$±
H2O2±ìPžÊìPOþ¿„…ÔÕ
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