全 文 ：·药理实验与临床观察·
Organic extract of Tripterygium hypoglaucum induced apoptosis
of HL-60 cells through NF-κB and mitochondrial signal ing
pathways as revealed by cDNA microarray technique
FANG Zhi-jun
1 , CAO Jia
2 , AO Lin
2 , ZHU ANG Wei-jian
3 ,
FANG Hong-xun
3 , XIAO Pei-gen
4 , YANG Meng-su
1, 3
( 1. Applied Research Centr e for Genomics Techno lo gy , City Univ ersity o f Hong Kong , Kow loon, China; 2. Mo lecula r
Toxicolog y Labo rato r y, Third M ilitar y Medical Univ e rsity , Chongqing 400038, China; 3. Bioac tiv e Products
Research Group, Depa rtment o f Biolog y& Chemistry , City Univ ersity o f Hong Kong , Kow loon, China;
4. Institute of M edicinal Plants Development , CAM S& PUMC, Beijing 100094, China)
　　 Abstract: Object　 To investiga te the signaling pa thw ays and mo lecular mechanism invo lv ed in the
apopto sis of human promyleocytic leukemia HL-60 cells induced by org anic ex tract of Tripterygium hy-
poglaucum ( T HH) ro ot. Methods　 Cell bio logical changes during the apopto sis induction w ere studied by
f low cy tometry. Mo lecula r mechanism involved in the apoptosis w as examined by cDN A microarray tech-
nique. Results　 Flow cy tometric study confirmed the induction of apoptosis by the organic ex t ract of
T HH. The gene expression profiles of HL-60 cells treated by THH were obtained using a cDN A microa r-
ray containing 3 000 human genes derived f rom a leukocy te cDN A libra ry. Six teen genes identi fied to be
di fferential ly expressed in HL-60 cells can be clustered into g roups related to apopto sis ( including Caspases
3 and Caspase 8) , cell proli feration, cell cycle control and di fferentia tion, and st ress response. Conclusion
The induced apopto sis of HL-60 cells by THH ex tract is associated wi th the up-regulation o f sev eral g enes
( such as N FKBIB, PRG1 and B2M ) related to N F-κB and mitochondrial signaling pathw ays.
Key words: Tripterygium hypoglaucum ( Lé v l. ) Lé v l. ex Hutch. ; apoptosis of HL-60 cells; mi tochon-
dria l signal
基因芯片分析显示昆明山海棠有机萃取液
经 NF-κB及线粒体信号传导途径诱导 HL-60细胞凋亡
方志俊 1 ,曹　佳 2 ,敖　琳2 ,庄为笕3 ,方宏勋 3* ,肖培根 4 ,杨梦苏 1, 3*
( 1. 香港城市大学基因组科技应用研究中心 ,香港 九龙 ; 2. 中国人民解放军第三军医大学 分子毒理学实验室 ,四川 重庆
400038; 3. 香港城市大学生物及化学系生物活性制品研究组 ,香港 九龙 ; 4. 中国医学科学院 中国协和医科大学药用植物研
究所 ,北京 100094)
摘　要 :目的　研究中草药昆明山海棠 Tripterygium hypoglaucum ( T HH)有机萃取液诱导人早幼粒白血病 HL-
60细胞凋亡过程的信号传导通道及分子机制。 方法　应用流式细胞仪研究了 THH有机萃取液诱导 HL-60细胞
的凋亡过程 ,并应用包含 3 000人类基因与 EST的基因芯片进行基因表达差异分析。结果　基因芯片杂交结果显
示有 16个基因表达发生大于 2倍的显著变化 ,这些基因同细胞生长 ,细胞周期调控 ,细胞分化 ,以及压力反应有
关。 部分基因在细胞凋亡过程中起关键作用 ,如 Caspase 3和 Caspase 8。 结论　 THH有机萃取液诱导 HL-60细
胞凋亡 ,该过程与 NF-κB和线粒体信号传导途径有关。
关键词: 昆明山海棠 ; HL-60细胞凋亡 ; 线粒体信号
中图分类号: R286. 91　　　文献标识码: A　　　文章编号: 0253 2670( 2003) 04 0334 05
Introduction
　　 The plants of Tripterygium Hook. f. ( Celas-
traceae ) have been used in tradi tional Chinese
medicine as remedies for cancer t reatment and as
·334· 中草药　 Chinese T raditional and Herbal Drug s　第 34卷第 4期 2003年 4月
收稿日期: 2002-09-02作者简介:方志俊 ,女 ,香港城市大学基因组科技应用研究中心博士研究生。 E-mai l: ccfong@ ci tyu. edu. hk通讯作者:方宏勋 ,教授 ,香港 ,九龙 ,香港城市大学生物及化学系。 杨梦苏 ,教授 ,香港 ,九龙 ,香港城市大学基因组科技应用研究中心。
Tel: 27888031　 Fax: 27887406　 E-mai l: bhw ffong@ ci tyu. edu. h k
an insecticide fo r hundreds of years
[ 1-4 ]
. Our prev i-
ous study has show n tha t the ro ot ex t ract of
Tripterygium hypoglaucum ( Lé v l. ) Lé vl. ex
Hutch. ( THH) can induce apoptosis in leukemia
cell line HL-60 cells and alter the expression pa t-
tern of c-M yc gene. The purpose o f this study is to
examine whether the org anic ex tracts of T HH may
induce apopto sis in HL-60 cells also and to further
investigate the mo lecula r mechanism of such induc-
tion process.
In the present study, f low cy tometry and c-
DN A microa rray techniques w ere used to investi-
ga te the cellular and molecular ef fects of T HH or-
ganic ex t ract on HL-60 cells. Flow cy tometry ha s
been used to detect and quantitate va rious aspects
o f cell death, including apopto tic o r necrotic
cells[5 ] . Apoptotic cells contain reduced DN A con-
tent and wi ll appea r as cells w ith low DN A stain-
abi li ty (“ sub-G1” peak) as compa red to that of G1
cells
[6 ] . cDN A micro array tech nique allow s moni-
to ring of the expression o f hundreds and thousands
o f genes simultaneously
[7, 8 ]
. In this study, a c-
DN A microarray containing 3 000 human genes de-
rived f rom a leukocyte cDN A library was used to
investigate the gene expression pro files of HL-60
cells upon T HH treatment.
Materials and methods
1. T HH prepa ra tion and cell culture. Organic ex-
tract of T HH was prepared acco rding to the pro-
cess show n in Fig. 1. The ex tract w as sto red at
dimethy lsulfo xide ( DM SO ) as a concentration of
20μg /μL. HL-60 cells ( American Type Culture
Col lection, MD, USA) w ere g row n at 37℃ in 5%
CO2 in RPM I-1640 supplemented wi th 1% antibio-
tic solution ( Invi t rog en) and 10% heat-inactiv ated
fetal bov ine serum ( Invi trog en) . For THH trea t-
ment , 2× 105 cells /mL w ere used and trea ted wi th
25 μg /mL o r 40 μg /m L of T HH ex tracts fo r
8 hours.
2. Flow cy tometry study. the control o r T HH-
treated cells 2— 3× 106 were collected by centri fu-
ga tion and w ashed twice by pho sphate-buffered
saline ( PBS) . The cell pellets w ere resuspended
gent ly in 1 mL of hypotonic propidium iodide solu-
Fig. 1 Process for preparation of organic extract of THH
tio n ( 50μg /m L) prepared in 0. 1% sodium ci trate
plus 0. 1% Triton X-100 and 100 mg /mL DNase-
free RNase A for DN A staining . Stained cel ls w ere
analy zed by ESP f low cytometry ( Coulter Elec-
tronic, U SA) a t ex ci tation 488 /emmision 600 nm.
Data w ere analy zed by cycle dist ribution so ftw are
( M odFi t LT version 2. 0, Veri ty So ftw are House,
USA) .
3. To tal RN A preparation and f luorescence label-
ing. HL-60 cells ( cont rol ) and trea ted w ith THH
( 40μg /mL, 8 h) w ere harv ested and co llected in a
50 mL conical tube by centrifug ation ( 1 000 r /min,
5 min) . Cells w ere lysated and to tal RN A iso lated
wi th T RIZO L reagent ( Invi t rogen) . The concen-
tration of total RN A was measured wi th a biopho-
tometer, and 500 ng o f each total RN A sample w as
used to run a 1% denatured agarose gel to v eri fy
quali ty. Same amount o f to tal RN A contro l o r
t rea ted to tal RN A were reverse t ranscripted to c-
DN A in the presence of tw o distinct f luo rescent
dyes, Cy3-dU T P and Cy5-dU T P, respectiv ely.
The labeled cDN A was purified using Microcon 30
( Millipo re) .
4. cDN A microarray hybridization. A cDN A mi-
croa rray w as prepa red by using 3 000 cDN A probes
ampli fied from a leukocyte cDN A library
( clontech) and array ed on g lass slides using a mi-
croa rrayer ( SPBIO, Hitachi ) . The labeled cDN As
w ere mixed and denatured at 100℃ for 2 minutes
and hybridized wi th the microa rra y a t 65℃
overnight. Micro array images were obtained by a
confocal f luorescence scanner ( ScanArray 4 000,
GSI Lumonics, U SA) and the scan results w ere
analy zed using ScanAnaly zer sof tw are ( Standford
Univ ersity, Cali fo rnia, USA) . Fluo rescence ratios
( Cy5 v s. Cy3) were used to determine the di ffer-
ential g ene expression lev els. Genes wi th ratio
·335·中草药　 Chinese T raditional and Herbal Drug s　第 34卷第 4期 2003年 4月
number above 2 or under 0. 5, i. e. induction or re-
pression o f 2 fo ld, w ere selected and subjected to
further analysis.
Result and discussion
1. Organic ex t ract of T HH induced apopto sis in
HL-60 cells. Flow cy tometry w as used to study
the ef fect of T HH organic ex tract on HL-60 cells.
Cells w ere t reated w ith 0 (cont rol) , 25 and 40μg /
m L of T HH ex tract fo r 8 hours. The propo rtion of
sub-G1 cells increased by 15 to 45 folds a fter 25
and 40μg /mL treatment, respectiv ely, as com-
pared to the untreated cells ( Table 1) . It has been
shown that the accumulation o f dying cells in this
“ sub-G1” hypodiploid peak is mainly due to a re-
duced DN A content and these cells are destined to
enter apoptosis
[5 ] . The data confi rmed that HL-60
cells w ere induced to enter apoptosis af ter T HH
treatment.
Table 1　 Proportion of sub-G1 phase in HL-60
cells after 8 h THH treatment
Treatment dose / (μg· mL- 1 ) Proportion of sub-G1 /%
0 0. 2
25 3. 1
40 9. 3
2. THH treatment changed gene expression pro-
fi les of HL-60 cells. To tal RN A from untreated
and T HH-trea ted HL-60 cells w ere used as tem-
plates fo r synthesis o f Cy3 ( g reen) and Cy5 ( red)
labeled cDN A probes, respectiv ely. The quali ty of
the tota l RN A was verified by gel elect ropho resis
( Fig . 2) . Fig. 3A show ed a typical microa rray im-
age fo llow ing the competi tive hybridization of the
tw o labeled cDN As wi th the microarray containing
3 000 probes. A scat tered plo t of Cy5 intensi ty
v ersus Cy3 intensi ty show ed the quali ty of hy-
bridiza tion to be rea sonable wi thout obvious bia s
( Fig. 3B ) . After preliminary da ta analysis, 16
genes w hich display ed a g rea ter than 2-fo ld dif fer-
ence betw een untreated and drug t reated samples
w ere identified. The accession numbers, functional
description, as w el l a s the Cy5 /Cy3 f luo rescence
ra tios of these genes w ere listed in Table 2. The
six teen differentially expressed genes can be classi-
fied into th ree main catego ries: g enes related to
apopto sis, g enes rela ted to cell cycle and dif ferenti-
500 ng of total RN A is olated f rom cont rol ( l an e 1) and THH-
treated ( 40 μg /m L, 8 h, lane 2 ) HL-60 cell s w ere elec-
tropho resed on a 1% formaldeh yd e denatured agarose gel
Fig. 2　 Quality of total RNA isolated from HL-60 cells
( A) To tal RN A w ere ext racted f rom both cont rol and treated
cel ls ( 40μg /m L of T HH wi th 8 h ) and revers e t ranscribed to
cDN A wi th Cy3 ( green ) and Cy5 ( red ) labeled, respect ively.
Labeled cDN A w ere mixed and hyb ridi zed to a microar ray. Im-
age w as ob tained using a confocal f luorescence las er s canner
( GSI 4000) .　 ( B) Scattered plot of Cy5 intensi ty versus Cy3
intensi ty.
Fig. 3　 cDNA microarray image showing gene
expression profiles of HL-60 cells
ation, and genes rela ted to stress response ( Table
2) . Both Caspases 3 and Caspase 8, impo rtant
common genes in executing the apopto sis process,
w ere upregulated, w hich is consistent wi th the ob-
serv ation by flow cytometry tha t HL-60 cells en-
tered activ e apopto sis pro cess following th e THH
trea tment.
3. THH-induced apopto sis involv ed N F-κBand mi-
to chondial pathw ays. Among the apopto sis related
genes, most are invo lv ed in tw o signaling path-
way s leading to apoptosis, including N F-κB signa l-
ing pathway and mitochondrial mediated signa ling
pa thw ay. Fo r example, NFKBIB, the gene encod-
ing the nuclea r facto r of kappa light po lypeptide
gene enhancer in B-cells inhibi to r beta, wa s up-
·336· 中草药　 Chinese T raditional and Herbal Drug s　第 34卷第 4期 2003年 4月
Table 2　 Dif ferentially expressed genes in HL-60 cells following THH treatment
Category Description of gen e
Acces sion
Number
Fold
Apoptosis
nuclear factor of kappa ligh t polypept ide gen e enhancer in B-cell s
inhibi tor, beta ( N FKBIB)
AI935157 4. 35
proteoglycan 1, s ecretory g ranule ( PRG1) NM 002727 3. 76
Caspase 8, apoptosi s-related cys teine protease BG529842 3. 15
solute carrier family 25 ( mitoch ond rial carrier; phosphate
carrier) , member 3
BG491863
　
3. 08
　
Capase 3, apoptosis-related cystein e protease
AU125557
　
2. 86
　
beta-2-microglobulin ( BM ) AV710740 2. 72
ATP synth as e, H+ t ranspo rting , mi toch ondria F0 complex,
s ubunit g
AV714814 2. 63
mi toch ondrial ribosomal protein S12 AF058761 2. 56
non-m etas tat ic cell s 5, p ro tein express ed in ( nucleoside-diph os-
phate kinase)
AL043778
　
0. 47
　
c-myc binding protein AL561551 0. 33
Cell cycle and
dif ferentiation
HIR his tone cell cycle regulation defectiv e homolog A ( S. cere-
visiae)
NM 003325
　
2. 18
　
ribos omal p ro tein L31 AW973154 2. 56
in terleukin 10 receptor, b eta BC001903 0. 48
cyclin B2 N87720 0. 32
St ress response heat sh ock 7× 104 protein 4 BE742483 2. 05
heat sh ock 9× 104 protein 1, beta BG336532 2. 13
regula ted by more than 4-fo ld. N FKBIB is the cen-
tral g ene in the N F-κB signaling regulation[9, 10 ] .
Pro teogly can 1, secreto ry g ranule ( PRG1) , an
early response gene in pancrea tic cancer cell regu-
la ted by P53 and N F-κB[9 ] , was up-regulated by
more than 3-fold. Beta-2 microg lobulin ( B2M ) as
the N F-κB ta rg et gene for immuno recepto r[ 9] was
also up-regula ted during THH treatment. On the
o ther hand, the up-regulation of AT P synth eses,
solute carrier family ( mitochondria l carrier; pho s-
pha te car rier ) member 3, and mitochondrial ribo-
somal pro tein S12, indicates the invo lv ement of the
mitochondrial mediated apoptotic pa thw ay. It wa s
w ell know n tha t apoptosis is an active, AT P-re-
quiring process
[ 11]
. Tw o genes were down regula t-
ed, including an NDP kinase pro tein expressed in
non-meta static cells 5, and cmyc binding protein.
Both are invo lv ed in the proliferation of cell s and it
is expected that their activi ties need to be at tenua t-
ed during apopto sis
[11 ]
.
　　 Several cell cycle rela ted genes, such as cyclin
B2 and interleukin 10 recepto r w ere down regula t-
ed af ter THH treatment , consisted wi th the fact
that the HL-60 cells w ere induced into apoptosis.
Two genes related to cell di fferentia tion, e. g. ri-
bo soma l pro tein L31 and a histone family g ene
w ere up regulated, w hich show ed that THH ex-
tracts may also induce HL-60 cell dif ferentiation.
In addi tion, sev eral st ress response genes, includ-
ing hea t shock 7× 104 pro tein 4 and 9× 104 pro tein
1, beta, w ere up regulated during T HH treat-
ment, w hich is a common response mechanism fo r
cells under drug treatment.
　　 In conclusion, f loe cy tometric study and the
up-regulation of Caspases 3 and Caspase 8 as re-
vealed by cDN A microa rray study has demonst rat-
ed that HL-60 cells entered active apoptosis pro-
cess following the T HH ex tract t rea tment. Several
genes related to cell apopto sis w ere up-regula ted,
mainly involved in N F-κB and mitochondrial sig-
naling pathway s.
　　 Acknowledgement: This wo rk is suppo rted by Applied
Resea rch G rant o f City Univ er sity o f Hong Kong .
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[1 ]　 Duan H Q, Takaishi Y, Imaku ra Y, et a l. Sesqui terpene al-
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[ 2 ]　 Yang M J, Cao J. Chromosomal composi tion of micronuclei
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·337·中草药　 Chinese T raditional and Herbal Drug s　第 34卷第 4期 2003年 4月
colchicine, detected by m ulticolo r FISH wi th cent romeric and
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草问荆总生物碱对大鼠脑内氨基酸类神经递质含量
和纹状体内乙酰胆碱含量的影响
季宇彬 1 ,高世勇 2
( 1. 哈尔滨商业大学药物研究所 博士后科研工作站 , 黑龙江 哈尔滨　 150076; 2. 哈尔滨商业大学 制药工程系 ,黑龙江 哈尔
滨　 150076)
摘　要:目的　研究草问荆总生物碱 ( TAEP)对大鼠脑内氨基酸类神经递质含量和纹状体内乙酰胆碱 ( Ach )含
量的影响 ,揭示其对中枢神经系统抑制作用的作用机制。 方法　采用双波长扫描定量法和豚鼠回肠生物测定法观
察大鼠脑内氨基酸类神经递质的含量和纹状体内 Ach含量。结果　 TAEP对大鼠脑内 4种氨基酸类神经递质 (谷
氨酸、甘氨酸、γ-氨基丁酸、天冬氨酸 )的含量均无影响 ,但可显著降低大鼠纹状体内 Ach的含量。结论　 TAEP对
中枢神经系统的抑制作用与脑内 4种氨基酸类神经递质 (谷氨酸、甘氨酸、γ-氨基丁酸、天冬氨酸 )的含量无关 ,而
是通过降低 Ach 的含量 ,进而影响多巴胺 -2 ( DA-2)受体达到的。
关键词: 草问荆总生物碱 ;氨基酸类神经递质 ;乙酰胆碱
中图分类号: R286. 1　　　文献标识码: A　　　文章编号: 0253 2670( 2003) 04 0338 03
Effect of total alkaloids of Equisetum pratense on amino acid
neurotransmitters and Ach of striatum in rat brain
JI Yu-bin, G AO Shi-yong
( 1. Postdocto ral Research Sta tion, Institute o f Materia Medica, Ha rbin Commercial Unive rsity , Harbin 150076, China;
2. Depa r tment o f Pha rmaceutical Engineering , Ha rbin Commercial Univ ersity , Harbin 150076, China )
Abstract: Object　 To study the effect of to tal alkaloids of Equisetum pratense Ehrh. ( TAEP) on the
contents o f amino acid neuro t ransmi t ters and Ach in rat brain to rev eal the mechanism o f T AEP inhibitory
action on the central nerv ous sy stem ( CN S) . Methods　 Contents o f amino acid neuro transmi tters and Ach
in rat brain w ere determined by double-w avelengh scan and GPI mensuration. Results　 T AEP could no t
influence four kinds of content of amino acid neurot ransmi tter ( glutamic acid, gly cin,γ-aminobuty ric acid,
aspar tic acid) , but TAEP could signi ficant ly lower the content of Ach in st ria tum of rat. Conclusion　 The
inhibi tion of T AEP to CN S is at tained by low ering the content of Ach in st riatum to affect DA-2 receptor,
and i t i s ir relev ant to the amino acid neurot ransmit ters ( g lutamic acid, g lycin,γ-aminobuty ric acid, aspa r-
tic acid) .
Key words: to tal alkaloids o f Equisetum pratense Ehrh. ( TAEP) ; amino acid neuro t ransmi t ters; Ach
·338· 中草药　 Chinese T raditional and Herbal Drug s　第 34卷第 4期 2003年 4月
收稿日期: 2002-10-19基金项目:黑龙江省科委自然科学基金资助项目 ( D01-05)作者简介:季宇彬 ( 1956— ) ,男 ,博士 ,教授 ,博士研究生导师 ,多年来一直致力于中药药理、肿瘤药理、分子药理学研究 , 1999年进入哈尔滨医科大学博士后科研流动站工作。