全 文 ：Inhibitory Effect of Sorbaria Sorbifolia on DEN-induced
Precancerous Hepatic Foci and Its Antioxidative
Activities in Rats
ZHANG Xue-wu 1 , 　ZHANG Xue-bin2 , 　QUAN Ji-shu1 , 　SHEN Ming-hua 1 , 　JIN Hai-ling 1
1.Department of Biochemistry &Molecular Biology , Yanbian University College of Medicine ,Yanji 133000 ,China
2.China-Japan Union Hospital , Jilin University , Changchun 130031 , China
　
Abstract:Objective　To investigate the effect of Sorbaria sorbifolia on diethylnitrosamine(DEN)induced pre-
cancerous hepatic foci and its antioxidative activities in rats.Methods 　According to the standard method ,75
rats were randomly divided into 3 groups:Group A(control),Group B (model),Group C (treatment), and 25
rats in each group.Hepatic precancerous lesions in rats were induced by the modified Solt and Farber protocol
and the rats were sacrificed after feeding Sorbaria sorbifolia extract for 6 weeks.The expressions of glutathine S-
transferase (GST-P),p53 protein were investigated by immunohistochemistry techniques.TNF-αwas measured
by the radioimmunoassay and antioxidative activities of SOD ,GSH-Px and the content of MDA were investigat-
ed by the colorimetric method in rat serum and mitochondria of liver cells.Resul ts 　GST-P stainting w as
86.7%positive in Group B and 28.6%positive in Group C;p53 protein was 53.3%in Group B and 38.1%
in Group C positive respectively.For the content of serum TNF-α, the activities of SOD and GSH-Px , and the
content of MDA in the serum and liver cells mitochondria , there were significant differences between Group B
and Group C.Conclusions　Sorbaria sorbifolia extract has inhibitory effect on DEN-induced precancerous he-
patic foci in rats and induces TNF-αproduction in rats.The antioxidative action is exhibited by the administra-
tion of Sorbaria sorbifolia extract in the early stage of chemical hepatocarcinogenesis in rat livers.
China J Cancer Prwv Treat ,2003 ,10(11):
Key words:Sorbaria sorbifolia pharmacology;liver neoplasms chemically induced;diethylnitrosamine toxicity;
liver neoplasms TCD therapy ;rats
珍珠梅提取物对二乙基亚硝胺所致大鼠肝脏癌前
病变的抑制作用及抗氧化活力的影响
张学武1 , 　张学斌2 , 　全吉淑1 , 　沈明花1 , 　金海玲1
1.延边大学医学院生物化学与分子生物学教研室 ,吉林 延吉 133000
2.吉林大学中日联谊医院 ,吉林 长春 130031
【摘要】　目的:研究珍珠梅对二乙基亚硝胺致大鼠肝脏癌前病变灶及抗氧化活力的抑制作用。方法:75
只大鼠随机分为A 、B 、C 3组:对照组 、模型组和治疗组 ,每组 25只。按略改良的Solt-Farber氏方法制作
大鼠肝脏癌前病变模型 ,用珍珠梅提取物饲养大鼠 6周后处死 ,通过免疫组织化学检测癌前病变组织
【基金项目】　吉林省延边朝鲜族自治州科技发展计划项目(2003YBKJ027)
【第一作者简介】　张学武(1973-),男 , 吉林省大安市人 ,讲师 ,硕士 ,主要从事中草药开发与肝脏生物化学的研究工作。
E-mail:zlz2913835@sina.com
·1137·肿瘤防治杂志 2003年 11月第 10卷第 11期　　CHINA J CANCER PREV TREAT , NOV.2003 ,Vol.10　No.11
DOI :10.16073/j.cnki.cjcpt.2003.11.005
谷胱甘肽S-转移酶(GST-P)和 p53蛋白的表达 ,放射免疫法检测血清肿瘤坏死因子(TNF-α)的含量 ,比
色分析法检测血清 、肝线粒体超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)的活性及丙二醛
(MDA)的含量。结果:B和C 组GST-P 的表达率为 86.7%和28.6%,p53的表达率为56%和36%,差异
均有显著意义 , P <0.05;B组血清 TNF-α的含量与 C 组比较 ,差异有显著意义 , P <0.05;B组血清 、肝
线粒体 SOD 、GSH-Px 活性及MDA含量与 C 组比较 ,差异均有显著意义 , P <0.05。结论:珍珠梅提取物
对 DEN所致大鼠癌前病变灶有抑制作用 ,可诱发 TNF-α的生成 ,并有抗氧化作用 。
肿瘤防治杂志 ,2003 ,10(11):
【关键词】　珍珠梅 药理学;肝肿瘤 化学诱导;二乙基亚硝胺 毒性;肝肿瘤 中药疗法;大鼠
【中图分类号】　R735.7　　　【文献标识码】　A　　　【文章编号】　1009-4571(2003)00-0000-00
　　Sorbaria sorbifolia is a kind of rose family plant
growing at the side of river and on the foot of hills in
East-North China.It also grows in Yunnan ,Guizhou , Si-
chuan ,Ningxia provinces ,China.We discovered that Sor-
baria sorbifolia extract had the protective effect on acute
hepatic injury by carton tetrachloride in rats[ 1] .In this pr-
esent study ,we report the inhibitory effect of Sorbaria sor-
bifolia on DEN-induced precancerous hepatic foci and its
antioxidative activities in rats.
1　Materials and methods
1.1　Materials
Diethylnitrosamine (DEN), 2-Acetylaminofluorene
(AAF)were obtained from Sigma Inc , kits for superoxide
dismutase (SOD), glutathione peroxidase (GSH-Px)and
malondialdehyde (MDA)were obtained from Institute of
Biotechnology , Nanjing Jiancheng ;GST-P antibody w as
purchased from Institute of Biotechnology , Fujian Xinda-
lu;p53 monoclonal antibody w as purchased from Institute
of Biotechnology , Tianjing TBD;kit for TNF-αwas pur-
chased from Institute of Biotechnology , Beijing Beifang.
Sorbaria sorbifolia harvested from the country of Yanji
City , Jilin Province ,China were identified by the authors.
It was dried , cut , made into powder and extracted for 24
hours with ethyl acetate and the extract was vacuum-con-
centrated (its mainly chemical constituents were flavonoid
compounds), and dried by speed vacuum.Male Wistar
rats , aged 6 weeks and weighing 180-200 g were used in
the experiments of hepatocarcinogenesis.Animals were
housed in group 5 animals in plastic cages with stainless-
steel gird tops at room temperature with a 12-hour light
dark cycle.
1.2　Induction of precancerous hepatic foci
Enzyme-altered hepatic foci and hyperplastic nodules
were induced by the modified protocol of Solt and Farb-
er
[ 2] .According to the standard method ,75 rats were ran-
domly divided into 3 groups:Group A (control),Group B
(model),Group C (treatment),25 rats in each group , and
treated as shown in Figure1.The rats in group B and C
were given a single ip.injection of DEN(200mg kg body
weight)dissolved in saline to initiate hepatocarxino-
genesis.After 2 weeks on a basal diet , the rats received
0.008%2-AAF for the following 6 weeks.Additionally ,
for the Group C , the Sorbaria sorbifolia treatment group ,
6.6 g kg Sorbaria sorbifolia was added together with 2-
AAF in the forage.Group A as a control group ,was intra-
peritoneally injected with saline instead of DEN and then
maintained on basal diet for 8 weeks.All rats of the ex-
perimental and control groups were subjected to two-thirds
partial hepatectomy (PH)at the third week.Rats in each
group were sacrificed for examination at the eighth week.
Saline ,0.9%NaC l ip , diethylnitrosamine(DEN);↑PH:Two-thi rds par-
tial hepatectomy;S acrifi ce; AAF:2-Acetylaminofluorene
Fig.1　Experimental protocol
1.3 　Immunohistochemical staining for GST-P and
p53
Rat liver slices were fixed with ice-cold acetone and
embedded in paraffin.Immunohistochemical staining for
GST-P w as performed by the ABC method using anti-
GST-P antibody , and immunohistochemical staining for
p53 was performed using monoclonaol antibody , respec-
tively .The number and the area of GST-P-positive hepatic
foci larger than 0.1 mm in diameter were analyzed by us-
ing the microscopic quantitative analyzer.
1.4　Determination of TNF-αcontent
·1138· 肿瘤防治杂志 2003年 11月第 10卷第 11期　　CHINA J CANCER PREV TREAT , NOV.2003 ,Vol.10　No.11
The content of TNF-αwas measured by the radioim-
munoassay method.
1.5 　Determination of the activities of SOD and
GSH-Px , and the content of MDA
SOD was measured by the xanthine oxidase colorim-
etric method;GSH-Px by the DTNB colorimetric method
and MDA by the thiobarbitric acid colorimetric method.
1.6　Statistical Analysis
Statistical analysis was made by using chi-square test
with the SPSS 10.0 windows , P <0.05 was considered
statistically significant.
2　Results
2.1　Effects of Sorbaria sorbifolia extract on DEN
induced enzyme altered hepatic foci in rats
Immunohistochemical investigation of expressions of
GST-P and p53 protein in each group were summarized in
Table 1.GST-P stainting in Group A was negative ,while
it was 86.7%positive in Group B and 28.6%positive in
Group C.Percentages of oncogene product p53 protein
w as zero in group A , while it was 53.3%in group B and
38.1% in Group C positive respectively.The number
(No cm2)and area(mm2 cm2)of GST-P-positive hepatic
foci in Group C significantly decreased compared with the
values of Group B , and these quantitative values were
shown in Table1.
Table 1　Effects of Sorbaria Sorbifolia
on the expressions of GST-P , p53 protein in
early stage of rat chemical hepatocarcinogenesis
Group n
GST-P positive
(%)
No.of foci
(No cm2)
Area of foci
(mm2 cm2)
p53 popsitive
(%)
A 21 Negative 0 　　0 　Negat ive
B 15 13(86.7%) 1.8±1.3 0.20±0.26 8(53.3%)
C 21 6(28.6%)b 0.4±0.7b 0.03±0.04 8(38.1%)a
a Compared with group B , P<0.05;b Compared with group B , P<0.01.
2.2　Effects of Sorbaria sorbifolia extract on rat se-
rum TNF-αin the early stage of chemical hepatocar-
cinogenesis
Rat serum TNF-αcontent in Group A , Group B and
Group C were 35.6±5.6 ,38.5±6.3 , and 46.7±7.2μmol
L , respectively .TNF-αincreased by the administration of
Sorbaria sorbifolia for 6 weeks in the early stage of rat chem-
ical hepatocarcinogenesis(P<0.05 ,B vs C ,Table 2).
Table 2　Effect of Sorbaria Sorbifolia
on rat serum TNF-αcontent in early
stage of rat chemical hepatocarcinogenesis( x±s)
Group n TNF-α(μmol L)
A 21 35.6±5.6a
B 15 38.5±6.3
C 21 46.7±7.2a
a Compared with Group B , P<0.05.
2.3　Effects of Sorbaria sorbifolia extract on antioxi-
dative activities in the early stage of rat chemical hep-
atocarcinogenesis
Effects of Sorbaria sorbifolia extract on rat serum and
liver mitochondria SOD ,GSH-Px activities and the cont-
ent MDA in the early stage of chemical hepatocarcino-
genesis were shown in Table 3.Both serum and mito-
chondria activities of SOD and GSH-Px decreased in
Group B compared with Group A , but rose again in
Group C by administering Sorbaria sorbifolia.The content
of MDA increased in Group B compared with Group A
and Group C.
3　Discussion
3.1　Effects of Sorbaria sorbifolia extract on DEN-
induced precancerous hepatic foci during chemical
hepatocarcinogenesis in rats
Table 3　Effects of Sorbaria sorbifolia extract on antioxidative
activities in the early stage of rat chemical hepatocarcinogenesis( x±s)
Group n
SOD
Serum(U L) Mitochondia[ NU (mgprot·mL)]
GSH-PX
Serum(U L) Mitochondia(U L)
MDA
Serum(nmol L) Mitochondia(nmol mgprot)
A 21 131±39a 153±56a 29±6a 35±10a 0.8±0.1a 1.2±0.3a
B 15 93±43 135±32 16±4 15±2 1.1±0.1 1.6±0.2
C 21 127±27a 144±45a 27±6a 36±16a 0.8±0.1a 0.9±0.4a
a Compared with Group B , P<0.05.
　　DEN as an initiator and AAF as a promoter , a modi-
fied system based on the Solt-Farber method was designed
to screen the medium-term bioassay of chemical induced
carcinogenesis.This screening system was used in this
study and successfully induced the precancerous GST-P-
positive hepatic foci and nodules.The results of the pr-
·1139·肿瘤防治杂志 2003年 11月第 10卷第 11期　　CHINA J CANCER PREV TREAT , NOV.2003 ,Vol.10　No.11
esent study demonstrated that Sorbaria sorbifolia extract
had an inhibitory effect on DEN-induced GST-P positive
precancerous hepatic foci in rats.We also studied the re-
lationship between the expression of GST-P and antionco-
gene p53 product in precancerous hepatic foci of rat liv-
ers.We studied p53 protein in precancerous hepatic of rat
livers.The results showed that 86.7% of GST-P was posi-
tive and 53.3% of p53 protein positive in Group B.
Meanwhile , the GST-P positive was 28.6% and p53 pro-
tein 38.1%in Group C.The results of our study indicat-
ed that GST-P and p53 appeared at the same time in the
early of chemical hapatocarcinogenesis.These results in-
dicated Sorbaria sorbifolia extract exhibited inhibitory ef-
fect on DEN induced precancerous hepatic foci in rats ad-
ministered with Sorbaria sorbifolia extract for 6 weeks dur-
ing chemical hepatocarcinogenesis.
3.2　Immunoactivating properties of Sorbaria sorbi-
folia
Macrophases are activated in a non-specific manner
when antigens or infectious agents invade the body , and
may secrete various cytokines (IL-1α β , IL-6 , TNF-α,
IFN-α β , etc.)and chemicals (NO ,O 2- , prostaglandins ,
etc.)for communication to other immune cells ,killing the
invading pathogens and inducing fundamental host
defense[ 3 , 4] .In our study , we observed the serum TNF-α
increased by administration of Sorbaria sorbifolia extract
in the early stage of rat chemical hepatocarcinognesis.
These observations suggest that the antihepatotoxic effect
of Sorbaria sorbifolia probably be due to its immunomodu-
lation.It may be related to that Sorbaria sorbifolia con-
tains polysaccharides.Our results indicate that Sorbaria
sorbifolia has immunoactivating properties.It may be one
of anticarcinogenic mechanisms of Sorbaria sorbifolia.
3.3　Antioxidative activities of Sorbaria sorbifolia
Superxiodes and free radicals that are produced dur-
ing utilization of oxygen in living tissues have a high reac-
tivity , and overproduction of superoxide has harmful action
and causes serous derangements , such as coronary arterio-
sclerosis and diabetes mellitus , as well as being associated
with aging and carcinogenesis.SOD ,GSH-Px are free rad-
ical scavengers and detoxication enzymes and MDA is a
lipid peroxidation product.SOD may be a major intracel-
lular enzyme against O
2
toxicity through catalysis of the
removal of O2
[ 5 , 6] .Recently , it w as reported that activity of
GSH-Px markedly decreased at an early stage of DEN in-
duced hepatocarcinogenesis in rats
[ 7] .
Our present study showed that both serum and mito-
chondria SOD and GSH-Px activities rose again in rats
administered Sorbaria sorbifolia extract , and increased the
content of MDA due to the formation of precancerous he-
patic foci decreased by the Sorbaria sorbifolia extract.
Therefore , our present study suggests that Sorbaria sorbifo-
lia should have a strong effect on the disorders caused by
free radical production in living tissue and the inhibitory
activity on senility.In short ,Sorbaria sorbifolia extract has
an antioxidative effect and it may be one of anticarcino-
genesis mechanisms of Sorbaria sorbifolia.
【REFERENCES】
[ 1] 　Jin HL ,Zhang XW ,Zhao H , et al.Protective effects of Sorbaria sorbi-
foli a ext ract on carton tetrachloride induced acute hepatic injury in rats
[ J] .World Chinese Journal of Digestology , 2002 , 10(7):783-786.
[ 2] 　Yin ZZ , Jin HL , Yin XZ , et al.Effect of Boschniakia rossica on expres-
sion of GST-P , p53 and p21ras proteins in early stage of chemical hep-
atocarcinogenesis and its anti-inflammatory activities in rats[ J] .World
J of Gastroenterology , 2000 , 8(6):812-818.
[ 3] 　Lee EJ ,Gerhold M ,Palmer MW , et al.p53 protein regulates the effects
of amifostine on apoptosis , cel l cycle progression , and cytoprotection
[ J] .Br J Cancer , 2003 , 88(5):754-759.
[ 4] 　Hamano K , Li TS , Takahashi M , et al.Enhan ced tumor necrosis factor-
alpha expression in small sized abdominal aortic aneurysms[ J] .World
J Surg , 2003 , 27(4):476-480.
[ 5] 　Barja G.Endogenous oxidative stress:relationship to aging , longevity
and caloric restriction[ J] .Ageing Res Rev , 2002 , 1(3):397-411.
[ 6] 　Iwamoto A , Egashi ra T , Takayama F , et al.Changes in f ree radical-re-
lated substances in plasma folling i schemia-reperfution in rat liver[ J] .
Pathophysiology , 2002 , 8(3):167-174.
[ 7] 　Lou XE , Zhou HJ , Huang HB.Lipid-peroxidantion damage of embryo
and placenta induced by artesunate in rat s[ J] .Journal of Zhejiang Uni-
versity (Medical S ciences), 2003 , 32(1):41-45.
收稿日期:2003-06-23　修回日期:2003-09-10
(编辑:黄一心)
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