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ETM-1324 He proofs

EXPERIMENTAL AND THERAPEUTIC MEDICINE 00: 0-00, 0000

Abstract. Obstructive jaundice is a condition caused by blockage of the flow of bile out of the liver. This results in an overflow of bile and its by-products into the blood, and bile excretion from the body is incomplete. Untreated, obstructive jaundice can lead to serious infection that spreads to other parts

of the body. We examined the protective effect of Yin Chen capillary artemisia polysaccharide on oxidative damage to the liver in growing rats with obstructive jaundice (OJ). Growing male Wistar rats (n=40, age 3-4 weeks) were randomly divided into four groups (n=10 in each group): normal control group, sham group, OJ group and OJ with Yin Chen polysaccharide treatment group (study group). The rats of the OJ group and the study group were subjected to common bile dust ligation,

while the sham group had the bile duct mobilized but not ligated. The rats of the study group recieved 5 ml/kg Yin Chen polysaccharide (0.5 g/ml) by intraperitoneal (i.p.) injection once daily while the other groups were administered 5 ml/kg saline by i.p. injection. After 4 weeks, the rats were sacrificed to obtain liver weight and to compute the liver coefficient. Additional measures included liver homogenate malondialde-hyde (MDA) and the activity levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT). The liver weight and liver coefficient of rats in the study group were lower than those in the OJ group and higher than those in the control and sham groups (P<0.05). Liver homogenate MDA content in the study group rats was lower than that in the OJ group and higher than that in the control and sham group (P<0.05). SOD, GSH-Px and CAT activities were higher in the

study group rats than those in the OJ group and lower than those in other groups (P<0.05). Yin Chen capillary artemisia polysaccharide protects the liver from oxidative damage and improves antioxidant defense in growing rats with obstructive jaundice.Introduction

Obstructive jaundice (OJ) refers to a type of jaundice caused by mechanical obstruction of the extrahepatic and intrahepatic bile ducts due to tumor, stones, inflammation and other causes. Gallbladder tissue damage is a major pathological manifest-ation of OJ (1). According to the literature, obstruction of the biliary tract can cause reduced cholate in the intestinal tract and decreased ability to clear endotoxin, which can directly induce apoptosis of liver cells (2). Obstruction of the biliary tract also can impair electron transport chain function of liver mitochondria and promote oxygen free radical genera -tion. Simultaneously, obstruction of the biliary tract reduces the activity of free radical scavenging enzymes in the liver, causing decreased free radical scavenging capacity and further induction of liver cell apoptosis.

Traditional Chinese medicine describes capillary artemisia as bitter, acrid and slightly cold, able to remove damp heat from the spleen, stomach, liver and gall bladder. It is considered an important drug for treating jaundice. Capillary artemisia impacts primary Qi of Shaoyang and affects the ascending vitality of liver. Its acridness can dispel stagnation of the liver and gallbladder (3). Studies show that capillary artemisia can protect the liver, relax the gallbladder and remove jaundice; this provides a pharmacological basis for treating liver disease with capillary artemisia (3-5). This study investigated the protective effects of capillary artemisia polysaccharide on oxidative injury to the liver using the common bile duct liga-tion method in growing rats.Materials and methods

Experimental animals and grouping. Healthy male Wistar rats (n=40, age 3-4 weeks old, weight 85-95 g) were randomized

Protective effects of capillary artemisia polysaccharide

on oxidative injury to the liver in rats

with obstructive jaundice

CHANG-SHENG HE 1, HONG-YI YUE 1, JIAN XU 1, FENG XUE 1,

JIAN LIU 1, YUAN-YUAN LI 2 and HONG-EN JING 1

Department of Hepatobiliary Surgery, Affiliated Hospital of Qinghai University, Xining, Qinghai 810001, P.R. China

DOI: 10.3892/etm_xxxxxxxx

313233343536373839404142434445464748495051525354555657585960616263646566676869707172

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Correspondence to: Dr Chang-Sheng He, Department of

Hepatobiliary Surgery, Jiaozhou Central Hospital of Qingdao, No. 29 Xuzhou Road, Qingdao, Shandong 266300, P.R. China E-mail: hechangsh@https://www.wendangku.net/doc/5d3306430.html,

Key words: Yin Chen, obstructive jaundice, oxidative damage,

polysaccharide

HE et al: CAPILLARY ARTEMISIA POLYSACCHARIDE 2

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120 into four groups (n=10 each): a normal control group, a sham

operation group, a OJ group and a OJ + capillaris polysaccha-

ride group or ‘study group’. The rats were kept in a quiet and

ventilated environment with a room temperature of 22-26?C

and relative humidity of 40-70%. All animals had free access

to a normal chow diet and water during the experiment.

Methods

Extraction of capillary artemisia polysaccharide. Dry powder

of crushed capillary artemisia was weighed and added to

water at a ratio of 1:20, then placed in a water bath at 80?C

for 3 h. The sample was centrifuged to remove filter residues

and harvest the supernatant. The supernatant was concen-

trated followed by addition of a 3-fold volume of anhydrous

ethanol. The sample was centrifuged to obtain the sediment.

Saline was added to prepare a 1% solution, and the sample was

deproteinized with 3% trichloroacetic acid and freeze-dried

to obtain crude polysaccharides (6). The phenol-sulfuric acid

method was used to determine polysaccharide with glucose as

the standard substance and a 721 spectrophotometer was used

for colorimetric analysis (7). The extracted capillary artemisia

polysaccharide was prepared as a 0.5 g/ml solution.

Preparation of a rat pup model of OJ. The rats were fasted for

12 and 4 h for food and drink, respectively. Rats were anesthe-

tized with ketamine (20 mg/kg) by intraperitoneal (i.p.)

injection, then cut along the mid-line of the upper abdomen to

the hepatoduodenal ligament. The common bile duct was

dissociated and ligated near the porta hepatis. Rats in the sham

operation group underwent common bile duct dissociation but

no ligation. The standard of a successful model preparation

was that the OJ rat pup model had total bilirubin and direct

bilirubin values (measured by blood sampling) that were

5-times higher than the normal control group two days after

the procedure (8). Rat pups in the study group were adminis-

tered 5 ml/kg capillary artemisia polysaccharide solution

(0.5 g/ml) via once-daily i.p. injection; the other groups were

given 5 ml/kg saline via once-daily i.p. injection. After

4 weeks, the animals were sacrificed and their livers were

rapidly removed, washed with ice-cold saline, blotted dry and

weighed. Liver weight was also expressed relative to grams

body weight, as the ‘liver coefficient’. A sample (0.2 g) was

taken from each liver, mixed with 0.2 mol/l phosphate buffer

into 10% tissue homogenate and centrifuged at 3000 rpm/min.

The super n atant was obtained, and xanthine oxidase super-

oxide was used to determine the activity level of superoxide

dismutase (SOD). The NADPH coupling method was used to

determine the activity of glutathione peroxidase (GSH-Px).

Visible light was used to determine the activity of catalase

(CAT) and thiobarbituric acid (TBA) was used to determine

the content of malondialdehyde (MDA) (9).

Statistical analysis. SPSS 13.0 software was used for statis-

tical analysis. The data are expressed as the means ± standard

deviation (SD). Single factor variance analysis was used to

compare the test results of each index across groups, followed

with pairwise comparison between groups using the Student-

Newman-Keuls test. The above analysis was performed as a

two-sided test; the test level α was 0.05 with a P-value of <0.05

considered statistically significant.

Results

Liver weight and liver coefficient. The liver weight and liver

coefficient in the normal control, sham operation, OJ and

study groups were: 7.96±0.26 g, 8.93±0.40%; 7.98±0.27 g,

8.92±0.46%; 9.03±0.26 g, 10.06±0.38%; and 8.32±0.44 g,

9.34±0.57%, respectively. The liver weight and liver coefficient

in the OJ model group were the highest followed by the study

group, the normal control group and the sham operation group

(Fig. 1). Liver weight and liver coefficient were statistically

significant among the groups (P<0.05). Liver weight and liver

coefficient for the study group were significantly reduced

compared to the OJ model group, but still higher than the

normal control group and the sham operation group (P<0.05).

MDA content in the liver homogenate. MDA content in the

liver homogenate was 0.48±0.01, 0.47±0.01, 0.64±0.05 and

Figure 1. Changes in (A) liver weight and (B) liver coefficient among the

groups.

Figure 2. Changes in MDA content in liver homogenate among the groups.

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operation group, OJ group and study group, respectively. MDA

content in the liver homogenate in the OJ model group was the

highest followed by the study group, normal control group and

sham operation group (Fig. 2). The difference in MDA content

was statistically significant among the groups (P<0.05). MDA

content in the study group was significantly reduced compared

to that in the OJ model group, but was still higher than the

content in the normal control group and sham operation group

(P<0.05).

Activity l evels o f S OD, G SH-Px a nd C AT i n t he l iver h omo g enate.

The activity of SOD, GSH-Px and CAT in the liver homogenate

in the normal control, sham operation, OJ and study groups,

respectively, were 185.61±2.82, 184.59±2.75, 132.56±11.51,

156.18±14.79 U/g prot (SOD); 577.12±9.21, 572.25±7.05,

370.74±18.37, 528.76±18.76 U/g prot (GSH-Px); 1031.80±64.08,

1014.07±64.65, 681.81±125.02, 861.66±32.48 U/g prot (CAT).

Among the groups, the difference in activity of SOD, GSH-Px

and CAT in the liver homogenate was statistically significant

(P<0.05). The activity levels of SOD, GSH-Px and CAT were

lowest in the OJ model group. The study group had higher

activities that the OJ group (P<0.05), and the normal control

group and sham operation group had the highest activities

(Fig. 3).

Discussion

Obstructive jaundice (OJ) is a jaundice caused by mechanical

obstruction of the bile duct caused by congenital malforma-

tions, inflammation, stones, tumors, parasitic disease and

cholestasis. Its main syndromes include tissue damage to the

liver and gallbladder, and a series of pathological and physi-

ological changes in various body systems. Furthermore, it can

cause dysfunction of intestinal mucous membrane, sepsis and

multiple organ failure (10).

In recent years, numerous studies concerning the mecha-

nisms of liver injury in OJ have been carried out and reveal

that reactive oxygen species (ROS) play an important role.

Oxygen radicals can peroxidize unsaturated fatty acids in

biomembranes to form lipid hydroxide (LPO). LPO can reduce

membrane fluidity, destroy membrane integrity, increase

membrane permeability, and cause cell swelling and increased

liver volume (11). MDA is one of the end-products of lipid

peroxidation and can be used as an index of lipid peroxidation

(12). Normal cells have a complex anti-ROS defense system

that includes enzyme-catalyzed and non-enzymatic methods

to reduce active oxygen radicals. Antioxidant enzymes include

SOD, GSH-Px and CAT. Non-enzymatic antioxidants include

cellulose, amino acids and metalloproteins. Cells produce

small amounts of active oxygen radicals under normal physi-

ological states that can be removed by intracellular antioxidant

enzymes. SOD acts upon the superoxide anion to generate

hydrogen peroxide which can be subsequently broken down

into H2O and O2 by CAT and GSH-Px (13,14). This sequence

reduces potential damage from hydrogen peroxide and

prevents hydrogen peroxide from generating more harmful

radicals (such as the hydroxide radical and alkoxy) through its

interactions with O2 (9).

Capillary artemisia is the dry, ground part of Artemisia

scoparia Waldst and Kit or Artemisia capillaris or feverfew. It

is harvested when its spring seedlings are 6-10 cm high or when

the autumn buds are growing. The stems are removed before

drying. The products harvested during spring are usually

called capillary wormwood, and those harvested in autumn

are called Artemisia capillaris (15). Capillary artemisia is

bitter, acrid and slightly cold, and moves accross channels of

the spleen, stomach, liver and gallbladder. Modern pharma-

cological studies have found that capillary artemisia has the

following functions – it relaxes the gallbladder, protects the

liver, reduces blood sugar, has antioxidant action and removes

heat, dampness and jaundice. It is widely used in the clinical

treatment of jaundice, hepatitis and other diseases (16).

In the present study, capillary artemisia polysaccharide

reduced liver weight, liver coefficient and MDA content as

well as increased the activity levels of SOD, GSH-Px and

CAT. These results indicate outstanding antioxidant and liver-

protective effects, and suggest that these antioxidant effects of

capillary artemisia may be a principal mechanism of effect.

The results were consistent with our hypothesis. Our findings Figure 3. Changes in (A) SOD, (B) GSH-Px and (C) CAT activity in the liver

homogenate among the groups.

HE et al: CAPILLARY ARTEMISIA POLYSACCHARIDE 4

1 2provide a theoretical basis for the clinical treatment of OJ

using capillary artemisia polysaccharide.

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