Accepted Manuscript Title: Protective effects of caffeic acid phenethyl ester against acute radiation-induced hepatic injury in rats Author: JianJun Chu Xiaojun Zhang Liugen Jin Junliang Chen Bin Du Qingfeng Pang PII: DOI: Reference:

S1382-6689(15)00039-3 http://dx.doi.org/doi:10.1016/j.etap.2015.01.020 ENVTOX 2192

To appear in:

Environmental Toxicology and Pharmacology

Received date: Revised date: Accepted date:

7-11-2014 27-1-2015 31-1-2015

Please cite this article as: Chu, J.J., Zhang, X., Jin, L., Chen, J., Du, B., Pang, Q.,Protective effects of caffeic acid phenethyl ester against acute radiationinduced hepatic injury in rats, Environmental Toxicology and Pharmacology (2015), http://dx.doi.org/10.1016/j.etap.2015.01.020 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Graphical Abstract (for review)

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Highlights 1. Caffeic acid phenethyl ester prevented radiation-induced hepatic injury in rat.

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2. Caffeic acid phenethyl ester reversed the serum levels of alanine

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aminotransferase and aspartate aminotransferase and decreased the number of apoptosis cells in rat liver induced by radiation.

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3. Caffeic acid phenethyl ester may be a new hepato-protective

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compound against radiation-induced hepatotoxicity.

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Protective effects of caffeic acid phenethyl ester against acute radiation-induced hepatic injury in rats JianJun Chu1, Xiaojun Zhang 1, Liugen Jin1, Junliang Chen2, Bin Du2,

Department of Oncology, the Affiliated Hospital of Jiangnan University,

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Qingfeng Pang 2,*

200 huihe Road, 214122 Wuxi, China

Wuxi Medical School, Jiangnan University, 1800 Lihu Road, 214122

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Wuxi, China; * corresponding author at:

China;

Tel.:

+86

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Wuxi Medical School, Jiangnan University, Wuxi, jiangsu 214122, PR 15052430172;

Fax:

+086-510-85329042;

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E-mail:[email protected]

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ABSTRACT Caffeic acid phenyl ester (CAPE) is a potent anti-inflammatory agent and it can eliminate the free radicals. The current study was intended to

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evaluate the protective effect of CAPE against the acute radiation-

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induced liver damage in rats. Male Sprague- Dawley rats were

intraperitoneally administered with CAPE (30 mg/kg) for three

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consecutive days before exposing them to a single dose of 30 Gy of

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β-ray irradiation to upper abdomen. We found that pretreatment with CAPE significantly decreased the serum levels of alanine

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aminotransferase and aspartate aminotransferase and increased the activity of superoxide dismutase and glutathione. Histological evaluation

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further confirmed the protection of CAPE against radiation- induced

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hepatotoxicity. TUNEL assay showed that CAPE pretreatment inhibited

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hepatocyte apoptosis. Moreover, CAPE inhibited the nuclear transport of NF- κB p65 subunit, decreased the level of tumor necrosis factor-α,

nitric oxide and inducible nitric oxide synthase. Taken together, these results suggest that pretreatment with CAPE offers protection against radiation-induced hepatic injury.

Keywords: Caffeic acid phenyl ester; Radiation; Liver; Apoptosis

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1. Introduction Radiation therapy (RT) is commonly applied to the cancerous tumor because of its ability to control cell growth. Almost 50% of patients with

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pelvic malignancies are candidates for radiation therapy (Timmerman et

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al., 2014). However, therapeutic radiation affects not only malignant

tumors but also surrounding normal tissues. RT for abdominal tumors

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often leads to radiation-induced liver disease (RILD), which is

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characterized by a pathologic change of venous occlusive disease (VOD) (Cheng et al., 2005). RILD is irreversible once the onset of radiation

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liver fibrosis and is life-threatening complication (Khozouz et al., 2008). Considering that RT can produce sever side effects, it is important to find

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new radioprotective substance to attenuate RILD. It is now widely

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accepted that oxidative stress induced by reactive oxygen species (ROS)

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is involved in the pathogenesis of RILD (Coleman et al., 2014). The interaction of ionizing radiation with water results in the generation of radical species. ROS are highly reactive and could diffuse to vital cellular targets like DNA, proteins and membrane, ultimately leading to cell death.

Since free radicals play a major role in the initiation and progression of radiation induced toxicity, antioxidants might offer protection against radiation induced damage. Recently several researches have been focused on the potential use of propolis as free radical scavengers to 4

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prevent oxidative damage (Aghel et al., 2014; Noronha et al., 2014). Propolis has properties of anti-virus, anti-inflammation and anti-oxidation. One of the major biologically active components of

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propolis is the caffeic acid phenethyl ester (CAPE). CAPE prevented the

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formation of ROS and lipid peroxidation against carbon tetrachloride or

cisplatin induced hepatic oxidative damage (Colakoglu et al., 2011; Kus

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et al., 2004; Kart et al., 2010) . However, no studies have evaluated the

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role of CAPE in radiation-induced hepatic injury. This study aimed to investigate the potential protective effects of CAPE on radiation

2.1. Chemicals

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2. Material and methods

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-induced hepatotoxicity in rats.

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The following reagents were purchased from the indicated sources:

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CAPE (Sigma-Aldrich, St. Louis, USA); TUNEL in situ detecting kit (KG Nanjing Ltd., China); TNF-α ELISIA KIT ((Duosets, R&D systems, Minneapolis, MN)); NF-κB p65 primary antibody and HRP-conjugated secondary antibody (Santa Cruz Biotechnology); GSH and SOD assay kit (Nanjing Jiancheng Bioengineering, China). All other reagents were of analytical grade and obtained commercially.

2.2. Animals Male Sprague-Dawley (5-6 week old) rats were provided by the 5

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experiment animal center of Jiangsu Institute of Schistosomiasis, key laboratory of Jiangsu province (Certificate NO. Su BKS 2007-003). Rats were housed under barrier conditions and kept at 22-25℃ with a 12h-

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light-dark cycle. Rats were allowed free access to food and water

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throughout the experimental period. All animal experiments were started after 1 week of acclimation and were performed in accordance with

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institutional guidelines.

2.3. Study design

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Thirty-two rats were randomly divided into four groups of eight each and intraperitoneally administered with the different treatments for 4

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days after radiation: (1) Control group, rats were given daily

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intraperitoneal (i.p.) injections of normal saline for 3 successive days; (2)

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CAPE group, rats were given CAPE(30 mg/kg per day, i.p.) for 3 successive days (Yiş et al., 2013); (3)RT group, rats administrated a

single 30 Gy fraction to upper abdomen(Du et al., 2010); (4) CAPE and RT group, rats were given CAPE(30 mg/kg per day, i.p.) for 3 successive days; three hours after the final administration, rats were treated a single 30 Gy fraction to upper abdomen.

2.4. Irradiation Rats were anesthetized with an intraperitoneal injection of 6

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pentobarbital sodium and subjected to a single dose irradiation of 30Gy fraction ionizing radiation to the upper abdomen, including the whole liver, through an accelerator (Varians, 2300CD23EX, American)

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producing 6-MV X-rays after simulation, absorbed dose rate being 200

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cGy/ min, the source-skin distance being 100 cm. After irradiation, the rats were placed in cages and had free access to water.

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Four days after radiation, all rats were sacrificed by cervical

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dislocation, and blood collected from the heart into plain centrifuge tube. The left hepatic lobe of each group from the same position were excised

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for light microscope and TUNEL assay, while the right hepatic lobe were

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GSH and SOD.

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douched thoroughly with saline for the determination of the activity of

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2.5. Analyses of serum markers The blood specimens were centrifuged at 3000rpm/min for 5 min. The

suctions of upper layer serum were obtained and the content of alanine aminotransferase(ALT) and aspartate aminotransferase (AST) were determined by using Automatic biochemical analyzer.

2.6. Determination of glutathione (GSH) activity, superoxide dismutase (SOD) activity in liver tissue The activities of GSH and SOD were measured using a commercially 7

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available kit(Jiancheng Biotech Ltd, Nanjing, China). GSH and SOD activities measurement were based on the instructions of the colorimetric

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method by the Beckman Coulter DU80.

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2.6. Measurement of nitric oxide (NO), inducible nitric oxide synthase (iNOS) levels in liver tissue

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The concentration of and the activity in liver tissue were determined

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by using an NO and NOS Kit (Jiancheng Biotech Ltd, Nanjing, China)

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according to the manufacture’ instructions.

2.7. Assay the level of serum tumor necrosis factor-α (TNF-α)

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The concentrations of TNF-α in serum were quantified by ELISA

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using the manufacturer’s suggested protocol (Duosets, R&D systems,

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Minneapolis, MN).

2.8. Histological assessment Formalin-fixed and paraffin-embedded tissue sections were cut at 5

μm and stained with hematoxylin and eosin for histological examination.

Tissues were examined under light microscopy by a blinded observer and were scored using a system described by Serafin et al., 2002.

2.9. Transmission electron microscopy microscope 8

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Hepatic specimens were fixed in 4% glutaral buffered and 1% osmium tetroxide in turn. They were then dehydrated through an ethanol series. Thin (0.2-1.0μm) and ultrathin (pale gold, 40-50 nm) sections

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were cut with a LKB4801 A ultramicrotome using a diamond knife. The

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thin sections were stained with uranyl acetate and Lead Citrate. They were examined with a ransmission electron microscopy microscope

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(Hitachi HD-2600).

2.10. TUNEL assay

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The number of apoptotic cells (5 microscope field in each section) in the liver was determined by the TUNEL method. The whole detecting

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process was undertaken strictly according to the TUNEL in situ detecting

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kit protocol ((KG Nanjing Ltd., Nanjing, China). The fluorescence

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microscope (Olympus IX51, ×200) was used to observe and photograph specimens. The cells with green fluorescent particles or fragments were deemed to apoptosis cells. Five microscope fields of each section were picked to calculate apoptosis index (AI) by using the formula: AI= account of positive cells/account of total cells×100%.

2.11. Western blot analysis The nucleoprotein was extracted with nucleoprotein extracted kit (KG Nanjing Ltd., Nanjing, China) according to the manufacturer’s 9

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instructions. The cell lysates were separated electrophoretically using 10% SDS-PAGE, and then the gel was transferred to nitrocellulose paper. The membranes was blocked with 5% nonfat milk in phosphate buffered

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saline containing 0.1% Tween-20 for 1 h at room temperature, washed in

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PBS-T four times, followed by incubation in a 1:1000 dilution of

primary antibody (Santa Cruz, CA) in PBS overnight at 4℃. After six

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washes in PBS, membranes were incubated for 1 h at room temperature

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in a 1: 5000 dilution of HRP-conjugated secondary antibody (Santa Cruz, CA). The blot was detected by the enhanced chemiluminescence

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detection system according to the recommended procedure.

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2.12. Statistical analysis

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Data were analyzed with SPSS 13.0 software and expressed as mean

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± SEM. Differences between groups were calculated by using one-way analysis of variance. Differences were considered to be significant for P values less than 0.05.

3. Results

3.1. Effect of CAPE on the content of serum ALT and AST The increased content of serum ALT and AST levels (66.6±14.1 and 176.4±22.3, respectively) were observed in RT group animals (P < 0.05). However, CAPE pretreatment significantly reduced serum ALT and 10

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AST levels at 4 days after radiation (Tab. 1, P < 0.05). 3.2. Effect of CAPE on antioxidant enzymes activities There was no significant difference (P>0.05) in the activity of SOD

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and GSH of control and CAPE group. The activity of SOD and GSH

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significantly decreased in RT group compared with those of control

changes as shown in Tab. 2 (P< 0.05).

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group and CAPE group (P

Protective effects of caffeic acid phenethyl ester against acute radiation-induced hepatic injury in rats.

Caffeic acid phenyl ester (CAPE) is a potent anti-inflammatory agent and it can eliminate the free radicals. The current study was intended to evaluat...
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