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Food and Chemical Toxicology 55 (2013) 60–69

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Food and Chemical Toxicology


journal homepage: www.elsevier.com/locate/foodchemtox

Protection of the flavonoid fraction from Rosa laevigata Michx fruit against
carbon tetrachloride-induced acute liver injury in mice
Shuai Zhang a,1, Binan Lu b,1, Xu Han a, Lina Xu a, Yan Qi a, Lianhong Yin a, Youwei Xu a, Yanyan Zhao a,
Kexin Liu a, Jinyong Peng a,c,⇑
a
College of Pharmacy, Dalian Medical University, 9 Western Lvshun South Road, Dalian 116044, China
b
Institute of Chinese Minority Traditional Medicine, Minzu University of China, 27 Zhongguancun South Avenue, Hadian District, Beijing 100081, China
c
Research Institute of Integrated Traditional and Western Medicine of Dalian Medical University, Dalian 116011, China

a r t i c l e i n f o a b s t r a c t

Article history: Protective effect of the total flavonoids (TFs) from Rosa laevigata Michx fruit against carbon tetrachloride
Received 9 October 2012 (CCl4)-induced hepatotoxicity in mice was investigated. Pretreatment with TFs significantly decreased
Accepted 21 December 2012 CCl4-induced elevation of serum aspartate transaminase (AST) and alanine transaminase (ALT) activities
Available online 29 December 2012
as well as the relative liver weight. Histopathological observation also revealed that TFs reduced the inci-
dence of liver lesions and improved hepatocyte abnormality. Moreover, oral administration of TFs signif-
Keywords: icantly enhanced antioxidant enzyme activities (superoxide dismutase, catalase and glutathione
Acute liver injury
peroxidase), increased the content of glutathione and decreased the content of malondialdehyde. Further
Carbon tetrachloride
Hepatoprotective effect
research indicated that TFs prevented the DNA fragmentation and mitochondrial ultrastructural altera-
Rosa laevigata Michx tions caused by CCl4 based on TUNEL and transmission electron microscopy (TEM) assays. Moreover, pre-
Total flavonoids treatment with TFs down-regulated the protein expressions of CYP2E1, iNOS, NF-jB, Bak and Caspase-3.
Quantitative Real-time PCR assay suggested that TFs markedly decreased the levels of TNF-a, Fas/FasL
and Bax gene expressions, and increased the level of Bcl-2. This is the first time to report the significant
hepatoprotective effect of TFs from R. laevigata Michx fruit against CCl4-induced liver injury in mice and
the action should be through reducing oxidative stress and suppressing inflammation and apoptosis.
Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Plants, especially to traditional herbal medicine, are rich source


of bioactive components that have desirable health benefits and ef-
Carbon tetrachloride (CCl4) is a well-known environmental bio- fects on the prevention of human diseases (Balunas and Kinghorn,
hazard, which can cause particularly toxic to the liver. CCl4-in- 2005). Many studies have been reported that herbal extracts or
duced hepatic injury, a classic experimental model, has been nature products have markedly protective effects against oxidative
extensively used to evaluate the potential of drugs and dietary stress by enhancing antioxidant enzyme activities to prevent CCl4-
antioxidants against the oxidative damage (Basu, 2003). Free radi- induced hepatotoxicity (Hung et al., 2006; Huang et al., 2012;
cals, such as trichloromethyl (CCl3 and/or CCl3OO) and oxygen- Nagalekshmi et al., 2011; Brito et al., 2012).
centered lipid radicals (LO and/or LOO), are pivotal in CCl4-induced Rosa laevigata Michx, a famous medicinal plant, belongs to the
hepatotoxicity, which are generated during CCl4 metabolism by Rosaceae family and the fruit of this plant has been widely used
hepatic cellular cytochrome P450 (Recknagel et al., 1989; Li in traditional Chinese medicine for a long history (Chinese Pharma-
et al., 2010). In addition, the activation of Kupffer cells also contrib- copoeia Commission, 2010). Multiple constituents including
utes to the liver injury through releasing both direct toxic products polysaccharose, flavonoids, saponins and triterpenes exist in
and cytokines which promote inflammatory response (Taniguchi R. laevigata Michx (Zhao et al., 2003; Chen and Zhang, 2005; Xiao
et al., 2004). The liver injury may be prevented or treated by block- and Liu, 2006; Wu et al., 2009). In previous reports, the fruit mainly
ing or retarding the process of oxidative stress and inflammation contained polyphenol substances, such as rutin, quercetin,
(Lin et al., 2012). kaempferol, luteolin, apigenin and liquiritigenin (Huang et al.,
2009; Li et al., 2012). The total flavonoids (TFs) extracted from
⇑ Corresponding author at: College of Pharmacy, Dalian Medical University, 9 the fruit were mainly flavone and flavonol (Chen and Zhang,
Western Lvshun South Road, Dalian 116044, China. Tel./fax: +86 411 8611 0411. 2005), and three aglucones including quercetin, kaempferide and
E-mail address: jinyongpeng2005@163.com (J. Peng). isorhamnetin have been investigated (Zhang et al., 2012). Our
1
These authors contributed same work to this paper and they are the co-first previous study have demonstrated TFs from R. laevigata Michx fruit
authors.

0278-6915/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.fct.2012.12.041
S. Zhang et al. / Food and Chemical Toxicology 55 (2013) 60–69 61

has powerful in vitro antioxidant activity (Liu et al., 2010), signifi- 2.4. Experimental design
cant hepatoprotective effect against paracetamol induced liver
After acclimation for 1 week, the animals were randomly divided into seven
damage in mice (Liu et al., 2011), and effective action against groups (n = 10). Group I (normal control) was given distilled water (10 ml/kg body
H2O2-induced damage in human umbilical vein endothelial cells weight). Group II (TFs control) received TFs (200 mg/kg, dissolved in water). Group
(Jia et al., 2012). III (model) was given distilled water. Groups IV, V and VI (low, medium and high
In the light of all these bases, the present study was to investigate dosage) were administrated TFs (50, 100 and 200 mg/kg, respectively, dissolved
in water). Group VII (positive control) was given silymarin (200 mg/kg, dissolved
the hepatoprotective effect of TFs on CCl4-induced hepatotoxicity in
in water). After the oral administration for 7 days, the animals were treated as de-
mice and then to explore the possible mechanisms of the action. scribed previously (Lu et al., 2012) with some modifications. Two hours after the fi-
nal administration, mice in Groups III–VII were injected intraperitoneally with 0.3%
(v/v) CCl4 (10 ml/kg, dissolved in olive oil), while the mice in Groups I and II re-
2. Materials and methods
ceived appropriate vehicle (olive oil, i.p.). Twenty-four hours after the CCl4 chal-
lenge followed by fasting, the animals were anesthetized for obtaining the blood
2.1. Reagents
and sacrificed to collect the livers. The fresh liver was weighed to calculate relative
liver weight (Relative liver weight (%) = liver weight/body weight  100). The right
Silymarin was obtained from Sigma Chemical Company (Milan, Italy). CCl4 was
lobe of the liver was fixed in 10% formalin to prepare paraffin sections and the
purchased from Kaixing Chemical Industry Co., Ltd. (Tianjin, China). Aspartate
remaining parts were stored at 80 °C for the other assays.
aminotransferase (AST), alanine aminotransferase (ALT), superoxide dismutase
(SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione (GSH) and mal-
ondialdehyde (MDA) detection kits were purchased from Nanjing Jiancheng Insti- 2.5. Serum biochemistry
tute of Biotechnology (Nanjing, China). Enhanced Bicinchoninic Acid (BCA)
Protein Assay Kit, Tissue Mitochondria Isolation Kit and Mitochondrial Membrane AST and ALT activities in serum were determined by using detection kits
Potential Assay Kit were obtained from Beyotime Institute of Biotechnology (Jiangsu, according to the manufacturer’s instructions to assess CCl4-induced hepatotoxicity.
China). Tissue Protein Extraction Kit was purchased from KeyGEN Biotech. Co., Ltd.
(Nanjing, China). 40 ,60 -Diamidino-2-phenylindole (DAPI), Tris and SDS were
purchased from Sigma (St. Louis, MO, USA). In Situ Cell Death Detection Kit, POD 2.6. Histopathological examination
was obtained from Roche Diagnostics (Germany). Hematoxylin, Histostain™-Plus
Kit and 3,30 -Diaminobenzidine tetrahydrochloride (DAB) Substrate Kit were Formalin-fixed specimens were embedded in paraffin and sectioned for 5 lm
provided by Zhongshan Golden Bridge Biotechnology (Beijing, China). RNAiso Plus, thickness according to the routine procedure. After hematoxylin and eosin (H&E)
PrimeScriptÒ RT reagent Kit with gDNA Eraser (Perfect Real Time) and SYBRÒ Premix staining, the slides were observed for conventional morphological evaluation under
Ex Taq™ II (Tli RNaseH Plus) were supplied by TaKaRa Biotechnology Co., Ltd. a light microscope (Nikon Eclipse TE2000-U, NIKON, Japan) and photographed at
(Dalian, China). Rabbit anti-CYP2E1 was obtained from US Biologicals. Rabbit anti- 100 magnification.
iNOS, rabbit anti-NF-jB, rabbit anti-Caspase-3, rabbit anti-Bak, mouse anti-GAPDH,
horseradish peroxidase-conjugated goat anti-rabbit IgG, and horseradish peroxi-
dase-conjugated goat anti-mouse IgG antibodies were provided by Proteintech Group, 2.7. Assay of antioxidant markers in liver tissue
Inc. (Chicago, USA).
Liver tissues were prepared to make 1:9 (w/v) homogenates with cold physio-
logical saline. The homogenate was centrifuged (2500g for 10 min at 4 °C) to collect
2.2. Herbal material and preparation of TFs supernatants for the subsequent determinations. The total protein concentration in
the liver tissue homogenate was measured by the reaction with BCA (Smith et al.,
The fruit of R. laevigata Michx was obtained from Yunnan Qiancaoyuan Pharma- 1985). SOD activity was estimated according to the previous method (Kono,
ceutical Company Co. Ltd. (Yunnan, China) and identified by Dr. Yunpeng Diao (Col- 1978). The determination of CAT activity was based on the method of Yao et al.
lege of Pharmacy, Dalian Medical University, Dalian, China). A voucher specimen (2006). GSH-Px activity was detected with 50 -dithio-bis-(2-nitrobenzoic acid) and
(DLMU, JYZ0-80426) has been deposited in the Herbarium of the College of Phar- the change in absorbance at 412 nm was measured (Hafeman et al., 1974). The con-
macy, Dalian Medical University (Dalian, China). The TFs from R. laevigata Michx tent of GSH was determined by the reaction with 50 -dithio-bis-(2-nitrobenzoic acid)
fruit was prepared according to the previous report (Zhang et al., 2012). Briefly, that produces a yellow complex with absorption maximum at 412 nm by the re-
dried R. laevigata Michx fruit (500 g) was crushed and extracted with 60% aqueous ported method (Yao et al., 2006). All values were normalized by the total protein
ethanol (4000 ml) for two times and 2 h for each under heat and reflux. After con- concentration of the same sample.
densed under low pressure at 60 °C, the extracted solution was added into a D101
macroporous resin column (Chemical Plant of Nankai University, Tianjin, China).
After elusion with water, the fraction eluted with 40% aqueous ethanol was evapo- 2.8. Assay of lipid peroxidation products
rated under reduced pressure at 60 °C to dryness, and the produced dry powders
were stored at 4 °C for subsequent experiments. Lipid peroxidation was detected by measuring the concentration of TBARS in li-
The content of TFs in the crude extract was determined by colorimetric method ver using the reported method (Hsu et al., 2009). Under the manufacturer’s instruc-
based on the previous methods (Chinese Pharmacopoeia Commission, 2010; Liu tions, the MDA formed in the liver was determined by reaction with thiobarbituric
et al., 2011) with some modifications. External standard calibration was used for acid (TBA) and the content of MDA was expressed as nmol/mg protein.
the quantitative analysis with rutin as the reference standard. A series of standards
of rutin in the range of 8–48 lg/ml were prepared in 40% ethanol solution to obtain
a linear response with correlation coefficient of 0.997 (n = 6). TFs (10 mg) was 2.9. DAPI staining
placed in a 25 ml volumetric flask with the solvent of 40% ethanol solution. 3 ml
TFs solution and 3 ml 40% ethanol solution were placed in another 25 ml volumetric Morphological assessment of nuclei was performed with DAPI staining. The par-
flask containing 1 ml of 5% (w/v) sodium nitrite and placed for 6 min, followed by affin sections were deparaffinized with xylene (two times, each for 15 min) and
reaction with 1 mL of 10% (w/v) aluminum nitrate to form a complex. Six minutes rehydrated with different concentration gradient of alcohol (100%, 90%, 80%, 70%
later, 10 ml of 4% (w/v) NaOH was added and the total volume was supplemented to and 60%) once for 5 min. Then the sections were incubated with DAPI (1 lg/ml)
25 ml with distilled water. Then the solution was mixed sufficiently and placed for for 8 min at room temperature, then washed with PBS and examined by fluores-
10 min at the room temperature. The absorbance was measured at 500 nm by UV– cence microscopy (Nikon Eclipse TE2000-U, NIKON, Japan).
Vis spectrophotometer (U-3010, Hitachi, Japan) and the percentage of TFs in the
crude extract was 81.5%.
2.10. Determination of mitochondrial membrane potential

2.3. Animals Mitochondria were isolated from liver tissue according to the manufacturer’s
instructions. Briefly, 100 mg liver tissue was obtained from the rats in control, mod-
Male Kunming mice (18–22 g) were purchased from the Experimental Animal el and TFs (200 mg/kg) groups after deep anesthesia. Then isolation reagent A (1 ml)
Center of Dalian Medical University (Dalian, China). The animals were maintained were mixed with the tissue and both were made into homogenates, which were
in a controlled environment under standard conditions with temperature centrifuged (600g for 5 min at 4 °C) to collect the supernatants. After the centrifuge
(21 ± 3 °C), relative humidity (55–70%), 12 h light/dark cycles and had access to of the supernatants at 11,000g for 10 min at 4 °C, the obtained supernatants were
standard chow diet (Xietong Organism Institute, Nanjing, China) and water ad libi- centrifuged at 12,000g for 10 min at 4 °C again, then the sediment were mitochon-
tum. The animal experiment was approved by the Ethical Committee (Number: dria and suspended in 40 ll PBS. All of the procedures were accomplished in iced
20120623) and the Laboratory Animal Center of Dalian Medical University. All pro- bath. After incubation with JC-1 dye in the dark for 5 min, mitochondrial membrane
cedures involving animals complied with the China National Institutes of Healthy potential was detected by the green and red fluorescence, observed and imaged
Guidelines for the Care and Use of Laboratory Animals. with a laser scanning confocal microscope (Leica, TCS SP5, Germany).
62 S. Zhang et al. / Food and Chemical Toxicology 55 (2013) 60–69

Table 1 2.11. Transmission electron microscopy (TEM)


Primer sequences used for Real-time PCR assay.
Fresh liver tissue was obtained from the rats in control, model and TFs (200 mg/
Name Sequence (50 ? 30 )a Genbankb kg) groups after deep anesthesia and fixed overnight at 4 °C in 2% glutaraldehyde.
GAPDH TGTGTCCGTCGTGGATCTGA NM_008084.2 The following treatment was carried out as described previously (Jia et al., 2012).
TTGCTGTTGAAGTCGCAGGAG The ultramicrotomies were observed and imaged with an electron microscope
TNF-a TATGGCCCAGACCCTCACA NM_013693.2 (JEM-2000EX, JEDL, Japan).
GGAGTAGACAAGGTACAACCCATC
Fas ACATGGACAAGAACCATTATGCTGA NM_007987.2
2.12. TUNEL assay
CTGGTTTGCACTTGCACTTGGTA
FasL CATGCAGCAGCCCATGAATTAC NM_010177.4
TUNEL assay was performed on paraffin-embedded sections from control, mod-
CTCTAGGCCCACAAGATGGACAG
el and TFs (200 mg/kg) groups according to the protocol of the kit. Briefly, the de-
Bcl-2 TGAAGCGGTCCGGTGGATA NM_177410.2
waxed course was prescribed above. After the incubation with Proteinase K
CAGCATTTGCAGAAGTCCTGTGA
solution (10–20 lg/ml in 10 mM Tris/HCl, pH 7.4–8.0) for 8 min, the fluorescein
Bax CAGGATGCGTCCACCAAGAA NM_007527.3
(green) labeled dUTP solution was added on the sections and incubated at 37 °C
CGTGTCCACGTCAGCAATCA
for 1 h. Then the sections were washed and photographed using fluorescence
a
Shown as forward primer followed by reverse primer. microscopy (magnification, 200). 50 ll converter-POD was added on the samples
b
GenBank Accession Number. and reacted at 37 °C for 30 min. Then the sections were washed and counterstained
with the DAB solution (5 ll 20  DAB, 1 ll 30% H2O2 and 94 ll PBS) and hematox-
ylin. Images were obtained using a light microscopy (magnification, 200). Ten

Fig. 1. Effects of the TFs on serum AST (A), ALT (B), relative liver weight (C) and histopathological examination (D) in CCl4-intoxicated mice. I: normal control; II: TFs control;
III: model; IV: TFs (50 mg/kg) + CCl4; V: TFs (100 mg/kg) + CCl4; VI: TFs (200 mg/kg) + CCl4; VII: silymarin (200 mg/kg) + CCl4. Relative liver weight (%) = liver weight/body
weight  100. Hematoxylin and eosin stained sections (magnification: 100) showed the injured areas (arrows). Values expressed as mean ± SD in each group. p < 0.05,

p < 0.01 vs. model, n = 10.
S. Zhang et al. / Food and Chemical Toxicology 55 (2013) 60–69 63

microscopic fields in each group were randomly selected for the count of positive were quantified by Real-time PCR with SYBRÒ Premix Ex Taq™ II (Tli RNaseH Plus)
cells. The extent of DNA damage was evaluated by the average number of positive and 7500 Real Time PCR System (Applied Biosystems, USA). The primers used for
cells (green spots) in the fluorescent images and the positive area in each DAB Real-time PCR are listed in Table 1. The GAPDH gene was amplified separately as
stained image. an internal control to normalize for specific gene expression in the samples. Fold
change between Groups III, IV, V, VI and Group I was calculated using the 2DDCt
2.13. Immunohistochemical analysis for CYP2E1 and iNOS method (Livak and Schmittgen, 2001).

The paraffin sections were deparaffinized and rehydrated as above and were 2.15. Western blotting assay
treated with 0.01 mol/l citrate (pH = 6.0) in a microwave oven for 15 min for anti-
gen retrieval. Then the following steps were done according to the instructions of Total protein, used for western blotting analysis, was isolated from the livers in
Histostain™-Plus and DAB substrate kits. Briefly, 3% H2O2 was used to block endog- Groups I, III, IV, V, VI using the Tissue Protein Extraction Kit on the base of the man-
enous peroxidase activity for 10 min and nonspecific protein binding was blocked ufacturer’s instructions. Briefly, liver tissues were collected and lysed in appropriate
by normal goat serum for 20 min. The treated slides were incubated in a moist cold lysis buffer contained 1 mM phenylmethylsulfonyl fluoride (PMSF). Then the
box at 4 °C overnight with rabbit anti-CYP2E1 and rabbit anti-iNOS antibodies mixture was centrifuged at 15,000g for 15 min at 4 °C and the total protein was ob-
(1:100, dilution), followed by incubation in biotin labeled goat anti-rabbit IgG tained in the supernatant. After the determination of protein contents, protein sam-
and horseradish peroxidase-conjugated streptavidin for 15 min respectively. DAB ple (6 mg/ml) was denatured by mixing with an equal volume of 2  sample
fluid including 1 ml distilled water, 50 ll H2O2 (20) and 50 ll DAB (20) was used loading buffer, and boiling at 100 °C for 5 min. An aliquot of the supernatant was
in color development, and counterstained by hematoxylin. Image was taken by a separated by electrophoresis with 10% SDS–PAGE and transferred onto a PVDF
light microscopy (magnification, 200). membrane (Millipore, USA). After blocking non-specific binding sites for 3 h with
5% dried skim milk dissolved in TTBS (10 mM TBS plus 1.0% Tween 20), the mem-
2.14. Quantitative Real-time PCR branes were individually incubated for overnight with rabbit anti-NF-jB (1:700
dilution), rabbit anti-Caspase-3 (1:700 dilution), rabbit anti-Bak (1:700 dilution),
Total RNA was isolated from liver tissue using RNAiso Plus reagent according to or mouse anti-GAPDH (1:1000 dilution). Then blots were incubated at room tem-
the manufacturer’s instructions. cDNA was synthesized using PrimeScriptÒ RT re- perature for 3 h with either the secondary goat anti-rabbit (1:1000 dilution) or
agent Kit with a TC-512 PCR system (TECHNE, UK). The levels of mRNA expression the goat anti-mouse (1:2000 dilution) IgG-horseradish peroxidase-conjugated anti-

Fig. 2. Effects of the TFs on the levels of SOD (A), CAT (B) GSH-Px (C), GSH (D) and MDA (E) in the livers in CCl4-intoxicated mice. I: normal control; II: TFs control; III: model;
IV: TFs (50 mg/kg) + CCl4; V: TFs (100 mg/kg) + CCl4; VI: TFs (200 mg/kg) + CCl4; VII: silymarin (200 mg/kg) + CCl4. Values expressed as mean ± SD in each group. p < 0.05,

p < 0.01 vs. model; #p < 0.05, ##p < 0.01 vs. normal control; n = 10.
64 S. Zhang et al. / Food and Chemical Toxicology 55 (2013) 60–69

bodies. Protein expression was detected by an enhanced chemiluminescence (ECL) administration of CCl4 caused severe hepatotoxicity, as indicated
method and photographed by Bio-Spectrum Gel Imaging System (UVP, USA). To
by the significant elevation of serum AST and ALT activities. How-
eliminate the variations due to protein quantity and quality, the data were adjusted
to GAPDH expression (IOD of objective protein versus IOD of GAPDH protein).
ever, TFs pretreatment prevented these elevations in a dose-
dependent manner and the extract at the dose of 200 mg/kg exhib-
2.16. Statistical analysis ited a significant decrease in the activities of these serum enzymes
compared with model (p < 0.01). Silymarin also significantly re-
Data were analyzed by conducting One-Way ANOVA using the SPSS software versed the alterations of the AST and ALT levels compared with
v.11.5 and expressed as mean and standard deviation (SD). Tukey in Post Hoc Multi-
the model group (p < 0.05). Consistent with these modifications,
ple Comparisons was used to examine statistical significance (p < 0.05 and p < 0.01)
between groups. relative liver weight was also reduced markedly in TFs treated
mice (Fig. 1C).
3. Results
3.1.2. Histopathological examination
3.1. Effects of TFs on CCl4-induced liver injury Hematoxylin and eosin stained sections are shown in Fig. 1D.
Normal liver lobular architecture and cell structure were shown
in normal control and TFs control groups. While the liver tissue
3.1.1. Serum AST and ALT activities of the mice treated with CCl4 alone showed apparent morphologi-
The effects of TFs pretreatment on the CCl4-induced increase of cal changes including large areas of extensive cell necrosis with
serum AST and ALT activities are shown in Fig. 1A and B. The loss of hepatocyte architecture around the blood vessels. Further-

Fig. 3. Representative micrographs of DAPI stained nuclei (magnification: 200), JC-1 labeled mitochondria (magnification: 400) and TEM (magnification: 25,000) in
normal control, model and TFs (200 mg/kg) + CCl4 groups. Fragmentation or condensation of nucleus in the liver tissues was shown in DAPI stained sections. Mitochondrial
membrane potential was detected by JC-1 staining. In the merged fluorescent images of mitochondria extracted from fresh liver tissue, the green fluorescence demonstrated
the mitochondria depolarization. The morphological alternations of nuclei and mitochondria (arrows) indicated in TEM images.
S. Zhang et al. / Food and Chemical Toxicology 55 (2013) 60–69 65

more, some condensed nuclei and massive inflammatory cells infil- the model, which were lightened by pretreatment with TFs
tration were observed in the injured area. However, the TFs pre- (200 mg/kg).
treatment significantly decreased the injured area, necrotic cells
and inflammatory infiltration. 3.3.2. Mitochondrial membrane potential (DWm)
After the extracted mitochondria from the livers of mice were
labeled with JC-1, red dot-like fluorescence reflecting JC-1 aggrega-
3.2. TFs suppressed CCl4-induced oxidative liver injury tion (J-aggregates) within the matrix was observed with high
DWm. While JC-1 could not pass through the impaired mitochon-
The levels of SOD, CAT, GSH-Px and GSH in livers (Fig. 2A–D) dria with low DWm and existed as monomer emitted green fluo-
showed significant decreases in the mice treated with CCl4 alone rescence. From the results of fluorescent images (Fig. 3), TFs
compared with normal control group (p < 0.01). However, the TFs (200 mg/kg) significantly increased the red fluorescence compared
pretreated animals showed a significant improvement towards with the model group and decreased the impaired mitochondria.
the control values. TFs at the dose of 200 mg/kg could obviously
elevate their levels (p < 0.01 or p < 0.05). MDA, a product of lipid
peroxidation, was detected to further evaluate the degree of oxida- 3.3.3. TEM
tive injury. As shown in Fig. 2E, significant increase of MDA content The results of TEM further revealed the status of DNA and mito-
was observed in the liver of mice exposed to CCl4 (p < 0.01), while chondria, showed in Fig. 3. Compared with the normal control,
the administration of TFs (100, 200 mg/kg) significantly decreased mitochondria valley disappearance, condensed nucleus, chromatin
the MDA levels in liver tissues compared with model (p < 0.01). condensation and margination were observed in CCl4-treated
Therefore, the TFs had markedly antioxidant activities and sup- hepatocyte. While the cellular morphology indicated that the TFs
pressed CCl4-induced oxidative liver injury. (200 mg/kg) could markedly decrease nuclear and mitochondrial
injury and exhibited a good integrity.

3.3. Effects of TFs on nuclear and mitochondrial injury 3.3.4. DNA fragmentation confirmed by TUNEL
Photomicrographs of TUNEL staining were presented in Fig. 4.
3.3.1. DNA stained by DAPI TUNEL-positive cells showed green fluorescence and brown after
After DAPI staining, the normal nuclei in liver tissues were fluorescein labeling and DAB staining respectively (Fig. 4A). Com-
round and emitted even blue fluorescence, shown in normal pared with model group, the administration of TFs (200 mg/kg)
control group (Fig. 3). Whereas, loss of nuclei, DNA fragments could obviously reduce the DNA fragmentation (p < 0.01) and had
and condensed nucleus were observed around the blood vessel in the potential to ameliorate the DNA damage (Fig. 4B and C).

Fig. 4. (A) TUNEL stained liver sections (magnification, 200) in normal control, model and TFs (200 mg/kg) + CCl4 groups. In fluorescent images, green fluorescence indicated
the positive cells. In 3,30 -Diaminobenzidine tetrahydrochloride and hematoxylin (DAB&H) stained images, the brown staining represented the positive areas. (B) Statistic
analysis of TUNEL fluorescent images. (C) Statistic analysis of DAB&H stained images. Values expressed as mean ± SD in each group. p < 0.01 vs. model. n = 7.
66 S. Zhang et al. / Food and Chemical Toxicology 55 (2013) 60–69

3.4. Effects of TFs on CYP2E1 and iNOS protein expressions and ALT levels by CCl4 suggested the hepatic structural damage
and they released into the circulation (Recknagel et al., 1989). How-
CYP2E1 and iNOS are important enzymes resulting in oxidative ever, the TFs pretreatment markedly reversed the alternations and
stress in CCl4 treated mice. Immunohistochemical analysis re- protected hepatocytes from CCl4 induced liver damage (Fig. 1A–C).
vealed that amounts of hepatic CYP2E1 and iNOS proteins in- Moreover, histopathological examination also indicated the protec-
creased considerably after CCl4 injection with the exhibition of tive effects of TFs by reducing the liver injury around centrilobular
brown staining (Fig. 5). In contrast, TFs (200 mg/kg) significantly area and TFs (200 mg/kg) could block the process of the ballooned
attenuated the increase in the levels of those proteins (p < 0.01) hepatocytes to necrosis or apoptosis (Shi et al., 1998). In contrast,
and effectively defended the livers against oxidative stress. the effect of TFs (200 mg/kg) was more notable than the others
and even better than silymarin. Silymarin had been demonstrated
to prevent CCl4-induced lipid peroxidation and hepatotoxicity in
3.5. Effects of TFs on the levels of TNF-a, NF-jB, Fas/FasL, Bcl-2, Bax,
mice by decreasing the metabolic activation of CCl4 and acting as a
Bak and Caspase-3
chain-breaking antioxidant (Lettéron et al., 1990; Muriel and Mou-
relle, 1990). The above results suggest that TFs have protective ef-
As shown in Fig. 6A, C, D and F, TFs pretreatment significantly
fects on CCl4 induced acute liver injury.
decreased CCl4-induced TNF-a, Fas/FasL and Bax gene expressions
The hepatic toxicity of CCl4 probably depends on the formation of
compared with the model group. Further investigation indicated
the trichloromethyl radical (CCl3), which interacts with oxygen to
that the level of Bcl-2 gene expression was markedly elevated by
form the more toxic CCl3OO (Shi et al., 1998). To prevent the damage
the administration of TFs (Fig. 6E). Furthermore, increases in the
caused by oxygen-free radicals, an antioxidant defense system
levels of NF-jB, Bak and Caspase-3 protein expressions resulted
including nonezymatic antioxidants (e.g., glutathione, vitamins C
from CCl4 treatment were markedly reversed by TFs administra-
and E) and enzymatic activities such as SOD, CAT, and GSH-Px plays
tion (Fig. 6B, G and H).
an important role. The change in antioxidant enzyme activities is rel-
evant to the ability of the liver to cope with oxidative stress during
4. Discussion CCl4 poisoning (Szymonik-Lesiuk et al., 2003). The present study
showed that SOD, CAT, GSH-Px and GSH levels were significantly
Administration of CCl4 is a well-established experimental model elevated in the liver after the treatment of TFs compared with the
of severe toxic liver injury (Weber et al., 2003) as evidenced by sig- model. Furthermore, MDA, an indicator of oxidative liver injury
nificantly increased serum AST and ALT activities as well as the rel- (Cesaratto et al., 2004), was also reduced in TFs-treated groups. All
ative liver weight in the present study. AST and ALT are normally of these indicated that TFs had prominent antioxidant activity
localized to the hepatic cytoplasm, while increases in serum AST against CCl4-induced oxidative liver damage.

Fig. 5. Effects of TFs (200 mg/kg) on the expressions of CYP2E1 and iNOS with the immunohistochemical method (magnification, 200). Statistic analysis was based on the
values of integrated optical density. Values expressed as mean ± SD in each group. p < 0.01 vs. model. n = 7.
S. Zhang et al. / Food and Chemical Toxicology 55 (2013) 60–69 67

Fig. 6. Effects of TFs on the gene (A: TNF-a, C: Fas, D: FasL, E: Bcl-2, F: Bax) and protein (B: NF-jB, G: Bak, H: Caspase-3) expressions of the liver tissue in normal control (I),
model (III), TFs (50 mg/kg) + CCl4 (IV); TFs (100 mg/kg) + CCl4 (V); TFs (200 mg/kg) + CCl4 (VI) groups. Values expressed as mean ± SD in each group. p < 0.05, p < 0.01 vs.
model. n = 7.

In acute liver injury induced by CCl4, the necrosis and apoptosis ture was destroyed with the loss of nuclei and amounts of frag-
of hepatocytes attributes to cell death in the centrilobular area (Shi mental and condensed nucleus were observed in Fig. 1D.
et al., 1998). In the H&E stained sections, the normal cellular struc- Furthermore, DAPI and TUNEL staining also indicated the produce
68 S. Zhang et al. / Food and Chemical Toxicology 55 (2013) 60–69

of DNA fragmentations (Figs. 4 and 5A) after CCl4 exposure. Con- system and blocking the CYP2El-mediated CCl4 activation. Further
densed and marginal nucleus, a hallmark of apoptosis, was ob- study indicated that the TFs inhibited inflammation response by
served in the TEM image (Fig. 3). However, pretreatment with decreasing pro-inflammatory cytokines and NF-jB express as well
TFs protected nuclei from nuclear loss and condensation as well as apoptosis by inhibiting the Fas/FasL and mitochondrial path-
as inhibiting DNA fragmentations, which was helpful to maintain ways. Further research to explicate the protective effects of TFs
the normal cellular structure and function against CCl4-induced in CCl4-induced liver injury is significant and will provide more
hepatotoxicity. exploitation of the protective effects.
Alterations in liver mitochondria as the consequence of poison-
ousness caused by CCl4 have been reported. The mitochondrial
Conflict of Interest
electron-transport chain is responsible for the activation of CC14
to free radicals, which further stimulate the occurrence of lipid
The authors declared that there are no conflicts of interest.
peroxidation (Tomasi et al., 1987). However, these toxic products
cause mitochondrial DNA depletion and damage as well as ultra-
structural alterations (Knockaert et al., 2012). Consisted with these Acknowledgements
reports, our study also indicated the mitochondrial injury includ-
ing the decrease of mitochondrial membrane potential and mito- This work was supported by funds of Science Foundation of Sci-
chondria valley disappearance (Fig. 3). A common event leading ence & Technology Bureau of Liaoning Province (No. 2009225009-
to both apoptosis and necrosis is mitochondrial permeabilization 2), the Program for Liaoning Excellent Talents in University (No.
and dysfunction (Malhi et al., 2006). However, the administration 2009R15), the Science and Technology Foundation of Dalian, China
of TFs to mice could protect the mitochondria from CC14-induced (No. 2008E11SF223) and the Academic Scholarship for Doctoral
damage, suggesting the inhibited effects of TFs on apoptosis and Candidates of Ministry of Education.
necrosis.
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