Preventive effects of the polysaccharide of Larimichthys crocea swim bladder on carbon tetrachloride (CCl4)-induced hepatic damage

Chinese Journal of Natural Medicines 2015, 13(7): 05210528

Chinese Journal of Natural Medicines

Preventive effects of the polysaccharide of Larimichth ys crocea swim bladder on carbon tetrachloride (CCl4 )-induced hepatic damage ZHAO Xin, QIAN Yu∆, LI Gui-Jie, TAN Jun* Department of Biological and Chemical Engineering, Chongqing University of Education, Chongqing 400067, China Available online 20 July 2015

[ABSTRACT] The aim of the present study was to determine the preventive effects of the polysaccharide of Larimichthys crocea swim bladder (PLCSB) on CCl4-induced hepatic damage in ICR mice. The in vitro preventive effects of PLCSB on CCl4-induced liver cytotoxic effect were evaluated in BRL 3A rat liver cells using the MTT assay. The serum levels of AST, ALT, and LDH in mice were determined using commercially available kits. The levels of IL-6, IL-12, TNF-α, and IFN-γ were determined using ELISA kits. The pathological analysis of hepatic tissues was performed with H and E staining, and the gene and protein expressions were determined by RT-PCR and Western blotting, respectively. PLCSB (20 μg·mL−1) could increase the growth of BRL 3A rat liver cells treated with CCl4. The serum levels of AST, ALT, and LDH were significantly decreased when the mice were treated with two doses of PLCSB, compared with the control mice (P < 0.05). PLCSB-treated groups also showed reduced levels of the serum pro-inflammatory cytokines IL-6, IL-12, TNF-α, and IFN-γ. PLCSB could decrease the liver weight, compared to the CCl4-treated control mice. The histopathology sections of liver tissues in the 100 mg·kg−1 PLCSB group indicated that the animals were recovered well from CCl4 damage, but the 50 mg·kg−1 PLCSB group showed necrosis to a more serious extent. The 100 mg·kg−1 PLCSB group showed significantly decreased mRNA and protein expression levels of NF-κB, iNOS, and COX-2, and increased expression of IκB-α compared with the CCl4-treated control group. In conclusion, PLCSB prevented from CCl4-induced hepatic damage in vivo. [KEY WORDS] Polysaccharide; Larimichthys crocea swim bladder; BRL 3A cells; Liver; Cytokine expression levels

[CLC Number] R965

[Document code] A

[Article ID] 2095-6975(2015)07-0521-08

Introduction Liver diseases include a broad spectrum of disorders, such as hepatitis, alcoholic liver disease, fatty liver disease, liver cirrhosis, and liver cancer, and most of these diseases cause liver tissue damage [1]. Many drugs are used for the treatment of hepatic damage; most of them are synthetic drugs and have side

[Received on] 26-Oct.-2014 [Research funding] This study was supported by Program for Innovation Team Building at Institutions of Higher Education in Chongqing (KJTD201325) and the Program for Innovative Research Team in Chongqing University of Education (No. KYC-cxtd03-20141002). [*Corresponding author] Tel: +86-23-62658256, E-mail: [email protected]. ∆ Co-first author These authors have no conflict of interest to declare. Published by Elsevier B.V. All rights reserved

effects [2]. New biological medicines have good effects on liver damage and are safe for use in humans [3]. The swim bladder is an important balancing organ in fish and the amount of polysaccharide in it is as much as 10% of its weight [4]. Larimichthys crocea Richardson (Sciaenidae) is one of the main commercial fish in the coastal waters of China [5], and the swim bladder is rich in protein, microelements, and vitamins [6]. Traditional Chinese medicine considers that it has good curative effect in various diseases including amnesia, insomnia, dizzy, anepithymia, and weakness after giving a birth [7]. Research has also suggested that L. crocea swim bladder could remove free radicals and prevent cancer [8]. Polysaccharides are important functional materials for pharmacy [9]. It is also established that the polysaccharides in swim bladders can promote wound healing and prevent infection and thrombus [10]. In vivo experiments have demonstrated that polysaccharide in Angelica sinensis and Taraxacum officinale seaweed could prevent hepatic damage [11-12]. Numerous lines of evidence have suggested that oxidative

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stress and inflammation are important in the etiology of liver disease, cardiovascular disease, and cancer [13]. As a result, carbon tetrachloride (CCl4), which produces reactive-free radicals when metabolized, has been widely used as a solvent to develop hepatic damage models in animals. CCl4 is able to increase lipid peroxidation on the cell membrane and alter enzyme activity, thereby inducing hepatic injury and necrosis [8]. Blood assays of ALT, AST, and LDH are standard measures of the hepatic damage levels [14]. Cytokines, including IL-6, IFN-γ, and TNF-α, are produced and released from numerous cells under certain physiological and pathological conditions. Cytokines may be centralized around this organ as it hosts cells that are highly susceptible to the action of the proteins [8]. Unlike sustained hepatocellular damage, acute hepatic damage is temporary, and ends up with the return of normal liver histology and function [15]. A stress situation may be induced in hepatocytes by hypatotoxins with the subsequent release of chemokines, followed by the accumulation of inflammatory cells and hepatocellular damage [16]. The NF-κB, IκB-α, iNOS, and COX-2 genes are responsible for the hepatic damage and deleterious effects in the liver, as a response to inflammatory stimuli [17]. In the present study, the preventive effects of the polysaccharide of L. crocea swim bladder (PLCSB) on hepatic damage were determined. The serum levels of AST, ALT, and LDH, and inflammation-related cytokine levels of IL-6, IL-12, TNF-α, and IFN-γ were used to determine the preventative effects of PLCSB on CCl4-induced hepatic damage in ICR mice. Liver tissue histology was also used to determine the preventative effects in vivo. The mRNA expressions of NF-κB, IκB-α, iNOS, and COX-2 in liver tissues were determined to explore the molecular mechanisms responsible for the preventative effects.

Materials and Methods Preparation of polysaccharide of Larimichthys crocea swim bladder (PLCSB) Wild Yellow Sea Larimichthys crocea were purchased from Shandong Province in China (Qingdao Jiazhijie Aquatic Products Co. Ltd., Qingdao, Shandong, China). The Larimichthys crocea were identified and authenticated by Dr. LI Gui-Jie in School of Food Science, Southwest University (Chongqing, China). Swim bladders of L. crocea (1 kg) were dried by freeze-drying, and the dried samples were crushed. Petroleum ether (3 L) was added to the samples and then reflux extraction was performed twice (1 h each time) at 60 ºC to remove the lipids. The residual materials were collected after filtration. Absolute ethanol (3 L) was added, and the reflux extraction conducted for 3 h. The residual materials without protein were filtered and combined. Finally, water (3 L) was added, and the materials were extracted at 60 ºC for 2 h and the filter liquid collected. The crude PLCSB was obtained after evaporation [18]. The extraction rate of crude

polysaccharide was 8.7%, and the crude polysaccharide was primrose yellow powder, and could be dissolved in water. In vitro testing in BRL 3A rat liver cells treated with PLCSB The BRL 3A rat liver cells were obtained from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). The cells were cultured in DMEM medium (Gibco Co., Birmingham, MI, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (Gibco) at 37 ºC in a humidified atmosphere containing 5% CO2 (Model 311 S/N29035; Forma, Waltham, MA, USA). The BRL 3A cells were seeded in a 96-well plate at a density of 1 × 104 cells·mL−1 (100 μL per well). After 24 h, the DMEM medium was discarded and new medium (100 μL) containing 2 mg·mL−1 of CCl4 was added to each well. The cells were cultured for 24 h, and then the medium solution was discarded. The medium containing 5, 10, or 20 μg·mL−1 of PLCSB was added and the cells were cultured for additional 24 h. After removing the medium, 100 μL of new medium contained MTT reagent (5 mg·mL−1) was added to each well and the plates were incubated for additional 4 h. Then the medium was discarded and dimethyl sulfoxide (100 μL per well) was added and mixed for 30 min. Finally, the absorbance at 540 nm was measured with an enzyme-linked immunosorbent assay reader (Model 680; Bio-Rad, Hercules, CA, USA) [19]. The cell growth rate was calculated as follows: Growth rate (%) = OD540 (CCl4 treated cells) / OD540 (Normal cells) × 100. Induction of hepatic damage in vivo Seven-week-old male ICR mice (n = 50) were purchased from the Experimental Animal Center of Chongqing Medical University (Chongqing, China). The animals were maintained in a specific pathogen-free facility (temperature 25±2 ºC, relative humidity 50%±5%; a 12-h/12-h light/dark cycle) with free access to a standard mice diet and tap water. The mice were divided into five groups consisting of ten mice each. The experimental design was as follows: the normal control group were administered distilled water for 14 days and a single dose of vehicle [0.2 mL·kg−1 body weight (BW) olive oil, p.o.]; the CCl4 control group received a 14-day repeated oral administration of distilled water, followed by oral gavage of CCl4 (0.2 mL·kg−1 bw dissolved in olive oil, 1 : 1, V/V) on the last day to induce hepatic damage; two PLCSB groups received 50 or 100 mg·kg−1 BW of PLCSB, and the positive control group received 14 days of 100 mg·kg−1 BW of silymarin (Shanghai Yuanye Bio-Technology Co., Ltd., Shanghai, China) dissolved in water, before the hepatic damage was induced in the same manner as above for the CCl4 control group. The mice were anesthetized at 24 h after the administration of CCl4 and sacrificed using CO2 [17]. Blood samples and livers were collected and preserved at −70 ºC until biological assays. The liver and body weights were determined. These experiments followed a protocol approved by the Animal Ethics Committee of Chongqing Medical University (Chongqing, China).

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Analysis of serum AST, ALT, and LDH levels AST and ALT levels in liver cells and mice serum were determined using the commercially available kits (Shanghai Institute of Biological Products Co., Ltd., Shanghai, China). LDH levels in serum were determined using a commercially available kit (Cayman Chemical Co., Ann Arbor, MI, USA) Analysis of inflammation-related cytokines in serum by ELISA For the serum cytokine assay, blood samples from the inferior vena cava were collected and centrifuged (3 000 r·min−1, 10 min, 4 ºC). The concentrations of pro-inflammatory-related cytokines of IL-6, IL-12, TNF-α, and IFN-γ were measured using an ELISA assay according to the manufacturer’s protocol (Biolegend ELISA MAX™ Deluxe kits; Biolegend, San Diego, CA, USA). Histological examination of liver tissues For histological investigations, liver tissues were fixed in 10% (V/V) buffered formalin for 24 h, dehydrated in ethanol, and then embedded in paraffin. Then, 4-μm-thick sections were prepared and stained with hematoxylin and eosin (H&E) for observation under an Olympus BX41 microscope (Olympus, Tokyo, Japan). CCl4 could cause liver damage, and the injured liver tissue had inflammatory cell infiltration around the hepatic central vein and liver cell necrosis. The hepatic damage was classified into three grades: Grade 1, showed less than 10%; Grade 2, 10%−50%, and grade 3, greater than 50%. RT-PCR for analysis of inflammation-related gene expression in liver tissues Total RNA from liver tissue was isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s recommendations. The RNA was digested with RNase-free DNase (Roche, Basel, Switzerland) for 15 min at 37 ºC and purified using an RNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. cDNA was synthesized from 2 μg of total RNA by incubation with avian myeloblastosis virus reverse transcriptase (GE Healthcare, Little Chalfont, Berkshire, England) with random hexanucleotides at 37 ºC for l h, according to the manufacturer’s instruction. The sequences of primers used to specifically amplify the genes of interest were as follows: 5'-CAC TTA TGG ACA ACT ATG AGG TCT CTG G-3' (forward) and 5'-CTG TCT TGT GGA CAA CGC AGT GGA ATT TTA GG-3' (reverse) for NF-κB; 5'-GCT GAA GAA GGA GCG GCT ACT-3' (forward) and 5'-TCG TAC TCC TCG TCT TTC ATG GA-3' (reverse) for IκB-α; 5'-AGA GAG

ATC GGG TTC ACA-3' (forward) and 5'-CAC AGA ACT GAG GGT ACA-3' (reverse) for iNOS; and 5'-TTA AAA TGA GAT TGT CCG AA-3' (forward) and 5'-AGA TCA CCT CTG CCT GAG TA-3' (reverse) for COX-2. GAPDH was amplified as an internal control gene with the following primers: 5'-CGG AGT CAA CGG ATT TGG TC-3' (forward) and 5'-AGC CTT CTC CAT GGT CGT GA-3' (reverse). The reaction condition was as follows: denature for 5 min at 95 ºC, anneal for 50 s at 58 ºC, ligate for 90 s at 72 ºC, cycling for 40 times, and ligate for 10 min at 72 ºC. The polymerase chain reaction (PCR) products were separated in 1.0% agarose gels and visualized with ethidium bromide staining [16]. Western blot analysis Total protein was obtained using RIPA buffer as described by Zhao et al. [16]. Protein concentrations were determined by using a Bio-Rad protein assay kit (Hercules, CA, USA). For western blot analysis, aliquots of the lysate containing 30−50 µg of protein were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electrotransfered onto a nitrocellulose membrane (Schleicher and Schuell, Keene, NH, USA). Primary antibodies against NF-κB, IκB-α, iNOS, and COX-2 were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The incubation with the horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology Inc. ) was for 1 h at room temperature. The blots were washed three times with PBS-T and then developed with enhanced chemiluminescence reagent (Amersham Life Sciences, Arlington Heights, IL, USA) as previously described [6]. Statistical analysis Data are presented as mean ± standard deviation (SD). Differences among the groups were assessed with one-way analysis of variance (ANOVA), followed by Duncan's multiple range test. P < 0.05 was considered statistically significant. SAS version 9.1 (SAS Institute Inc., Cary, NC, USA) was used for statistical analyses.

Results Effects of PLCSB on the growth of BRL 3A rat liver cells The growth of BRL 3A rat liver cells after CCl4 treatment (control cells) was only 53.7% of the control (Table 1). PLCSB could prevent the CCl4-induced damage; the growth rates were increased to 59.3%, 64.2%, and 73.1% of the control, after treatment with PLCSB at 5, 10, and 20 μg·mL−1, respectively.

Table 1 Growth of normal and CCl4-trerated BRL 3A rat liver cells and the effects of PLCSB on the CCl4 –treated cells

CCl4 treated

Treatment

Value OD540

Normal cells (untreated)

0.531 ± 0.008 a

100

Control cells

0.285 ± 0.011 e

53.7

5 μg·mL−1 PLCSB

0.315 ± 0.006 d

59.3

10 μg·mL PLCSB

0.341 ± 0.012

c

64.2

20 μg·mL−1 PLCSB

0.388 ± 0.007 b

73.1

−1

a~e

Growth rate (%)

Values with different letters in the same column mean the value of one group was significantly different from others (P < 0.05) according to Duncan's multiple range test

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Effects of PLCSB on the levels of AST, ALT, and n 3A rat liver cells and mouse sera As shown in Table 2, the normal BRL 3A cells without CCl4 treatment showed the lowest levels of AST, ALT,

and LDH, and the CCl 4-treated control cells showed the highest levels. The AST, ALT, and LDH levels of CCl4 + PLCSB treated cells were higher than that of the normal cells, but lower than that of the CCl4control cells.

Table 2 AST, ALT, and LDH levels of CCl4-induced cytotoxic BRL 3A rat liver cells treated with polysaccharide of Larimichthys crocea swim bladder (PLCSB) AST /(IU·L−1)

Treatment

LDH /(IU·L−1)

58.7 ± 5.2 E

1025.7 ± 63.8 e

87.6 ± 12.1 e

Normal cells (untreated)

CCl4 treated

ALT /(IU·L−1)

a

4358.9 ± 189.1 a

Control cells

1257.6 ± 52.6

5 μg·mL−1 PLCSB

1052.3 ± 39.8 b

802.1 ± 24.0 B

3864.7 ± 155.1 b

c

C

3208.7 ± 133.6 c

−1

10 μg·mL PLCSB

831.2 ± 31.3

20 μg·mL−1 PLCSB

553.6 ± 28.7 d

897.5 ± 28.4

A

671.5 ± 20.3

438.7 ± 24.8 D

2371.5 ± 46.9 d

a~e, A-E, a-e

Values with different letters in the same column mean the value of one group was significantly different from others (P < 0.05) according to Duncan's multiple range test

As shown in Table 3, the serum AST, ALT, and LDH levels in CCl 4-treated mice were the highest among all the groups; although the serum AST, ALT, and LDH levels in mice treated with different concentrations of PLCSB were higher than that of the normal group, but

significantly lower than that of the CCl4-treated mice. Silymarin, as the positive control, reduced the ensyme levels. The AST, ALT, and LDH levels in the 100 mg·kg−1 PLCSB group were significantly lower than the 100 mg·kg−1 silymarin group.

Table 3 Serum AST, ALT, and LDH levels of CCl4-induced hepatic damage mice treated with polysaccharide of Larimichthys crocea swim bladder (PLCSB) Treatment

AST /(IU·L−1)

Normal (untreated)

237.2 ± 38.0 e

ALT /(IU·L−1)

LDH /(IU·L−1)

137.2 ± 22.6 E a

5871.3 ± 246.5 a

Control (CCl4 treated)

1922.8 ± 183.1

Silymarin (100 mg·kg−1)

543.7 ± 31.2 c

556.2 ± 26.1 C

2417.3 ± 156.2 c

712.9 ± 40.3 b

886.0 ± 51.2 B

3855.0 ± 131.1 b

d

D

1761.3 ± 112.8 d

50

PLCSB (mg·kg−1)

100

423.7 ± 21.8

1538.2 ± 121.5

1233.6 ± 122.7 e A

387.6 ± 17.6

a~e, A-E, a-e

Values with different letters in the same column mean the value of one group was significantly different from others (P < 0.05) according to Duncan's multiple range test.

Effects of PLCSB on serum cytokine levels of IL-6, IL-12, TNF-α, and IFN-γ in mice As shown in Table 4, the serum cytokine IL-6, IL-12, TNF-α, and IFN-γ levels in mice in the 50 and 100 mg·kg−1 PLCSB-treated groups were significantly lower than those of the CCl4 control group. The reductions observed the 100 mg·kg−1 PLCSB group were 72.1%, 46.8%,

44.3%, and 57.7%, respectively, compared with the CCl4 control group. The levels of these pro-inflammatory cytokines in mice treated with 100 mg·kg −1 PLCSB and silymarin were similar to that of the normal group, and the effects in the 100 mg·kg−1 PLCSB-treated mice were significantly better than that in the 100 mg·kg−1 silymarintreated mice.

Table 4 Cytokine IL-6, IL-12, TNF-α, and IFN-γ levels of CCl4-induced hepatic damage mice treated with polysaccharide of Larimichthys crocea swim bladder (PLCSB) IL-6 /(pg·mL−1)

Treatment

IL-12 /(pg·mL−1)

e

TNF-α /(pg·mL−1)

22.6 ± 4.1 E

40.8 ± 5.2

Control (CCl4 treated)

211.7 ± 15.7 a

731.0 ± 31.7 A

81.2 ± 9.8 a

73.7 ± 5.1 A

Silymarin (100 mg·kg−1)

87.2 ± 17.3 c

401.7 ± 28.5 C

51.5 ± 3.3 c

40.4 ± 2.2 C

b

B

b

57.2 ± 2.6 B

45.2 ± 1.7 d

31.2 ± 1.6 D

50

119.3 ± 12.3

100

59.0 ± 6.2 d

554.3 ± 11.9

388.6 ± 16.8 D

36.1 ± 4.4

IFN-γ /(pg·mL−1)

e

Normal (untreated)

PLCSB (mg·kg−1)

209.1 ± 22.4

E

66.2 ± 1.9

a~e, A-E, a~e, A-E

Values with different letters in the same column mean the value of one group was significantly different from others (P < 0.05) according to Duncan's multiple range test

Effects of PLCSB on liver and body weights of CCl4-treated mice As shown in Table 5, the mean liver weight of the normal

mice was the lowest among all the groups. The mean liver weight in the 100 mg·kg−1 PLCSB group was close to that of

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the normal mice, wand much lower than that of the CCl4-treated mice. The body weight of the mice showed the opposite trend, the normal mice showed the highest body weight, and the 100 mg·kg−1 PLCSB-treated mice showed a slightly lower body weight than the normal mice. The index of liver weight/body weight in the control mice was higher than any other groups. The index of the 100 mg·kg−1 PLCSB-treated mice was slightly higher than that of the normal mice but much lower than that of the CCl4-treated mice.

Effects of PLCSB on the liver tissues in mice with CCl4- induced hepatic damage As shown in Figure 1, the histological tissue sections of the normal group showed normal liver histological morphology after H&E staining. The CCl4-induced histopathological changes in the liver were observed, with significant degeneration and necrosis of hepatocytes in the centrilobular region and perivenular inflammatory infiltrates.

Table 5 Liver and body weights of mice by polysaccharide of Larimichthys crocea swim bladder (PLCSB) treatment Treatment

Liver weight /g

Body weight /g

Liver weight / Body weight (%)

Normal (untreated)

1.62 ± 0.22 c

34.53 ± 2.31 A

4.68 ± 0.32 e

Control (CCl4 treated)

2.12 ± 0.31 a

29.42 ± 2.06 C

7.18 ± 0.55 a

−1

1.87 ± 0.26

b

B

5.82 ± 0.53 c

50

2.03 ± 0.17 ab

31.77 ± 1.28 BC

6.38 ± 0.28 b

100

1.76 ± 0.32 b

32.79 ± 1.41 B

5.35 ± 0.75 d

Silymarin (100 mg·kg ) PLCSB /(mg·kg−1)

32.03 ± 1.55

a~e, A-E, a-e

Values with different letters in the same column mean the value of one group was significantly different from others (P < 0.05) according to Duncan's multiple range test, the same letter mean there was no significantly different in related group. a~e vs Liver weight values; A-E vs Body weight values; a~e Liver weight / Body weight LDH values

The sections from the CCl4 treated group showed widespread areas of congestion and hemorrhages in the centrilobular zone, as well as necrosis involving all the hepatocytes in the centrilobular zone (Grade 3 damage). The 50 mg·kg−1 PLCSB group showed moderate congestion and hemorrhages in the area around the centrilobular vein and extending into the midzonal cells (Grade 2 damage), with

the majority of lobules being affected. The areas of confluent necrosis were limited to the liver cells surrounding the centrilobular vein. The tissue sections of the 100 mg·kg−1 PLCSB and silymarin groups appeared significantly tissue damage (Grade 1 damage). The 100 mg·kg−1 PLCSB group showed more normal tissues than the silymarin group mice.

Fig. 1 Histology images of liver tissues in mice with CCl4-induced hepatic damage (200 × magnification)

Effects of PLCSB on the mRNA and protein expression levels of NF-κB, IκB-α, iNOS, and COX-2 in the liver tissues As shown in Figs. 2 and 3, the mRNA and protein expression of NF-κB was reduced in liver tissues treated with PLCSB and silymarin. PLCSB and silymarin significantly modulated the expression of genes associated with inflammation. The mRNA and protein expressions of NF-κB were decreased, while IκB-α mRNA levels were increased. Addition-

ally, the mRNA and protein expressions of COX-2 and iNOS were decreased in the PLCSB treated goups.. These findings indicate that PLCSB could prevent hepatic damage by increasing anti-inflammatory activities, and PLCSB showed better effects than silymarin at equivalent dose levels.

Discussion

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CCl4 could increase the serum hepaticenzymes and lead

ZHAO Xin, et al. / Chin J Nat Med, 2015, 13(7): 521528

to some liver cytotoxic diseases, such as fibrosis, cirrhosis, and hepatic carcinoma [20]. The MTT assay is a simple colorimetric assay for assessing cell viability [21], and could serve as a preliminary index of CCl4-induced liver cytotoxic effects in BRL 3A rat liver cells. AST and ALT are enzymes located in liver cells that leak out into the general circulation when the liver cells are injured.

AST is located in many body tissues, including the heart, muscle, kidney, brain, and lung tissues. ALT is located predominately in the liver, with lesser quantities located in the kidneys, heart, and skeletal muscles [22]. LDH is an enzyme located in numerous body tissues, including the liver, where elevated levels of LDH may indicate liver damage. Researchers have reported that the serum AST and ALT levels in

Fig. 2 Effects of PLCSB on the mRNA expression levels of NF-κB, IκB-α, iNOS, and COX-2 in liver tissues of mice. Fold-ratio: gene expression / GAPDH × control numerical value (control fold ratio: 1). a–eValues with different letters over the bars mean the value of one group was significantly different from others (P < 0.05) according to Duncan's multiple range test, the same letter mean there was no significantly different in related group

Fig. 3 Effects of PLCSB on the protein expression levels of NF-κB, IκB-α, iNOS, and COX-2 in liver tissues of mice. Fold-ratio: gene expression / β-actin × control numerical value (control fold ratio: 1). a–eValues with different letters over the bars mean the value of one group was significantly different from others (P < 0.05) according to Duncan's multiple range test, the same letter mean there was no significantly different in related group

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CCl4-treated rats are markedly increased, compared with the normal group, indicating that liver damage was significantly induced by CCl4 [23]. Our results indicated that PLCSB appeared to have preventive effects on CCl4 –induced hepatic damage. The levels of serum cytokines, including IL-6, IL-12, and TNF-α, in patients with inflammatory diseases are higher than that in healthy individuals [24]. Cytokine receptors and the inflammatory cytokines, such as IL-6, IL-12, TNF-α, and IFN-γ, play pathogenic roles in gastric disease, and lower levels of those cytokines were also indicative of an improved gastric ulcer preventive effect [25-26]. IL-6 is an interleukin that functions as a pro-inflammatory and an anti-inflammatory cytokine, [27]. IL-6 is secreted by T cells and macrophages to stimulate an immune response, particularly during tissue damage, which leads to inflammation [28]. IL-12, through IFN-γ-dependent induction of the anti-angiogenic factors interferon-inducible protein (IP) 10 and the monokine induced by gamma interferon (MIG), contributes to inflammation eradication [29]. TNF-α is a cytokine involved in systemic inflammation, and is a member of a group of cytokines that stimulate the acute phase reaction [30]. In the present study, the levels of IL-6, IL-12, TNF-α, and IFN-γ in the CCl4-hepatic damage mice were markedly decreased by PLCSB treatment, indicating that PLCSB showed preventive effects on hepatic damagein a dose- dependent manner. The liver weight and the ratio of liver weight/body weight are important indices for the degree of hepatic injury. Ou’yang et al. have reported that CCl4-treated mice show the higher liver weight and the ratio of liver weight/body weight than the normal mice [31]. In the present study, the PLCSB-treated mice showed reduced the liver weight and the ratio of liver weight/body weight, compared with CCl4-treated mice. Histopathology is an important clinical standard for the diagnosis of hepatic damage [32]. In addition, histopathological examination of rat liver sections has been reported as an effective method for evaluation of hepatoprotective activity in a CCl4-induced hepatic damage [33]. In the present study, PLCSB exerted a preventive effect against CCl4-induced hepatic damage, based on the histopathological findings. The NF-κB, IκB-α, COX-2, and iNOS genes in the tissue could be used as biomarkers to monitor viscera damage. NF-κB is one of the most ubiquitous transcription factors, and regulates the expression of genes required for cellular proliferation, inflammatory responses, and cell adhesion [34]. NF-κB is present in the cytosol, where it binds to the inhibitory protein IκB. Following its induction by a variety of agents, NF-κB is released from IκB and translocates to the nucleus where it binds to the NF-κB binding sites in the promoter regions of target genes [35]. Following inflammatory stimuli, both COX-2 and iNOS have been reported to induce deleterious effects in the liver [17]. iNOS and COX-2 could boost inflammatory responses in early stages [36]. The expression of COX-2 in hepatic injury is strong. In addition, the expression of COX-2 proteins in hepatitis was also obvious. When hepatocirrhosis is healing, expression of COX-2 will decrease [37]. iNOS are widely spread in macrophages, neutrophile granu-

locytes, T-lymphocytes, vascular smooth muscle cells, etc. In the digestive system, NO produced by cNOS can play a role of cytoprotection, while excessive NO produced by iNOS palys a role of cytotoxicity [17]. Inflammatory stimuli elicit the synthesis of iNOS and COX-2 proteins with similar time courses, which suggests that the two systems may interact [38]. In the present study, PLCSB was shown to be efficacious in the prevention of hepatic damage and inflammation. Swim bladder has been historically used as a traditional medicine [39]. It has recently been reported that it ameliorates different pathological conditions of inflammation, and it also could effectively improve the functions of platelet, capillary vessel, and clotting factors [38]. Polysaccharides are the main constituents of swim bladder, there are only a few studies of the polysaccharide fraction of swim bladder, the preventive effects of PLCSB on lupus nephritis, colon carcinogenesis and gastric ulcer have been reported [4, 6, 40-41]. In the present study, its preventive effects on liver damage was researched for the first time, demonstrating that that PLCSB had stronger protective effects against liver damage than silymarin, a drug used to treat liver diseases.

Conclusions The preventive effects of PLCSB on hepatic damage were evaluated through various in vitro and in vivo models, including assay of AST, ALT, and LDH levels, assay for serum cytokine levels of IL-6, IL-12, TNF-α, and IFN-γ, histology test, and RT-PCR and Western blotting assays for the inflammatory related genes of NF-κB, IκB-α, iNOS, and COX-2. Our results revealed that PLCSB prevented CCl4-induced hepatic damage, indicating that PLCSB may represent a potentially useful medicine for the treatment or prevention of hepatic damage.

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Cite this article as: ZHAO Xin, QIAN Yu, LI Gui-Jie, TAN Jun. Preventive effects of the polysaccharide of Larimichthys crocea swim bladder on carbon tetrachloride (CCl4)-induced hepatic damage [J]. Chinese Journal of Natural Medicines 2015, 13(7): 521-528.

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