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Protective effect of C-phycocyanin against carbon tetrachloride-induced hepatocyte damage in vitro and in vivo
Chemico-Biological Interactions 185 (2010) 94–100
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Protective effect of C-phycocyanin against carbon tetrachloride-induced hepatocyte damage in vitro and in vivo Yu Ou ∗ , Shan Zheng, Lin Lin, Qizhou Jiang, Xuegan Yang School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China
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Article history: Received 30 December 2009 Received in revised form 23 February 2010 Accepted 6 March 2010 Available online 12 March 2010 Keywords: C-phycocyanin (C-PC) Carbon tetrachloride (CCl4 ) Human hepatocyte cell line L02 Hepatocyte damage
a b s t r a c t This study focused on the hepatoprotective activity of C-phycocyanin (C-PC) against carbon tetrachlorideinduced hepatocyte damage in vitro and in vivo. In in vitro study, human hepatocyte cell line L02 was used. C-PC showed its capability to reverse CCl4 -induced L02 cells viability loss, alanine transaminase (ALT) leakage and morphological changes. C-PC also showed the following positive effects: prevent the CCl4 -induced overproduction of intracellular reactive oxygen species (ROS) and malondialdehyde (MDA); prevent changes in superoxide dismutase (SOD) activity; and reduce glutathione (GSH) level. In vivo, CPC showed its capability to decrease serum ALT and aspartate transaminase (AST) levels in CCl4 -induced liver damage in mice. The histological observations supported the results obtained from serum enzymes assays. C-PC also showed the following effects in mice liver: prevent the CCl4 -induced MDA formation and GSH depletion; prevent SOD and glutathione peroxidase (GSH-Px) activity; and prevent the elevation of transforming growth factor-beta1 (TGF-1) and hepatocyte growth factor (HGF) mRNAs. Both the in vitro and in vivo results suggested that C-PC was useful in protecting against CCl4 -induced hepatocyte damage. One of the mechanisms is believed to be through C-PCs scavenging ability to protect the hepatocytes from free radicals damage induced by CCl4 . In addition, C-PC may be able to block inflammatory infiltration through its anti-inflammatory activities by inhibiting TGF-1 and HGF expression. © 2010 Elsevier Ireland Ltd. All rights reserved.
The liver is an important organ for the detoxification and deposition of endogenous and exogenous substances. Liver disease is considered to be a serious health problem. An evidence suggested that reactive oxygen species (ROS) are known to play a crucial role in liver disease’s pathology and progression [1]. Experimentally induced cirrhotic response in animals by CCl4 is shown to be superficially similar to human cirrhosis of the liver. Thus, CCl4 -induced hepatic damage has been extensively used in the experimental models to evaluate the therapeutic potential of drugs [2]. Among the various mechanisms involved in the hepatotoxic effect of CCl4 , one is oxidative damage through free radical generation [3,4]. The antioxidant property is claimed to be one of the mechanisms of hepatoprotective effect [5]. A major defense mechanism for prevention and treatment of liver damage comprises of decreasing lipid peroxidation and reducing the reactive metabolites production, which is indicated by the increased levels of endogenous antioxidant enzymes such as SOD, catalase, and GSH-Px. C-PC has been proven to have radical scavenging properties and antioxidant activities [6,7]. In our study, we wanted to investigate whether C-PC has a protective effect against CCl4 -induced hepato-
∗ Corresponding author. Tel.: +86 25 83371248; fax: +86 25 83220372. E-mail address: [email protected] (Y. Ou). 0009-2797/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2010.03.013
cyte damage in vitro and in vivo. We also wanted to elucidate the mechanisms underlying these effects. 1. Materials and methods 1.1. Preparation of C-PC from Spirulina maxima C-PC, a biliprotein found in blue green algae, was extracted and purified from Spirulina maxima. The process of extraction and purification of C-PC mainly included homogenization, centrifugation, precipitation with ammonium sulphate, DEAE-Sepharose Fast Flow chromatography, hydroxylatite chromatography, and Sephacryl S-200 chromatography [8]. 1.2. Cell culture and treatments Human hepatocyte cell line L02 was cultured in RPMI-1640 medium supplemented with 10% FBS, 100 units/ml of penicillin, and 100 mg/ml of streptomycin in a water-saturated atmosphere of 5% CO2 at 37 ◦ C. We refer to the above culture medium as the base culture medium. Cells were seeded in multi-well plates or flasks. Cells are cultured in the base culture medium for 24 h. Then the cells are divided into 6 groups that would finally become the normal group, the model group, and 4 drug groups. To produce the
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normal group, cells were cultured in the base culture medium for 48 h. To produce the model group, cells were cultured in the base culture medium supplemented with 10 mmol/L CCl4 (final concentration) for 6 h and then back to the base culture medium for 48 h. To produce the drug groups, cells were cultured in the base culture medium supplemented with 10 mmol/L CCl4 (final concentration) for 6 h and then cultured in the base culture medium supplemented with C-PC for 48 h. The concentration of C-PC for the 4 drug groups is as follows: 31 g/ml, 62 g/ml, 125 g/ml, and 250 g/ml. The 4 drug groups are named after their concentration, i.e. C-PC(31), C-PC(62), C-PC(125), and C-PC(250) groups respectively. 1.3. Cell viability and morphological studies Cell proliferation was assessed by the MTT assay [9]. After the cell treatments described in the previous section, 10 l of 5 mg/ml methylthiazol tetrazolium (MTT) was added to each well, and incubation proceeded at 37 ◦ C for 4 h. The formazan granules obtained were then dissolved in 100 l DMSO, and absorbance at 570 nm was detected with an ELISA plate reader (Multiskan Mk3, Finland). Absorbance of L02 cells in the normal group was measured as 100% cell survival, and the percentage of cell survival was then calculated for each group. Morphological alteration of L02 cells was observed under a light microscope (OlympusIX51, Japan) stained with Wright–Giemsa dye solution in situ. 1.4. Measurement of ROS in L02 cells To measure ROS generation, a fluorometric assay using intracellular oxidation of 2,7-dichlorohydrofluoroscein diacetate (H2 DCFDA) was performed [10]. Cells were incubated with 40 M H2 DCFDA for 15 min. At the end of H2 DCFDA incubation, cells were washed with PBS, lysed with 1 M NaOH. Aliquots were then transferred to the black well plate. Then the fluorescence of dichlorofluoroscein (DCF), the oxidized product of H2 DCFDA, was measured using a microplate spectrofluorometer with excitation and emission wavelengths of 485 nm and 530 nm, respectively. Protein assays were performed using Lowry method. The visual image of ROS generation in cells was made using a fluorescent microscope with an excitation wavelength of 485 nm and an emission wavelength of 530 nm.
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1.6. Biochemical analysis The activities of ALT, AST, SOD or GSH-Px and the levels of MDA and GSH in serum, liver or cell culture supernatant were determined using commercial kits (Nanjing Jiangcheng Bioengineering Institute, Nanjing, China) according to the enclosed guidelines. Protein content was measured using Lowry method with bovine serum albumin as a standard. 1.7. Histopathologic analysis The samples were stained with hematoxylin–eosin (HE) and examined under light microscope (Olympus, Japan) for general histopathology examination. 1.8. Reverse transcription-polymerase chain reaction (RT-PCR) The total RNA in the liver was isolated using a total RNA isolation kit (Shanghai Sangon Biological Engineering Technology & Services Co., Ltd.). The total RNA (1.0 g) was reversetranscribed using an oligo(dT) 18mer as a primer and M-MLV reverse transcriptase (Promega) to produce the cDNAs. PCR was performed using the selective primers for TGF-1 (sense: 5 CTGTCCAAACTAAGGCTCGC-3 , antisense: 5 -AGCCCTGTATTCCGTCTCCT-3 ), HGF (sense: 5 -TACAGGGGAACCAGCAATACC-3 , antisense: 5 -TGGCTCCCAGAAGATATGACG-3 ) and glyceraldehyde3-phosphate dehydrogenase (GAPDH) as an internal control (sense: 5 -CATCACCATCTTCCAGGAGC-3 , antisense: 5 -TAAGCAGTTGTTGTTGCAGG-3 ). PCR was performed for 35 cycles using the following conditions: denaturation at 94 ◦ C for 1 min, annealing at 55 ◦ C for 1 min, and elongation at 72 ◦ C for 1 min. The band intensities of the amplified DNAs were compared after visualization. 1.9. Statistical analysis of the data All results were presented as mean ± SD, “Student’s” t-test was used for statistical analysis and statistical significance was defined as P < 0.05 or P < 0.01. 2. Results 2.1. C-PC attenuated CCl4 -induced cytotoxicity and cell morphological changes in L02 cells
1.5. Animals and their treatments Male ICR mice (18–22 g) were obtained from the Comparative Medical Center of Yangzhou University (Yangzhou, China), and were allowed one week to be quarantined and acclimated prior to experimentation. The mice were maintained on a 12 h light/dark cycles in a temperature and humidity controlled room. The animals were randomly divided into five groups with each consisting of 12 mice. The 5 groups are the normal group, the model group, and the 3 test groups. Mice in the 3 test groups were treated with C-PC at a dose of 400, 200 and 100 mg/kg body weight for seven consecutive days (once per day), while mice in normal group and model group were treated with physiological saline. The mice were injected with CCl4 (10 ml/kg i.p. of 0.5% CCl4 solution in olive oil) one hour after the sixth administration, except for the normal group, which was given only olive oil injection. The animals were sacrificed 48 h after the CCl4 and olive oil treatment, respectively. Blood was collected into non-heparinized Eppendorf tubes and centrifuged (2000 × g, 10 min, 4 ◦ C). Serum was aspirated and stored at −20 ◦ C until assayed as described below. The liver was also removed and stored at liquid nitrogen until use. An extra sample of liver was excised and fixed in 10% formalin solution for histopathologic analysis.
The MTT assay was performed to assess the toxicity of CCl4 in L02 cells and to determine the influence of C-PC on CCl4 toxicity in these cells. Cells treated with 10 mmol/L CCl4 only showed evident reduced cell viability. This cytotoxic effect was significantly attenuated by co-treatment with C-PC in a dose-dependent manner. Cells treated with C-PC from 31 g/ml to 250 g/ml showed evident difference in cell viability from that of normal cells (Fig. 1). To characterize cell morphological changes induced by CCl4 , L02 cells were examined after Wright–Giemsa stain by the light microscope. L02 cells treated with 10 mmol/L CCl4 only exhibited morphological changes such as disruption of nuclear envelope and cytoplasm (Fig. 2B). These morphological changes in CCl4 treated cells were attenuated by co-treatment with 31–250 g/ml in a dose-dependent manner. Cells treated with 250 g/ml C-PC appeared healthy, with regularity in shape, normal nuclei, and well developed cell-to-cell contact (Fig. 2C). 2.2. C-PC prevented CCl4 -induced changes in biochemical parameters of L02 cells 10 mmol/L CCl4 caused evident hepatocyte toxicity, as evidenced by the significant elevation of ALT activities in the
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Table 1 C-PC prevented CCl4 -induced changes in activities of ALT and SOD, levels of GSH and MDA in L02 cells. Groups
ALT (U/106 cell)
SOD (units/mg protein)
MDA (nmol/mg protein)
GSH (g/mg protein)
Normal Model C-PC(31) C-PC(62) C-PC(125) C-PC(250)
5.01 ± 0.23 12.6 ± 1.5# 9.75 ± 0.50* 8.18 ± 1.4* 6.99 ± 1.28** 5.13 ± 0.48**
4.07 ± 0.59 2.32 ± 0.27# 2.49 ± 0.38 3.07 ± 0.15* 3.52 ± 0.03** 3.76 ± 0.07**
2.55 ± 0.13 4.85 ± 0.30# 4.14 ± 0.07* 3.51 ± 0.24** 2.82 ± 0.24** 2.39 ± 0.23**
49.4 ± 2.8 35.9 ± 1.2# 36.5 ± 0.8 38.0 ± 1.5* 42.3 ± 1.0** 45.3 ± 2.5**
The data are expressed in mean ± S.D. of at least three independent experiments. *P < 0.05 and **P < 0.01 compared with the corresponding value for model group. # P < 0.01 compared with the corresponding value for normal control group.
Fig. 1. C-PC attenuated CCl4 -induced cytotoxicity in L02 cells. Cell viability was determined by MTT assay. The cells were treated with or without 10 mmol/ L CCl4 for 6 h and then cultured in the presence or absence of C-PC for 48 h. Data were shown as means ± S.D. (n = 6). ## P < 0.01 vs normal group, **P < 0.01 vs model group.
Fig. 3. Effects of C-PC on the level of ROS in L-02 induced by CCl4 damage. ## P < 0.01, model group compared with normal group; * P < 0.05, ** P < 0.01, C-PC groups compared with model group.
Table 2 Effects of C-PC on serum ALT and AST in CCl4 -intoxicated mice.
culture supernatant. However, co-treatment with C-PC prevented CCl4 -induced release of cellular AST into the medium in a dosedependent manner (Table 1). The L02 cells treated with CCl4 only showed decrease in the SOD activity and the GSH level, but significant increase in the MDA level. Co-treatment with C-PC prevented CCl4 -induced changes in the SOD activity and the levels of GSH and MDA (Table 1).
Group
ALT (U/ml)
AST (U/ml)
Normal Model C-PC(100) C-PC(200) C-PC(400)
12.76 ± 156.9 ± 92.38 ± 57.85 ± 46.11 ±
16.41 ± 98.01 ± 41.53 ± 33.56 ± 23.58 ±
3.81 29.9# 23.0* 11.9* 9.01*
3.99 19.4# 6.45* 3.81* 5.45*
All values are means ± SD (n = 12). # P < 0.01 compared with normal group. * P < 0.01 compared with CCl4 group.
2.3. C-PC inhibited CCl4 -induced accumulation of free radicals in L02 cells
2.4. Effect of C-PC on CCl4 -induced liver damage in mice
To further explore the biochemical basis contributing to protective effects of C-PC on L02 cell, CCl4 -induced free radical generation and free radical scavenging capacity of C-PC were examined. Intracellular ROS was detected by fluorescein dye 2 ,7 -H2 DCFDA. L02 cells treated with CCl4 only showed increase in intracellular ROS as shown by increased fluorescence intensity (Fig. 3). Co-treatment with C-PC reduced intracellular ROS in a dose-dependent manner. These findings were further confirmed by fluorescent microscopy (Fig. 4).
The effect of C-PC on CCl4 -induced hepatotoxicity was summarized in Table 2. A significant elevation of serum ALT and AST activities in CCl4 -treated group as compared to the normal group (P < 0.01) indicated CCl-induced damage to the hepatic cells. Mice in the test groups (treated with C-PC) reduced the activities of serum ALT and AST, as compared to the model group (treated with CCl4 ) (P < 0.01). The effect was found to be dose-dependent, and the magnitude of the standard deviation (SD) in all test groups indicated the individual variability in drug response.
Fig. 2. Morphological changes in L02 cells treated with CCl4 . The cells were examined under the light microscope after Wright–Giemsa stain. (A) Normal L02 cells; (B) L02 cells treated with 10 mmol/ L CCl4 ; (C) L02 cells co-treatment with 250 g/ml C-PC.
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Fig. 4. The generation of ROS in L02 cells. DCF fluorescence was detected by fluorescent microscope.
The histological observations basically supported the results obtained from serum enzymes assays. There were no pathological changes in healthy control livers which showed normal architecture (Fig. 5A). Histopathological examination of livers challenged with CCl4 showed vacuole formation and inflammatory infiltration. The hepatocytes were markedly edematous and the cytoplasm of hepatocytes was loose (Fig. 5B). The liver sections of mice pre-
treated with the C-PC before CCl4 challenge revealed that C-PC was able, in a dose-dependent manner, to prevent the development of histopathological changes, which exhibited areas of normal liver architecture and patches of inflammatory infiltration and necrotic hepatocytes (Fig. 5C–E). Livers of mice pre-treated with the highest dose (400 mg/kg) showed well-preserved architecture (Fig. 5E).
Fig. 5. Photomicrographs of liver sections stained with hematoxylin and eosin (×100). (A) Hepatic tissue of control mice, showing normal appearance. (B) Hepatic tissue of CCl4 -treated mice. (C) Hepatic tissue of C-PC (100 mg/kg) and CCl4 -treated mice. (D) Hepatic tissue of C-PC (200 mg/kg) and CCl4 -treated mice. (E) Hepatic tissue of C-PC (400 mg/kg) and CCl4 -treated mice.
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Table 3 Effects of C-PC on liver MDA, GSH levels and SOD, GSH-Px activities in CCl4 -intoxicated mice. Group
MDA (nmol/mg protein)
GSH (g/mg protein)
SOD (U/mg protein)
GSH-Px (U/mg protein)
Normal Model PC(100) PC(200) PC(400)
2.8 ± 0.11 8.0 ± 0.31## 3.2 ± 0.13** 2.3 ± 0.07** 2.1 ± 0.06**
36 ± 1.2 15 ± 0.7## 25 ± 2.3** 28 ± 1.8** 34 ± 2.0**
62 ± 2.6 21 ± 1.9## 26 ± 2.2 44 ± 3.1** 45 ± 3.3**
83.8 ± 1.7 55.7 ± 2.4## 68.8 ± 3.1* 70.7 ± 2.5** 71.9 ± 1.4**
All values are means ± SD (n = 12). ## P < 0.01 compared with normal group. * P < 0.05, ** P < 0.01 compared with CCl4 group.
Fig. 6. The levels of mRNA for TGF-1 and HGF were examined by RT-PCR. Expression of GAPDH mRNA was used as the internal standard. (A) Level of TGF-1 and HGF mRNAs. (Lane 1) normal group, (lane 2) CCl4 group, (lane 3) 100 mg/kg C-PC + CCl4 group and (lane 4) 200 mg/kg C-PC + CCl4 group. (B) Quantitative analysis of mRNA by densitometry scanning.
2.5. Lipid peroxidation MDA level is widely used as a marker of free radical mediated lipid peroxidation damage. We measured MDA levels in the livers. The results are shown in Table 3. MDA levels in the CCl4 treated group were significantly higher than that in the normal group (P < 0.01). MDA levels in the C-PC treated groups were significantly lower than that in the model group (P < 0.01). These findings indicated that the free radicals being released in the liver were effectively scavenged when treated with C-PC. 2.6. Hepatic antioxidant activities SOD, GSH-Px, and GSH were measured as an index of antioxidant status of tissues. Significantly lower liver GSH, SOD, and GSH-Px activities were observed in the model group as compared to the normal group. There was a significant increase of SOD and GSH-Px activity in the drug groups as compared to the model group. Treatment with CCl4 also significantly decreased the GSH levels in the liver as compared to the normal group. By contrast, administration with C-PC significantly increased the GSH levels as compared to the CCl4 -treated group (Table 3). 2.7. Effect of C-PC on TGF-ˇ1 mRNA and HGF expression in mice liver We detected the expression of TGF-1 and HGF at the mRNA levels in mice liver using RT-PCR (Fig. 6). GAPDH was used as an internal control. Compared to the normal group, the level of TGF1 and HGF mRNAs were significantly increased after treated with CCl4 . 200 mg/kg C-PC pretreatment significantly decreased CCl4 induced TGF-1 mRNA upregulation, but no significant change
was found between the low C-PC dosage (100 mg/kg) drug group and the model group (P > 0.05). Unlike TGF-1, 100 mg/kg and 200 mg/kg C-PC both evidently down-regulated HGF mRNA levels (P < 0.01).
3. Discussion CCl4 is a xenobiotic that produces hepatotoxicity in various experimental animals. CCl4 is metabolized by hepatic microsomal cytochrome P450 (CYP) 2E1 to form a reactive trichloromethyl (CCl3 · ) radical and/or a trichloromethyl peroxyl (CCl3 OO· ) radical. Both radicals are capable of binding to DNA, lipids, proteins, or carbohydrates, leading to lipid peroxidation, cell necrosis, excessive deposition of collagen in liver, and liver fibrosis [2]. Some studies have demonstrated that an important mechanism of the hepatoprotective effects may be related to an antioxidant capacity to scavenge reactive oxygen species [11,12]. C-PC is a potent free radical scavenger and an antioxidant agent. However, little information is available about the effects of C-PC on oxidatively damaged hepatocyte in vitro. In this study, co-incubation with C-PC for 48 h significantly attenuated oxidative stress in human hepatocyte cell line L02 induced by CCl4 in a concentrationdependent manner. Our results showed that CCl4 inhibited the growth of L02 cells, and induced cell death. The L02 cells treated with CCl4 were also associated with increased intercellular ROS, MDA and decreased GSH levels, changes in activity of intercellular SOD, as well as activity of ALT in culture medium. These CCl4 induced changes were, to a large extent, prevented by co-treatment with C-PC. Our results also indicated that C-PC exerted its protective effect intra-cellularly, rather than extra-cellularly, reacting with CCl4 in the culture medium.
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It has been reported that intraperitoneal (i.p.) administration (200 mg/kg) of a single dose of C-PC (from Spirulina platensis) to rats, one or three hours prior to CCl4 challenge, significantly reduced the hepatotoxicity [13]. In our study, a single dose of C-PC (from S. maxima) could not protect mice against CCl4 -induced liver damage (data not shown), while pretreatment with C-PC for seven consecutive days evidently attenuated CCl4 -induced liver damage in mice, evidenced by decreased serum activities of ALT and AST. The hepatic cells consist of higher concentrations of AST and ALT in cytoplasm. Higher concentrations of AST also exist in mitochondria. Due to the damage caused to hepatic cells, the leakage of plasma causes an increased level of hepatospecific enzymes in serum in vivo. The elevated serum enzyme such as AST and ALT are indicative of cellular leakage and functional integrity of cell membrane in the liver. The present study showed that C-PC significantly reduced CCl4 -induced serum ALT and AST levels. The biochemical observations are supported by the histopathological examination of the mice liver. Lipid peroxidation is one of the principal causes of CCl4 induced liver damage [14,15] and is mediated by the free radical derivatives of CCl4 . The elevation of MDA levels in the liver implies that an enhanced peroxidation causes tissue damage and breakdown of the antioxidant defense mechanisms and thus prevents the formation of superabundant free radicals [16]. In our study, C-PC administration caused a significant decrease in MDA levels as compared to the CCl4 -treated group, suggesting that C-PC could protect against CCl4 -induced lipid peroxidation in mice. In contrast to the toxic consequences of CCl4 metabolism via the CYP2E1 pathway, the detoxification pathway involves GSH conjugation of trichloromethyl-free radicals [17]. GSH plays a key role in eliminating the reactive toxic metabolites of CCl4 . Our results also showed that C-PC pretreatment significantly inhibited the CCl4 -induced depletion of hepatic GSH. SOD is an effective defense enzyme that catalyses the dismutation of superoxide anions into hydrogen peroxide (H2 O2 ). GSH-Px is an important enzyme in the liver and converted the reduction of H2 O2 and hydroperoxides to non-toxic products [11]. Lipid peroxides or ROS easily inactivate these antioxidant enzymes, which results in reduced activities of these enzymes in CCl4 toxicity [18]. In the present study, the hepatic antioxidant activities were significantly decreased in CCl4 -intoxicated mice compared with the mice in the normal group, implying increased oxidative damage to the liver. Administration of C-PC to CCl4 -treated mice could significantly elevate the activity of SOD and GSH-Px, suggesting that it has the ability to restore these enzymes’ activities in CCl4 -damaged liver. It has been revealed that TGF-1 down-regulates the glutathione synthesis [19] and the expression levels and activity of antioxidant enzymes, including glutathione-s-transferase, superoxide dismutase and glutathione peroxidase [20]. Mice treated with CCl4 exhibited a significantly increased level of liver-associated TGF-1 mRNA at 24 h and 48 h, respectively, as compared to the mice in the normal group [21]. The present study also showed increased level of TGF-1 mRNA in the liver of CCl4 -treated mice, compared with normal mice. 200 mg/kg C-PC pretreatment significantly decreased the elevated level of TGF-1 in CCl4 -intoxicated mice. These findings suggest that the inhibitory effects of C-PC on liver damage might be related to its action on the production of TGF-1. HGF was first identified in the serum of rats that underwent partial hepatectomy [22]. Physiologically, HGF has an important role as an organotrophic factor responsible for the regeneration of liver, kidney and lung [23]. Beside this physiological effect, it is known that HGF is produced by mesenchymal cells in the body in response to injuries of distant organ [24]. HGF mRNA
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and HGF activity increase markedly in the liver of rats after various liver injuries such as hepatitis, ischaemia, physical crush, and partial hepatectomy [25]. The number of studies on HGF level in the liver of CCl4 -treated mice is limited. In our study, HGF mRNA levels in the liver of CCl4 -intoxicated mice were found statistically significantly higher compared to those in the normal group (P < 0.01). This result suggests that HGF in the liver is significantly correlated with liver injury progression and may be a predictor of liver injury. Administration of C-PC inhibited liver damage induced by CCl4 , resulting in decreased HGF level in the liver. In conclusion, the results of this study demonstrate that the CPC was effective in preventing CCl4 -induced damage to hepatocyte in vitro and in vivo. The hepatoprotective effects of C-PC may be due to inhibited lipid peroxidation and effective recovery of the antioxidative defense system. In addition, C-PC may be able to block inflammatory infiltration through its anti-inflammatory activities by inhibiting TGF-1 and HGF expression in CCl4 -induced hepatic damage. References [1] P. Vitaglione, F. Morisco, N. Caporaso, et al., Dietary antioxidant compounds and liver health, Crit. Rev. Food Sci. Nutr. 44 (2004) 575–586. [2] C.F. Tsai, Y.W. Hsu, W.K. Chen, et al., Hepatoprotective effect of electrolyzed reduced water against carbon tetrachloride-induced liver damage in mice, Food Chem. Toxicol. 47 (8) (2009) 2031–2036. [3] L.D. DeLeve, N. Kaplowitz, Mechanisms of drug-induced liver disease, Gastroenterol. Clin. North Am. 24 (1995) 787–810. [4] S.K. Natarajan, S. Thomas, S. Ramamoorthy, et al., Oxidative stress in the development of liver cirrhosis: a comparison of two different experimental models, J. Gastroenterol. Hepatol. 21 (2006) 947–957. [5] A.D. Bhatt, N.S. Bhatt, Indigenous drugs and liver disease, Indian J. Gastroenterol. 15 (1996) 63–67. [6] B.B. Vadiraja, K.M. Madyastha, C-phycocyanin: a potent peroxyl radical scavenger in vivo and in vitro, Biochem. Biophys. Res. Commun. 275 (2000) 20– 25. ˜ [7] J.E. Pinero Estrada, P. Bermejo Bescós, A.M. Villar del Fresno, Antioxidant activity of different fractions of Spirulina platensis protean extract, Farmaco 156 (2000) 497–500. [8] Y. Ou, J. Dong, W.T. Wu, Purification and characterization of Phycobiliprotein from Aphanothece halophytica, Pharm. Biotechnol. 11 (1) (2004) 37–41. [9] Y. Ou, P. Geng, G.Y. Liao, et al., Intracellular GSH and ROS levels may be related to galactose-mediated human lens epithelial cell apoptosis: role of recombinant hirudin variant III, Chem-Biol. Interact. 179 (2009) 103–109. [10] E.J. Park, K. Park, Oxidative stress and pro-inflammatory responses induced by silica nanoparticles in vivo and in vitro, Toxicol. Lett. 184 (1) (2009) 18– 25. [11] S.R. Naik, V.S. Panda, Antioxidant and hepatoprotective effects of Ginkgo biloba phytosomes in carbon tetrachloride-induced liver damage in rodents, Liver Int. 27 (2007) 393–399. [12] G. Mehmetc¸ik, G. Ozdemirler, N. Koc¸ak-Toker, et al., Effect of pretreatment with artichoke extract on carbon tetrachloride-induced liver damage and oxidative stress, Exp. Toxicol. Pathol. 60 (6) (2008) 475–480. [13] B.B. Vadiraja, N.W. Gaikward, K.M. Madyastha, Hepatoprotective effect of Cphycocyanin: protection for carbon tetrachloride and R-(+) Pulegone-mediated hepatotoxicity in rats, Biochem. Biophys. Res. Commun. 249 (1998) 428– 431. [14] S. Basu, Carbon tetrachloride-induced lipid peroxidation: eicosanoid formation and their regulation by antioxidant nutrients, Toxicology 189 (2003) 113– 127. [15] M.K. Manibusan, M. Odin, D.A. Eastmond, Postulated carbon tetrachloride mode of action: a review, J. Environ. Sci. Health. C: Environ. Carcinog. Ecotoxicol. Rev. 25 (2007) 185–209. [16] Y.W. Hsu, C.F. Tsai, W.K. Chen, et al., Protective effects of seabuckthorn (Hippophae rhamnoides L.) seed oil against carbon tetrachloride-induced hepatotoxicity in mice, Food Chem. Toxicol. 47 (2009) 2281–2288. [17] K.J. Leea, J.H. Choia, T. Khanala, et al., Protective effect of caffeic acid phenethyl ester against carbon tetrachloride-induced hepatotoxicity in mice, Toxicology 248 (2008) 18–24. [18] Y.S. Yang, T.H. Ahn, J.C. Lee, et al., Protective effects of Pycnogenol on carbon tetrachloride-induced hepatotoxicity in Sprague-Dawley rats, Food Chem. Toxicol. 46 (2008) 380–387. [19] K. Arsalane, C.M. Dubois, T. Muanza, et al., Transforming growth factor-beta1 is a potent inhibitor of glutathione synthesis in the lung epithelial cell line A549: transcriptional effect on the GSH rate-limiting enzyme gammaglutamylcysteine synthetase, Am. J. Respir. Cell Mol. Biol. 17 (1997) 599–607. [20] Y. Kayanoki, J. Fujii, K. Suzuki, et al., Suppression of antioxidative enzyme expression by transforming growth factor-beta 1 in rat hepatocytes, J. Biol. Chem. 269 (1994) 15488–15492.
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Protective effect of C-phycocyanin against carbon tetrachloride-induced hepatocyte damage in vitro and in vivo Yu Ou ∗ , Shan Zheng, Lin Lin, Qizhou Jiang, Xuegan Yang School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China
a r t i c l e
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Article history: Received 30 December 2009 Received in revised form 23 February 2010 Accepted 6 March 2010 Available online 12 March 2010 Keywords: C-phycocyanin (C-PC) Carbon tetrachloride (CCl4 ) Human hepatocyte cell line L02 Hepatocyte damage
a b s t r a c t This study focused on the hepatoprotective activity of C-phycocyanin (C-PC) against carbon tetrachlorideinduced hepatocyte damage in vitro and in vivo. In in vitro study, human hepatocyte cell line L02 was used. C-PC showed its capability to reverse CCl4 -induced L02 cells viability loss, alanine transaminase (ALT) leakage and morphological changes. C-PC also showed the following positive effects: prevent the CCl4 -induced overproduction of intracellular reactive oxygen species (ROS) and malondialdehyde (MDA); prevent changes in superoxide dismutase (SOD) activity; and reduce glutathione (GSH) level. In vivo, CPC showed its capability to decrease serum ALT and aspartate transaminase (AST) levels in CCl4 -induced liver damage in mice. The histological observations supported the results obtained from serum enzymes assays. C-PC also showed the following effects in mice liver: prevent the CCl4 -induced MDA formation and GSH depletion; prevent SOD and glutathione peroxidase (GSH-Px) activity; and prevent the elevation of transforming growth factor-beta1 (TGF-1) and hepatocyte growth factor (HGF) mRNAs. Both the in vitro and in vivo results suggested that C-PC was useful in protecting against CCl4 -induced hepatocyte damage. One of the mechanisms is believed to be through C-PCs scavenging ability to protect the hepatocytes from free radicals damage induced by CCl4 . In addition, C-PC may be able to block inflammatory infiltration through its anti-inflammatory activities by inhibiting TGF-1 and HGF expression. © 2010 Elsevier Ireland Ltd. All rights reserved.
The liver is an important organ for the detoxification and deposition of endogenous and exogenous substances. Liver disease is considered to be a serious health problem. An evidence suggested that reactive oxygen species (ROS) are known to play a crucial role in liver disease’s pathology and progression [1]. Experimentally induced cirrhotic response in animals by CCl4 is shown to be superficially similar to human cirrhosis of the liver. Thus, CCl4 -induced hepatic damage has been extensively used in the experimental models to evaluate the therapeutic potential of drugs [2]. Among the various mechanisms involved in the hepatotoxic effect of CCl4 , one is oxidative damage through free radical generation [3,4]. The antioxidant property is claimed to be one of the mechanisms of hepatoprotective effect [5]. A major defense mechanism for prevention and treatment of liver damage comprises of decreasing lipid peroxidation and reducing the reactive metabolites production, which is indicated by the increased levels of endogenous antioxidant enzymes such as SOD, catalase, and GSH-Px. C-PC has been proven to have radical scavenging properties and antioxidant activities [6,7]. In our study, we wanted to investigate whether C-PC has a protective effect against CCl4 -induced hepato-
∗ Corresponding author. Tel.: +86 25 83371248; fax: +86 25 83220372. E-mail address: [email protected] (Y. Ou). 0009-2797/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2010.03.013
cyte damage in vitro and in vivo. We also wanted to elucidate the mechanisms underlying these effects. 1. Materials and methods 1.1. Preparation of C-PC from Spirulina maxima C-PC, a biliprotein found in blue green algae, was extracted and purified from Spirulina maxima. The process of extraction and purification of C-PC mainly included homogenization, centrifugation, precipitation with ammonium sulphate, DEAE-Sepharose Fast Flow chromatography, hydroxylatite chromatography, and Sephacryl S-200 chromatography [8]. 1.2. Cell culture and treatments Human hepatocyte cell line L02 was cultured in RPMI-1640 medium supplemented with 10% FBS, 100 units/ml of penicillin, and 100 mg/ml of streptomycin in a water-saturated atmosphere of 5% CO2 at 37 ◦ C. We refer to the above culture medium as the base culture medium. Cells were seeded in multi-well plates or flasks. Cells are cultured in the base culture medium for 24 h. Then the cells are divided into 6 groups that would finally become the normal group, the model group, and 4 drug groups. To produce the
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normal group, cells were cultured in the base culture medium for 48 h. To produce the model group, cells were cultured in the base culture medium supplemented with 10 mmol/L CCl4 (final concentration) for 6 h and then back to the base culture medium for 48 h. To produce the drug groups, cells were cultured in the base culture medium supplemented with 10 mmol/L CCl4 (final concentration) for 6 h and then cultured in the base culture medium supplemented with C-PC for 48 h. The concentration of C-PC for the 4 drug groups is as follows: 31 g/ml, 62 g/ml, 125 g/ml, and 250 g/ml. The 4 drug groups are named after their concentration, i.e. C-PC(31), C-PC(62), C-PC(125), and C-PC(250) groups respectively. 1.3. Cell viability and morphological studies Cell proliferation was assessed by the MTT assay [9]. After the cell treatments described in the previous section, 10 l of 5 mg/ml methylthiazol tetrazolium (MTT) was added to each well, and incubation proceeded at 37 ◦ C for 4 h. The formazan granules obtained were then dissolved in 100 l DMSO, and absorbance at 570 nm was detected with an ELISA plate reader (Multiskan Mk3, Finland). Absorbance of L02 cells in the normal group was measured as 100% cell survival, and the percentage of cell survival was then calculated for each group. Morphological alteration of L02 cells was observed under a light microscope (OlympusIX51, Japan) stained with Wright–Giemsa dye solution in situ. 1.4. Measurement of ROS in L02 cells To measure ROS generation, a fluorometric assay using intracellular oxidation of 2,7-dichlorohydrofluoroscein diacetate (H2 DCFDA) was performed [10]. Cells were incubated with 40 M H2 DCFDA for 15 min. At the end of H2 DCFDA incubation, cells were washed with PBS, lysed with 1 M NaOH. Aliquots were then transferred to the black well plate. Then the fluorescence of dichlorofluoroscein (DCF), the oxidized product of H2 DCFDA, was measured using a microplate spectrofluorometer with excitation and emission wavelengths of 485 nm and 530 nm, respectively. Protein assays were performed using Lowry method. The visual image of ROS generation in cells was made using a fluorescent microscope with an excitation wavelength of 485 nm and an emission wavelength of 530 nm.
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1.6. Biochemical analysis The activities of ALT, AST, SOD or GSH-Px and the levels of MDA and GSH in serum, liver or cell culture supernatant were determined using commercial kits (Nanjing Jiangcheng Bioengineering Institute, Nanjing, China) according to the enclosed guidelines. Protein content was measured using Lowry method with bovine serum albumin as a standard. 1.7. Histopathologic analysis The samples were stained with hematoxylin–eosin (HE) and examined under light microscope (Olympus, Japan) for general histopathology examination. 1.8. Reverse transcription-polymerase chain reaction (RT-PCR) The total RNA in the liver was isolated using a total RNA isolation kit (Shanghai Sangon Biological Engineering Technology & Services Co., Ltd.). The total RNA (1.0 g) was reversetranscribed using an oligo(dT) 18mer as a primer and M-MLV reverse transcriptase (Promega) to produce the cDNAs. PCR was performed using the selective primers for TGF-1 (sense: 5 CTGTCCAAACTAAGGCTCGC-3 , antisense: 5 -AGCCCTGTATTCCGTCTCCT-3 ), HGF (sense: 5 -TACAGGGGAACCAGCAATACC-3 , antisense: 5 -TGGCTCCCAGAAGATATGACG-3 ) and glyceraldehyde3-phosphate dehydrogenase (GAPDH) as an internal control (sense: 5 -CATCACCATCTTCCAGGAGC-3 , antisense: 5 -TAAGCAGTTGTTGTTGCAGG-3 ). PCR was performed for 35 cycles using the following conditions: denaturation at 94 ◦ C for 1 min, annealing at 55 ◦ C for 1 min, and elongation at 72 ◦ C for 1 min. The band intensities of the amplified DNAs were compared after visualization. 1.9. Statistical analysis of the data All results were presented as mean ± SD, “Student’s” t-test was used for statistical analysis and statistical significance was defined as P < 0.05 or P < 0.01. 2. Results 2.1. C-PC attenuated CCl4 -induced cytotoxicity and cell morphological changes in L02 cells
1.5. Animals and their treatments Male ICR mice (18–22 g) were obtained from the Comparative Medical Center of Yangzhou University (Yangzhou, China), and were allowed one week to be quarantined and acclimated prior to experimentation. The mice were maintained on a 12 h light/dark cycles in a temperature and humidity controlled room. The animals were randomly divided into five groups with each consisting of 12 mice. The 5 groups are the normal group, the model group, and the 3 test groups. Mice in the 3 test groups were treated with C-PC at a dose of 400, 200 and 100 mg/kg body weight for seven consecutive days (once per day), while mice in normal group and model group were treated with physiological saline. The mice were injected with CCl4 (10 ml/kg i.p. of 0.5% CCl4 solution in olive oil) one hour after the sixth administration, except for the normal group, which was given only olive oil injection. The animals were sacrificed 48 h after the CCl4 and olive oil treatment, respectively. Blood was collected into non-heparinized Eppendorf tubes and centrifuged (2000 × g, 10 min, 4 ◦ C). Serum was aspirated and stored at −20 ◦ C until assayed as described below. The liver was also removed and stored at liquid nitrogen until use. An extra sample of liver was excised and fixed in 10% formalin solution for histopathologic analysis.
The MTT assay was performed to assess the toxicity of CCl4 in L02 cells and to determine the influence of C-PC on CCl4 toxicity in these cells. Cells treated with 10 mmol/L CCl4 only showed evident reduced cell viability. This cytotoxic effect was significantly attenuated by co-treatment with C-PC in a dose-dependent manner. Cells treated with C-PC from 31 g/ml to 250 g/ml showed evident difference in cell viability from that of normal cells (Fig. 1). To characterize cell morphological changes induced by CCl4 , L02 cells were examined after Wright–Giemsa stain by the light microscope. L02 cells treated with 10 mmol/L CCl4 only exhibited morphological changes such as disruption of nuclear envelope and cytoplasm (Fig. 2B). These morphological changes in CCl4 treated cells were attenuated by co-treatment with 31–250 g/ml in a dose-dependent manner. Cells treated with 250 g/ml C-PC appeared healthy, with regularity in shape, normal nuclei, and well developed cell-to-cell contact (Fig. 2C). 2.2. C-PC prevented CCl4 -induced changes in biochemical parameters of L02 cells 10 mmol/L CCl4 caused evident hepatocyte toxicity, as evidenced by the significant elevation of ALT activities in the
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Table 1 C-PC prevented CCl4 -induced changes in activities of ALT and SOD, levels of GSH and MDA in L02 cells. Groups
ALT (U/106 cell)
SOD (units/mg protein)
MDA (nmol/mg protein)
GSH (g/mg protein)
Normal Model C-PC(31) C-PC(62) C-PC(125) C-PC(250)
5.01 ± 0.23 12.6 ± 1.5# 9.75 ± 0.50* 8.18 ± 1.4* 6.99 ± 1.28** 5.13 ± 0.48**
4.07 ± 0.59 2.32 ± 0.27# 2.49 ± 0.38 3.07 ± 0.15* 3.52 ± 0.03** 3.76 ± 0.07**
2.55 ± 0.13 4.85 ± 0.30# 4.14 ± 0.07* 3.51 ± 0.24** 2.82 ± 0.24** 2.39 ± 0.23**
49.4 ± 2.8 35.9 ± 1.2# 36.5 ± 0.8 38.0 ± 1.5* 42.3 ± 1.0** 45.3 ± 2.5**
The data are expressed in mean ± S.D. of at least three independent experiments. *P < 0.05 and **P < 0.01 compared with the corresponding value for model group. # P < 0.01 compared with the corresponding value for normal control group.
Fig. 1. C-PC attenuated CCl4 -induced cytotoxicity in L02 cells. Cell viability was determined by MTT assay. The cells were treated with or without 10 mmol/ L CCl4 for 6 h and then cultured in the presence or absence of C-PC for 48 h. Data were shown as means ± S.D. (n = 6). ## P < 0.01 vs normal group, **P < 0.01 vs model group.
Fig. 3. Effects of C-PC on the level of ROS in L-02 induced by CCl4 damage. ## P < 0.01, model group compared with normal group; * P < 0.05, ** P < 0.01, C-PC groups compared with model group.
Table 2 Effects of C-PC on serum ALT and AST in CCl4 -intoxicated mice.
culture supernatant. However, co-treatment with C-PC prevented CCl4 -induced release of cellular AST into the medium in a dosedependent manner (Table 1). The L02 cells treated with CCl4 only showed decrease in the SOD activity and the GSH level, but significant increase in the MDA level. Co-treatment with C-PC prevented CCl4 -induced changes in the SOD activity and the levels of GSH and MDA (Table 1).
Group
ALT (U/ml)
AST (U/ml)
Normal Model C-PC(100) C-PC(200) C-PC(400)
12.76 ± 156.9 ± 92.38 ± 57.85 ± 46.11 ±
16.41 ± 98.01 ± 41.53 ± 33.56 ± 23.58 ±
3.81 29.9# 23.0* 11.9* 9.01*
3.99 19.4# 6.45* 3.81* 5.45*
All values are means ± SD (n = 12). # P < 0.01 compared with normal group. * P < 0.01 compared with CCl4 group.
2.3. C-PC inhibited CCl4 -induced accumulation of free radicals in L02 cells
2.4. Effect of C-PC on CCl4 -induced liver damage in mice
To further explore the biochemical basis contributing to protective effects of C-PC on L02 cell, CCl4 -induced free radical generation and free radical scavenging capacity of C-PC were examined. Intracellular ROS was detected by fluorescein dye 2 ,7 -H2 DCFDA. L02 cells treated with CCl4 only showed increase in intracellular ROS as shown by increased fluorescence intensity (Fig. 3). Co-treatment with C-PC reduced intracellular ROS in a dose-dependent manner. These findings were further confirmed by fluorescent microscopy (Fig. 4).
The effect of C-PC on CCl4 -induced hepatotoxicity was summarized in Table 2. A significant elevation of serum ALT and AST activities in CCl4 -treated group as compared to the normal group (P < 0.01) indicated CCl-induced damage to the hepatic cells. Mice in the test groups (treated with C-PC) reduced the activities of serum ALT and AST, as compared to the model group (treated with CCl4 ) (P < 0.01). The effect was found to be dose-dependent, and the magnitude of the standard deviation (SD) in all test groups indicated the individual variability in drug response.
Fig. 2. Morphological changes in L02 cells treated with CCl4 . The cells were examined under the light microscope after Wright–Giemsa stain. (A) Normal L02 cells; (B) L02 cells treated with 10 mmol/ L CCl4 ; (C) L02 cells co-treatment with 250 g/ml C-PC.
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Fig. 4. The generation of ROS in L02 cells. DCF fluorescence was detected by fluorescent microscope.
The histological observations basically supported the results obtained from serum enzymes assays. There were no pathological changes in healthy control livers which showed normal architecture (Fig. 5A). Histopathological examination of livers challenged with CCl4 showed vacuole formation and inflammatory infiltration. The hepatocytes were markedly edematous and the cytoplasm of hepatocytes was loose (Fig. 5B). The liver sections of mice pre-
treated with the C-PC before CCl4 challenge revealed that C-PC was able, in a dose-dependent manner, to prevent the development of histopathological changes, which exhibited areas of normal liver architecture and patches of inflammatory infiltration and necrotic hepatocytes (Fig. 5C–E). Livers of mice pre-treated with the highest dose (400 mg/kg) showed well-preserved architecture (Fig. 5E).
Fig. 5. Photomicrographs of liver sections stained with hematoxylin and eosin (×100). (A) Hepatic tissue of control mice, showing normal appearance. (B) Hepatic tissue of CCl4 -treated mice. (C) Hepatic tissue of C-PC (100 mg/kg) and CCl4 -treated mice. (D) Hepatic tissue of C-PC (200 mg/kg) and CCl4 -treated mice. (E) Hepatic tissue of C-PC (400 mg/kg) and CCl4 -treated mice.
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Table 3 Effects of C-PC on liver MDA, GSH levels and SOD, GSH-Px activities in CCl4 -intoxicated mice. Group
MDA (nmol/mg protein)
GSH (g/mg protein)
SOD (U/mg protein)
GSH-Px (U/mg protein)
Normal Model PC(100) PC(200) PC(400)
2.8 ± 0.11 8.0 ± 0.31## 3.2 ± 0.13** 2.3 ± 0.07** 2.1 ± 0.06**
36 ± 1.2 15 ± 0.7## 25 ± 2.3** 28 ± 1.8** 34 ± 2.0**
62 ± 2.6 21 ± 1.9## 26 ± 2.2 44 ± 3.1** 45 ± 3.3**
83.8 ± 1.7 55.7 ± 2.4## 68.8 ± 3.1* 70.7 ± 2.5** 71.9 ± 1.4**
All values are means ± SD (n = 12). ## P < 0.01 compared with normal group. * P < 0.05, ** P < 0.01 compared with CCl4 group.
Fig. 6. The levels of mRNA for TGF-1 and HGF were examined by RT-PCR. Expression of GAPDH mRNA was used as the internal standard. (A) Level of TGF-1 and HGF mRNAs. (Lane 1) normal group, (lane 2) CCl4 group, (lane 3) 100 mg/kg C-PC + CCl4 group and (lane 4) 200 mg/kg C-PC + CCl4 group. (B) Quantitative analysis of mRNA by densitometry scanning.
2.5. Lipid peroxidation MDA level is widely used as a marker of free radical mediated lipid peroxidation damage. We measured MDA levels in the livers. The results are shown in Table 3. MDA levels in the CCl4 treated group were significantly higher than that in the normal group (P < 0.01). MDA levels in the C-PC treated groups were significantly lower than that in the model group (P < 0.01). These findings indicated that the free radicals being released in the liver were effectively scavenged when treated with C-PC. 2.6. Hepatic antioxidant activities SOD, GSH-Px, and GSH were measured as an index of antioxidant status of tissues. Significantly lower liver GSH, SOD, and GSH-Px activities were observed in the model group as compared to the normal group. There was a significant increase of SOD and GSH-Px activity in the drug groups as compared to the model group. Treatment with CCl4 also significantly decreased the GSH levels in the liver as compared to the normal group. By contrast, administration with C-PC significantly increased the GSH levels as compared to the CCl4 -treated group (Table 3). 2.7. Effect of C-PC on TGF-ˇ1 mRNA and HGF expression in mice liver We detected the expression of TGF-1 and HGF at the mRNA levels in mice liver using RT-PCR (Fig. 6). GAPDH was used as an internal control. Compared to the normal group, the level of TGF1 and HGF mRNAs were significantly increased after treated with CCl4 . 200 mg/kg C-PC pretreatment significantly decreased CCl4 induced TGF-1 mRNA upregulation, but no significant change
was found between the low C-PC dosage (100 mg/kg) drug group and the model group (P > 0.05). Unlike TGF-1, 100 mg/kg and 200 mg/kg C-PC both evidently down-regulated HGF mRNA levels (P < 0.01).
3. Discussion CCl4 is a xenobiotic that produces hepatotoxicity in various experimental animals. CCl4 is metabolized by hepatic microsomal cytochrome P450 (CYP) 2E1 to form a reactive trichloromethyl (CCl3 · ) radical and/or a trichloromethyl peroxyl (CCl3 OO· ) radical. Both radicals are capable of binding to DNA, lipids, proteins, or carbohydrates, leading to lipid peroxidation, cell necrosis, excessive deposition of collagen in liver, and liver fibrosis [2]. Some studies have demonstrated that an important mechanism of the hepatoprotective effects may be related to an antioxidant capacity to scavenge reactive oxygen species [11,12]. C-PC is a potent free radical scavenger and an antioxidant agent. However, little information is available about the effects of C-PC on oxidatively damaged hepatocyte in vitro. In this study, co-incubation with C-PC for 48 h significantly attenuated oxidative stress in human hepatocyte cell line L02 induced by CCl4 in a concentrationdependent manner. Our results showed that CCl4 inhibited the growth of L02 cells, and induced cell death. The L02 cells treated with CCl4 were also associated with increased intercellular ROS, MDA and decreased GSH levels, changes in activity of intercellular SOD, as well as activity of ALT in culture medium. These CCl4 induced changes were, to a large extent, prevented by co-treatment with C-PC. Our results also indicated that C-PC exerted its protective effect intra-cellularly, rather than extra-cellularly, reacting with CCl4 in the culture medium.
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It has been reported that intraperitoneal (i.p.) administration (200 mg/kg) of a single dose of C-PC (from Spirulina platensis) to rats, one or three hours prior to CCl4 challenge, significantly reduced the hepatotoxicity [13]. In our study, a single dose of C-PC (from S. maxima) could not protect mice against CCl4 -induced liver damage (data not shown), while pretreatment with C-PC for seven consecutive days evidently attenuated CCl4 -induced liver damage in mice, evidenced by decreased serum activities of ALT and AST. The hepatic cells consist of higher concentrations of AST and ALT in cytoplasm. Higher concentrations of AST also exist in mitochondria. Due to the damage caused to hepatic cells, the leakage of plasma causes an increased level of hepatospecific enzymes in serum in vivo. The elevated serum enzyme such as AST and ALT are indicative of cellular leakage and functional integrity of cell membrane in the liver. The present study showed that C-PC significantly reduced CCl4 -induced serum ALT and AST levels. The biochemical observations are supported by the histopathological examination of the mice liver. Lipid peroxidation is one of the principal causes of CCl4 induced liver damage [14,15] and is mediated by the free radical derivatives of CCl4 . The elevation of MDA levels in the liver implies that an enhanced peroxidation causes tissue damage and breakdown of the antioxidant defense mechanisms and thus prevents the formation of superabundant free radicals [16]. In our study, C-PC administration caused a significant decrease in MDA levels as compared to the CCl4 -treated group, suggesting that C-PC could protect against CCl4 -induced lipid peroxidation in mice. In contrast to the toxic consequences of CCl4 metabolism via the CYP2E1 pathway, the detoxification pathway involves GSH conjugation of trichloromethyl-free radicals [17]. GSH plays a key role in eliminating the reactive toxic metabolites of CCl4 . Our results also showed that C-PC pretreatment significantly inhibited the CCl4 -induced depletion of hepatic GSH. SOD is an effective defense enzyme that catalyses the dismutation of superoxide anions into hydrogen peroxide (H2 O2 ). GSH-Px is an important enzyme in the liver and converted the reduction of H2 O2 and hydroperoxides to non-toxic products [11]. Lipid peroxides or ROS easily inactivate these antioxidant enzymes, which results in reduced activities of these enzymes in CCl4 toxicity [18]. In the present study, the hepatic antioxidant activities were significantly decreased in CCl4 -intoxicated mice compared with the mice in the normal group, implying increased oxidative damage to the liver. Administration of C-PC to CCl4 -treated mice could significantly elevate the activity of SOD and GSH-Px, suggesting that it has the ability to restore these enzymes’ activities in CCl4 -damaged liver. It has been revealed that TGF-1 down-regulates the glutathione synthesis [19] and the expression levels and activity of antioxidant enzymes, including glutathione-s-transferase, superoxide dismutase and glutathione peroxidase [20]. Mice treated with CCl4 exhibited a significantly increased level of liver-associated TGF-1 mRNA at 24 h and 48 h, respectively, as compared to the mice in the normal group [21]. The present study also showed increased level of TGF-1 mRNA in the liver of CCl4 -treated mice, compared with normal mice. 200 mg/kg C-PC pretreatment significantly decreased the elevated level of TGF-1 in CCl4 -intoxicated mice. These findings suggest that the inhibitory effects of C-PC on liver damage might be related to its action on the production of TGF-1. HGF was first identified in the serum of rats that underwent partial hepatectomy [22]. Physiologically, HGF has an important role as an organotrophic factor responsible for the regeneration of liver, kidney and lung [23]. Beside this physiological effect, it is known that HGF is produced by mesenchymal cells in the body in response to injuries of distant organ [24]. HGF mRNA
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and HGF activity increase markedly in the liver of rats after various liver injuries such as hepatitis, ischaemia, physical crush, and partial hepatectomy [25]. The number of studies on HGF level in the liver of CCl4 -treated mice is limited. In our study, HGF mRNA levels in the liver of CCl4 -intoxicated mice were found statistically significantly higher compared to those in the normal group (P < 0.01). This result suggests that HGF in the liver is significantly correlated with liver injury progression and may be a predictor of liver injury. Administration of C-PC inhibited liver damage induced by CCl4 , resulting in decreased HGF level in the liver. In conclusion, the results of this study demonstrate that the CPC was effective in preventing CCl4 -induced damage to hepatocyte in vitro and in vivo. The hepatoprotective effects of C-PC may be due to inhibited lipid peroxidation and effective recovery of the antioxidative defense system. In addition, C-PC may be able to block inflammatory infiltration through its anti-inflammatory activities by inhibiting TGF-1 and HGF expression in CCl4 -induced hepatic damage. References [1] P. Vitaglione, F. Morisco, N. Caporaso, et al., Dietary antioxidant compounds and liver health, Crit. Rev. Food Sci. Nutr. 44 (2004) 575–586. [2] C.F. Tsai, Y.W. Hsu, W.K. Chen, et al., Hepatoprotective effect of electrolyzed reduced water against carbon tetrachloride-induced liver damage in mice, Food Chem. Toxicol. 47 (8) (2009) 2031–2036. [3] L.D. DeLeve, N. Kaplowitz, Mechanisms of drug-induced liver disease, Gastroenterol. Clin. North Am. 24 (1995) 787–810. [4] S.K. Natarajan, S. Thomas, S. Ramamoorthy, et al., Oxidative stress in the development of liver cirrhosis: a comparison of two different experimental models, J. Gastroenterol. Hepatol. 21 (2006) 947–957. [5] A.D. Bhatt, N.S. Bhatt, Indigenous drugs and liver disease, Indian J. Gastroenterol. 15 (1996) 63–67. [6] B.B. Vadiraja, K.M. Madyastha, C-phycocyanin: a potent peroxyl radical scavenger in vivo and in vitro, Biochem. Biophys. Res. Commun. 275 (2000) 20– 25. ˜ [7] J.E. Pinero Estrada, P. Bermejo Bescós, A.M. Villar del Fresno, Antioxidant activity of different fractions of Spirulina platensis protean extract, Farmaco 156 (2000) 497–500. [8] Y. Ou, J. Dong, W.T. Wu, Purification and characterization of Phycobiliprotein from Aphanothece halophytica, Pharm. Biotechnol. 11 (1) (2004) 37–41. [9] Y. Ou, P. Geng, G.Y. Liao, et al., Intracellular GSH and ROS levels may be related to galactose-mediated human lens epithelial cell apoptosis: role of recombinant hirudin variant III, Chem-Biol. Interact. 179 (2009) 103–109. [10] E.J. Park, K. Park, Oxidative stress and pro-inflammatory responses induced by silica nanoparticles in vivo and in vitro, Toxicol. Lett. 184 (1) (2009) 18– 25. [11] S.R. Naik, V.S. Panda, Antioxidant and hepatoprotective effects of Ginkgo biloba phytosomes in carbon tetrachloride-induced liver damage in rodents, Liver Int. 27 (2007) 393–399. [12] G. Mehmetc¸ik, G. Ozdemirler, N. Koc¸ak-Toker, et al., Effect of pretreatment with artichoke extract on carbon tetrachloride-induced liver damage and oxidative stress, Exp. Toxicol. Pathol. 60 (6) (2008) 475–480. [13] B.B. Vadiraja, N.W. Gaikward, K.M. Madyastha, Hepatoprotective effect of Cphycocyanin: protection for carbon tetrachloride and R-(+) Pulegone-mediated hepatotoxicity in rats, Biochem. Biophys. Res. Commun. 249 (1998) 428– 431. [14] S. Basu, Carbon tetrachloride-induced lipid peroxidation: eicosanoid formation and their regulation by antioxidant nutrients, Toxicology 189 (2003) 113– 127. [15] M.K. Manibusan, M. Odin, D.A. Eastmond, Postulated carbon tetrachloride mode of action: a review, J. Environ. Sci. Health. C: Environ. Carcinog. Ecotoxicol. Rev. 25 (2007) 185–209. [16] Y.W. Hsu, C.F. Tsai, W.K. Chen, et al., Protective effects of seabuckthorn (Hippophae rhamnoides L.) seed oil against carbon tetrachloride-induced hepatotoxicity in mice, Food Chem. Toxicol. 47 (2009) 2281–2288. [17] K.J. Leea, J.H. Choia, T. Khanala, et al., Protective effect of caffeic acid phenethyl ester against carbon tetrachloride-induced hepatotoxicity in mice, Toxicology 248 (2008) 18–24. [18] Y.S. Yang, T.H. Ahn, J.C. Lee, et al., Protective effects of Pycnogenol on carbon tetrachloride-induced hepatotoxicity in Sprague-Dawley rats, Food Chem. Toxicol. 46 (2008) 380–387. [19] K. Arsalane, C.M. Dubois, T. Muanza, et al., Transforming growth factor-beta1 is a potent inhibitor of glutathione synthesis in the lung epithelial cell line A549: transcriptional effect on the GSH rate-limiting enzyme gammaglutamylcysteine synthetase, Am. J. Respir. Cell Mol. Biol. 17 (1997) 599–607. [20] Y. Kayanoki, J. Fujii, K. Suzuki, et al., Suppression of antioxidative enzyme expression by transforming growth factor-beta 1 in rat hepatocytes, J. Biol. Chem. 269 (1994) 15488–15492.
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