Cell Biochem Biophys DOI 10.1007/s12013-014-0313-x
ORIGINAL PAPER
Hepatoprotective Effect of Collagen Peptides From Cod Skin Against Liver Oxidative Damage In Vitro and In Vivo Yantao Han • Jing Xie • Hui Gao • Yunqiu Xia Xuehong Chen • Chunbo Wang
•
Ó Springer Science+Business Media New York 2014
Abstract The objective of this study was to investigate the hepatoprotective effect of cod skin collagen peptides (CSCP), isolated from fishing industrial by-products, in vitro and in vivo. Effect of CSCP on cell proliferation of normal and H2O2-damaged Chang liver cells was determined by MTT assay in vitro. Two animal models, CCl4induced and acetaminophenum-induced acute hepatotoxicity, were established to assess the hepatoprotective effect of CSCP. Liver weight index, serum ALT and AST, antioxidant enzymes, and lipid peroxidation product were used as the markers of liver toxicity. The cell viability in the H2O2-treated Chang liver cells was remarkably increased when pretreated with CSCP from 100 to 1,000 lg/ml in a dose-dependent manner. CSCP pretreatment also alleviated the CCL4-induced liver index loss, while no marked changes were found in acetaminophenum-treated mice. Furthermore, CSCP pulled down serum ALT and AST level, increased the activities of SOD and CAT, and decreased MDA in both murine models of acute liver toxicity. Pretreatment with CSCP protected liver tissue against oxidative injure in vivo and in vitro. The underlying mechanism might involve enhancement in the activities of antioxidant enzymes and reduction in the lipid peroxidation.
Y. Han J. Xie (&) H. Gao Y. Xia X. Chen C. Wang Department of Pharmacology, Medical College of Qingdao University, Qingdao 266071, Shandong, China e-mail: [email protected]
Keywords Cod skin collagen peptides Hepatoprotective effect Oxidative damage
Introduction Collagen has been widely used in food, cosmetic, biomedical, and pharmaceutical industries due to its excellent biocompatibility and biodegradability, and weak antigenicity [1]. Typically, collagen is isolated from the skins of land-based animals, such as cows and pigs. However, because of the risks of bovine spongiform encephalopathy (BSE) and foot-and-mouth disease (FMD) associated with land-based animals, fish has been considered a safer resource of collagen in recent years [2, 3]. Moreover, using by-products of fish processing such as skins, scales, bones, and swim bladders as resources for collagen also produced extra profit for the industry [4, 5]. As a source of biologically active peptides, fish collagen peptides have been reported to have a variety of health benefits including antioxidative activity [6, 7], antimicrobial activity [8], lipid-lowering effect [9], wound healing [10], beneficial effects on skin [11, 12], and bone or joint health [13]. Majority of the studies on fish collagen peptides focused on their antioxidative activity, regardless of different genus and characters of fish collagen [6, 7, 14]. Our previous study has revealed cod skin collagen peptides (CSCP) could scavenge hydroxyl radical, superoxide radical, ABTS radical, and DPPH radical effectively (not published), which led to the hypothesis that CSCP could act as a potential candidate for the therapy of diseases associated with oxidative stress, such as free radical species-induced liver damage. Therefore, the present study was aimed to investigate the hepatoprotective potential of CSCP in vitro and in vivo.
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Materials and Methods Chemicals Cod skin collagen peptide (CSCP) was donated by Fusheng Co. Ltd (Qingdao, Shandong, China) with 85.8 % of the molecular weight distributed between 500 and 3,000 Da. The composition and molecular weight of CSCP were listed in Table 1. The drugs were dissolved in distilled water for all the experiments. Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco (USA). Alanine aminotransferase (ALT), aspartate aminotransferase (AST), malondialdehyde (MDA), superoxidedismutase (SOD), glutathioneperoxidase (GSH-Px), and catalase (CAT) assay kits were purchased from Nanjing Jiancheng Bioengineering Research Institute (Nanjing, Jiangsu, China); dimethyl diphenyl bicarboxylate (DDB) was purchased from Yantai Luyin Pharmaceutical Co., Ltd (Yantai, Shandong, China); all other chemicals were of analytical grade. Cell Culture and Treatment Chang liver cells, an immortalized normal human hepatocyte line, were purchased from Chinese Academy of Sciences Cell bank and maintained in DMEM with 10 % FBS and 100 lg/ml penicillin–streptomycin in a humidified incubator at 37 °C and 5 % CO2.
(i) control group: cells were sham treated; (ii) H2O2 group: cells were treated with 200 lM H2O2; (iii) CSCP-treated groups: Cells pretreated with CSCP (gradient concentrations from 1 and 30 mg/ml) for 2 h before 200 lM H2O2 was added. The cells were continued to culture for 24 h before the cell viability assay. Cell viability was determined using methyl thiazoldiphenyl-tetrazolium bromide (MTT) assay. Briefly, cells were washed and incubated with 0.5 mg/ml MTT at 37 °C for 4 h following drug treatment. The formazan crystals were extracted and dissolved to dimethyl sulfoxide (DMSO) at room temperature. Absorbance was measured at a wavelength of 490 nm using a Molecular Devices VERSAmax microplate reader (Molecular devices, Sunnyvale, CA, USA). Animals Male Kunming mice (20 ± 2 g), SPF, were obtained from the Animal Department of Institute for drug control (Qingdao, China) and were allowed to quarantine and acclimate for a week prior to experimentation. The study protocol was approved by the ethical committee of our institution. All mice were housed under controlled conditions with temperature of 25 ± 2 °C, relative humidity of 60 ± 10 %, room air changes 12–18 times/h, and a 12-h light/dark cycle. Animals were allowed free access to food and water. Animal Grouping and Treatment
MTT Assay Firstly, cell proliferation was assessed on normal Chang liver cells by adding different concentrations of CSCP into the cells and cultured for 24 h. To assess the effect of CSCP on oxidative damaged cells, Chang liver cells were grouped as following: Table 1 Composition and molecular weight of CSCP Composition (%) Water
1.8
Ash
0.4
Protein
97.6
Hydroxyproline
6.1 Molecular weight (%)
[5,000 Da 3,000–5,000 Da
6.9 18.0
1,000–3,000 Da
40.6
500–1,000 Da
27.2
\500 Da
7.3
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Two acute hepatotoxic models were established by treating mice with CCl4 and acetaminophenum, respectively. For each model, the animals were divided into six groups with 10 mice in each group. For CCl4-induced acute hepatotoxicity model, animals were grouped and treated as follows: (i) control group: saline intragastrically for 10 days; (ii) CCl4-induced acute hepatotoxicity group: saline for 10 days ? CCl4 (100 mg/kg, ip. 0.2 % dissolved in peanut oil); (iii) DDB-treated group (positive control): DDB (200 mg/kg/day) for 10 days ? CCl4 (100 mg/kg, ip.); (iv) (v) (vi) low-, medium- and high-dosage CSCP-treated groups: CSCP (1 g, 2 g, 4 g/kg, respectively) for 10 days ? CCl4 (100 mg/kg, ip.). For acetaminophenum-induced acute hepatotoxicity model, animals were grouped and treated the same way as the CCl4 model except that they were treated with acetaminophenum (150 mg/kg) by oral administration. At the final stage of the experiment, the animals were fast overnight and sacrificed on the next day by cervical dislocation immediately after withdrawal of blood from the retrobulbar vessels. Afterward, the whole blood was centrifuged and serum samples were collected for liver
Cell Biochem Biophys
function tests. The whole liver was dissected out and washed immediately with ice-cold saline to remove as much blood as possible. After weighted, liver samples was immediately stored at -80 °C for future analysis. Calculation of Liver Index Livers were taken out right after the mice were scarified, and were gently cleaned off blood then weighted. The liver index was represented as mouse liver weight (g)/mouse weight (100 g). Liver Function Test Serum level of ALT and AST were measured using commercially available kits (Nanjing Jiancheng Bioengineering Research Institute, Nanjing, China) according to the manufacturer’s instructions. Estimation of Antioxidant Enzymes and Lipid Peroxidation Product Liver tissue samples were homogenized on ice in Tris–HCl (5 mmol/l containing 2 mmol/l EDTA, pH 7.4), and then centrifuged at 1,0009g for 15 min at 4 °C. The supernatants were used immediately for the assays of SOD, CAT, GSH, and MDA. All these enzymes were determined using commercially available kits (Nanjing Jiancheng Bioengineering Research Institute, Nanjing, China) according to the manufacturer’s instructions.
Following H2O2 treatment, cell viability showed a significant decrease compared with the control group and CSCP pretreatment effectively protected Chang liver cells against the H2O2-induced cell viability loss at the dosage from 100 to 1,000 lg/ml. The protective effect of CSCP was in a dose-dependent manner and after CSCP pretreatment the cell viabilities was increased to the level above the control group, as shown in Fig. 1b. Effects of CSCP on Liver Index in Mice As shown in Fig. 2a, liver index showed a significant decrease in the model group treated with CCL4 compared to the control group, indicating that the liver tissue was seriously damaged by CCL4 treatment. The CSCP pretreatment at the medium and high dosage, as well as DDB pretreatment, alleviated the CCL4-induced liver index loss. In contrast, as shown in Fig. 2b, liver index in the acetaminophenum-induced model had only a slight decrease (P [ 0.05) compared with the control group. Moreover, the liver index had no marked change following the CSCP and DDB treatment. Effects of CSCP on Serum ALT and AST in Mice
Statistical analysis was performed using SPSS13.0 software. One-way analysis of variance (ANOVA) was used to compare the means among different groups and p values of \0.05 were considered statistically significant.
The serum ALT and AST level were examined to further evaluate the liver injury of mice. In both model groups, the activities of ALT and AST were dramatically increased compared to the control group. When the two models were compared, CCL4 treatment resulted in a much higher level of ALT and AST than acetaminophenum. DDB pretreatment could pull down ALT and AST in both models. The medium and high dosage of CSCP pretreatment attenuated the acetaminophenum-induced increase in ALT and AST activities. Only the high dosage of CSCP pretreatment was effective to attenuate the CCL4-induced elevation in ALT and AST activities (Fig. 3a, b).
Results
Effects of CSCP on Antioxidant Enzyme and Lipid Peroxidation in Mice
Statistical Analysis
Effects of CSCP on Cell Proliferation in Normal and Oxidative Damaged Cell Effect of CSCP on normal Chang liver cell growth was shown in Fig. 1a. Cell growth was not affected by CSCP at dose lower than 10,000 lg/ml, indicating that CSCP was safe to Chang liver cells at dose lower than 10,000 lg/ml. Furthermore, following treatment with CSCP at 1,000 and 10,000 lg/ml, the cell viabilities was markedly increased compared with the non-CSCP-treated group. The result indicated that at the dose between 1,000 and 10,000 lg/ml CSCP could enhance the cell viability.
Liver injury induced by CCL4 and acetaminophenum provoked a significant reduction of SOD and CAT activity, as well as a decline of GSH content. However, MDA, one of the most important products of membrane lipid oxidation, was remarkably increased after CCL4 and acetaminophenum treatment (Figs. 4, 5). In the CCL4-induced liver injury, DDB and high dosage of CSCP pretreatment significantly increased the activities of SOD and CAT and decrease MDA. The medium dosage of CSCP was only effective on SOD and MDA, whereas the low dosage of CSCP had no influence to the antioxidants and MDA (Fig. 4). In contrast, in the acetaminophenum-induced liver
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Cell Biochem Biophys Fig. 1 CSCP increased cell viability in Chang liver cells. a Cell viability in Chang liver cells after concentration gradients of CSCP pretreatment. The cells were pretreated with CSCP and the cell viability was assessed by MTT 24 h later. Compared with the previous group, *P \ 0.05, **P \ 0.01. b Cell viability in H2O2-treated Chang liver cells after concentration gradients of CSCP pre-treatment. The cells were pre-treated with CSCP for 2 h before 200 lM H2O2 was administrated to cells. Cell viability was assessed by MTT 24 h later. Compared with the control group, *P \ 0.05; compared with the H2O2-treated group, ##P \ 0.01
injury model, both of DDB and CSCP at all experimental dosages enhanced SOD, CAT, and GSH, and reduced MDA (Fig. 5).
Discussion Marine species comprise approximately half of the total global biodiversity and the sea offers an enormous resource for nutritional or pharmaceutical applications. In recent years, marine originated collagen earned more and more attention due to biological safety, excellent biocompatibility, low antigenicity, high biodegradability, and cell growth potential [2, 3, 15]. However, few studies have investigated the biological activities of the collagen from marine animals. CSCP, compounds of hydroxyproline-rich collagen peptides (mainly 500–3,000 Da), were derived from cod skin which has been the wastage and pollution in the fish-processing industry. In the present study, the hepatoprotective effect of CSCP was examined in vitro and in vivo. We for the first time revealed the hepatoprotective effect of fish collagen peptides. Prior to the animal experiment, the Chang liver cell was chosen as a normal human hepatocyte line to assess whether CSCP could alleviate the H2O2-induced liver damage. Our results indicated that CSCP was safe to Chang liver
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cells at the dose lower than 10,000 lg/ml while CSCP at dosage higher than 10,000 lg/ml reduced the cell viability. CSCP was considered safe as the absorption dosage of collagen was far below 10,000 lg/ml. Furthermore, without H2O2 treatment, 1,000 to 10,000 lg/ml CSCP could effectively enhance the cell viability, which suggested that CSCP has the potential to improve liver function. Following H2O2 treatment, CSCP at the dose of 100 to 1,000 lg/ml protected Chang liver cells against the H2O2induced cell viability loss in a dose-dependent manner. Moreover, CSCP pretreatment restored the cell viabilities to the level higher than control. Taken together, these results demonstrated that CSCP could protect against the free radical-induced liver damage in vitro. In the animal experiment, two different liver damaged models were established in mice: the CCL4-induced model and the acetaminophenum-induced model. The hepatotoxic effect of CCL4 is caused by the conversion of CCL4 by the cytochrome-P450 mixed function enzyme in the smooth endoplasmic reticulum of the liver to the highly reactive CCL3, which yielded the free radical attack to liver to initiate peroxidate degradation of membrane lipids [16]. On the other hand, acetaminophenum is a well-known antipyretic and analgesic agent, which is harmless at therapeutic doses but can produce fatal hepatic necrosis at high doses. Acetaminophenum is also metabolized by
Cell Biochem Biophys
Fig. 2 CSCP enhanced liver index in acute liver injured mice. a Liver index in CCL4-induced acute liver damage in mice. The mice were oral administrated with 1, 2, 4 g CSCP, respectively, for 10-days. Two hours after CSCP pretreatment, animals received 100 mg/kg CCl4 by intraperitoneal injection. 16 h later, the mice were scarified and the livers were taken out to calculate the liver index. b Liver index in acetaminophenum-induced acute liver damage in mice. The mice were treated as above and the liver damage was induced by 150 mg/kg acetaminophenum oral administration at the same time point. Compared with the control group, *P \ 0.05; compared with the model group, #P \ 0.05
Fig. 3 CSCP elevated ALT and AST in acute liver injured mice. a The activities of ALT and AST in CCL4-induced acute liver damage in mice. The mice were scarified to take the eyeballs off for blood. The serum was separated and assessed. b The activities of ALT and AST in acetaminophenum-induced acute liver damage in mice. Compared with the control group, **P \ 0.01; compared with the model group, #P \ 0.05, ##P \ 0.01
cytochrome P450 and high dose of acetaminophenum forms the highly reactive N-acetyl-p-benzoquinone imine (NAPQI), which cannot be detoxified by enough GSH and harms the liver tissue [17]. Both of these two models represented free radical damage to liver, and the serum ALT and AST activities were remarkably increased and AST/ALT [1 in both models, indicating the severe impairment of liver function. Furthermore, the liver index was calculated to assess the liver injury. In the CCL4treated model, was significantly dropped, while in the acetaminophenum-treated model the liver index was not significantly changed. Therefore, we took the CCL4-treated model for severely damaged liversevere and the acetaminophenum-treated model as moderately damaged liver. Pretreated with gradient concentration of CSCP, the liver index of the CCL4-damaged groups was increased while that of the acetaminophenum-damaged groups remained unchanged. The results might be explained by the insensitivity of the acetaminophenum-induced moderate
damage to CSCP. In the CCL4-damaged groups, the medium- and high- dosage of CSCP treatment, as well as the DDB treatment, markedly enhanced the liver index compared to the model group, which indicated that the medium- and high- dosage of CSCP pretreatment could alleviate the CCL4-induced severe damage to the liver in mice. The serum ALT and AST activities were determined after the liver index assay. We found the ALT and AST activities in the CCL4-treated model mice were much higher than those in the acetaminophenum-treated model mice, which was in line with the results of liver index to further demonstrate that the CCL4-induced liver toxicity was more severe than the acetaminophenum-induced liver toxicity. Moreover, our result showed that in both models, CSCP pretreatment, as well as DDB treatment, could attenuate the elevated ALT and AST activities induced by CCL4 or acetaminophenum. We found that the high-dose of CSCP was effective in the CCL4-induced liver damage,
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Fig. 4 CSCP modulated the redox states in CCL4-induced acute liver damage in mice. a–d represented the activities of SOD, CAT, GSH and MDA in liver tissue in mice. Compared with the control group, **P \ 0.01; compared with the model group, #P \ 0.05, ##P \ 0.01
Fig. 5 CSCP modulated the redox states in acetaminophenum-induced acute liver damage in mice. a–d represented the activities of SOD, CAT, GSH and MDA in liver tissue in mice. Compared with the control group, **P \ 0.01; compared with the model group, #P \ 0.05, ##P \ 0.01
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while in acetaminophenum-induced liver damage, both of the medium- and high- dosage of CSCP could alleviate the liver injury. The effects of CSCP on antioxidant enzymes and lipid peroxidation products were further explored in liver tissue. After CCL4 or acetaminophenum treatment, the activities of antioxidant enzymes SOD, CAT, and GSH were remarkably suppressed while the lipid peroxidation product MDA was markedly enhanced, suggesting an increased oxidative stress in liver tissue. In the acetaminophenumtreated model, CSCP at all three doses as well as DDB were effective on restoring the redox balance by increasing the activities of antioxidant enzymes and decreasing MDA. In contrast, in the CCL4-induced model, only the high-dose of CSCP and DDB pretreatment partially restored the activities of antioxidant enzymes and MDA (medium dose of CSCP only effective in SOD and MDA). These results suggested that CSCP might yield its hepatoprotective effect through balancing the redox state in liver. In summary, CSCP pretreatment protected liver tissue against oxidative injury in vivo and in vitro. The mechanism might involve enhancing the activities of antioxidant enzymes and reducing the lipid peroxidation.
6.
7.
8.
9.
10.
11.
12. Acknowledgments This work was supported by Agricultural Science and Technology Program of Qingdao (Grant No.14-2-3-50-nsh). Conflict of interest The authors declare that there is no conflict of interests regarding the publication of this paper.
13.
References 14. 1. Shoulders, M. D., & Raines, R. T. (2009). Collagen structure and stability. Annual Review of Biochemistry, 78, 929–958. 2. Go´mez-Guille´n, M. C., Gime´nez, B., Lo´pez-Caballero, M. E., & Montero, M. P. (2011). Functional and bioactive properties of collagen and gelatin from alternative sources: A review. Food Hydrocolloids, 25, 1813–1827. 3. Kaewdang, O., Benjakul, S., Kaewmanee, T., & Kishimura, H. (2014). Characteristics of collagens from the swim bladders of yellowfin tuna (Thunnus albacares). Food Chemistry, 15(155), 264–270. 4. Senevirathne, M., & Kim, S. K. (2012). Utilization of seafood processing by-products: Medicinal applications. Advances in Food and Nutrition Research, 65, 495–512. 5. Jayathilakan, K., Sultana, K., Radhakrishna, K., & Bawa, A. S. (2012). Utilization of byproducts and waste materials from meat,
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poultry and fish processing industries: A review. Journal of Food Science and Technology, 49(3), 278–293. Wang, B., Wang, Y. M., Chi, C. F., Luo, H. Y., Deng, S. G., & Ma, J. Y. (2013). Isolation and characterization of collagen and antioxidant collagen peptides from scales of croceine croaker (Pseudosciaena crocea). Marine Drugs, 11(11), 4641–4661. Jia, J., Zhou, Y., Lu, J., Chen, A., Li, Y., & Zheng, G. (2010). Enzymatic hydrolysis of Alaska pollack (Theragra chalcogramma) skin and antioxidant activity of the resulting hydrolysate. Journal of the Science of Food and Agriculture, 90(4), 635–640. Go´mez-Guille´n, M. C., Lo´pez-Caballero, M. E., de Lo´pez de Lacey, A., Alema´n, A., Gime´nez, B., & Montero, P. (2010). Antioxidant and antimicrobial peptide fractions from squid and tuna skin gelatin. In E. Le Bihan & N. Koueta (Eds.), Sea byproducts as a real material: New ways of application (p. 89e115). Kerala: Transworld Research Network Signpost. Saito, M., Kiyose, C., Higuchi, T., Uchida, N., & Suzuki, H. (2009). Effect of collagen hydrolysates from salmon and trout skins on the lipid profile in rats. Journal of Agriculture and Food Chemistry, 57(21), 10477–10482. Zhang, Z., Wang, J., Ding, Y., Dai, X., & Li, Y. (2011). Oral administration of marine collagen peptides from Chum Salmon skin enhances cutaneous wound healing and angiogenesis in rats. Journal of the Science of Food and Agriculture, 91(12), 2173–2179. Zhuang, Y., Hou, H., Zhao, X., Zhang, Z., & Li, B. (2009). Effects of collagen and collagen hydrolysate from jellyfish (Rhopilema esculentum) on mice skin photoaging induced by UV irradiation. Journal of Food Science, 74(6), H183–H188. Chai, H. J., Li, J. H., Huang, H. N., Li, T. L., Chan, Y. L., Shiau, C. Y., et al. (2010). Effects of sizes and conformations of fishscale collagen peptides on facial skin qualities and transdermal penetration efficiency. Journal of Biomedicine and Biotechnology, 2010, 757301. Yamada, S., Nagaoka, H., Terajima, M., Tsuda, N., Hayashi, Y., & Yamauchi, M. (2013). Effects of fish collagen peptides on collagen post-translational modifications and mineralization in an osteoblastic cell culture system. Dental Materials Journal, 32(1), 88–95. Chalamaiah, M., Dinesh Kumar, B., Hemalatha, R., & Jyothirmayi, T. (2012). Fish protein hydrolysates: Proximate composition, amino acid composition, antioxidant activities and applications: A review. Food Chemistry, 135(4), 3020–3038. Yamamoto, K., Igawa, K., Sugimoto, K., Yoshizawa, Y., Yanagiguchi, K., Ikeda, T., et al. (2014). Biological safety of fish (tilapia) collagen. BioMed Research International, 2014, 630757. Gnanadesigan, M., Ravikumar, S., & Inbaneson, S. J. (2011). Hepatoprotective and antioxidant properties of marine halophyte Luminetzera racemosa bark extract in CCL(4) induced hepatotoxicity. Asian Pacific Journal of Tropical Medicine, 4(6), 462–465. Fakurazi, S., Sharifudin, S. A., & Arulselvan, P. (2012). Moringa oleifera hydroethanolic extracts effectively alleviate acetaminophen-induced hepatotoxicity in experimental rats through their antioxidant nature. Molecules, 17(7), 8334–8350.
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ORIGINAL PAPER
Hepatoprotective Effect of Collagen Peptides From Cod Skin Against Liver Oxidative Damage In Vitro and In Vivo Yantao Han • Jing Xie • Hui Gao • Yunqiu Xia Xuehong Chen • Chunbo Wang
•
Ó Springer Science+Business Media New York 2014
Abstract The objective of this study was to investigate the hepatoprotective effect of cod skin collagen peptides (CSCP), isolated from fishing industrial by-products, in vitro and in vivo. Effect of CSCP on cell proliferation of normal and H2O2-damaged Chang liver cells was determined by MTT assay in vitro. Two animal models, CCl4induced and acetaminophenum-induced acute hepatotoxicity, were established to assess the hepatoprotective effect of CSCP. Liver weight index, serum ALT and AST, antioxidant enzymes, and lipid peroxidation product were used as the markers of liver toxicity. The cell viability in the H2O2-treated Chang liver cells was remarkably increased when pretreated with CSCP from 100 to 1,000 lg/ml in a dose-dependent manner. CSCP pretreatment also alleviated the CCL4-induced liver index loss, while no marked changes were found in acetaminophenum-treated mice. Furthermore, CSCP pulled down serum ALT and AST level, increased the activities of SOD and CAT, and decreased MDA in both murine models of acute liver toxicity. Pretreatment with CSCP protected liver tissue against oxidative injure in vivo and in vitro. The underlying mechanism might involve enhancement in the activities of antioxidant enzymes and reduction in the lipid peroxidation.
Y. Han J. Xie (&) H. Gao Y. Xia X. Chen C. Wang Department of Pharmacology, Medical College of Qingdao University, Qingdao 266071, Shandong, China e-mail: [email protected]
Keywords Cod skin collagen peptides Hepatoprotective effect Oxidative damage
Introduction Collagen has been widely used in food, cosmetic, biomedical, and pharmaceutical industries due to its excellent biocompatibility and biodegradability, and weak antigenicity [1]. Typically, collagen is isolated from the skins of land-based animals, such as cows and pigs. However, because of the risks of bovine spongiform encephalopathy (BSE) and foot-and-mouth disease (FMD) associated with land-based animals, fish has been considered a safer resource of collagen in recent years [2, 3]. Moreover, using by-products of fish processing such as skins, scales, bones, and swim bladders as resources for collagen also produced extra profit for the industry [4, 5]. As a source of biologically active peptides, fish collagen peptides have been reported to have a variety of health benefits including antioxidative activity [6, 7], antimicrobial activity [8], lipid-lowering effect [9], wound healing [10], beneficial effects on skin [11, 12], and bone or joint health [13]. Majority of the studies on fish collagen peptides focused on their antioxidative activity, regardless of different genus and characters of fish collagen [6, 7, 14]. Our previous study has revealed cod skin collagen peptides (CSCP) could scavenge hydroxyl radical, superoxide radical, ABTS radical, and DPPH radical effectively (not published), which led to the hypothesis that CSCP could act as a potential candidate for the therapy of diseases associated with oxidative stress, such as free radical species-induced liver damage. Therefore, the present study was aimed to investigate the hepatoprotective potential of CSCP in vitro and in vivo.
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Materials and Methods Chemicals Cod skin collagen peptide (CSCP) was donated by Fusheng Co. Ltd (Qingdao, Shandong, China) with 85.8 % of the molecular weight distributed between 500 and 3,000 Da. The composition and molecular weight of CSCP were listed in Table 1. The drugs were dissolved in distilled water for all the experiments. Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco (USA). Alanine aminotransferase (ALT), aspartate aminotransferase (AST), malondialdehyde (MDA), superoxidedismutase (SOD), glutathioneperoxidase (GSH-Px), and catalase (CAT) assay kits were purchased from Nanjing Jiancheng Bioengineering Research Institute (Nanjing, Jiangsu, China); dimethyl diphenyl bicarboxylate (DDB) was purchased from Yantai Luyin Pharmaceutical Co., Ltd (Yantai, Shandong, China); all other chemicals were of analytical grade. Cell Culture and Treatment Chang liver cells, an immortalized normal human hepatocyte line, were purchased from Chinese Academy of Sciences Cell bank and maintained in DMEM with 10 % FBS and 100 lg/ml penicillin–streptomycin in a humidified incubator at 37 °C and 5 % CO2.
(i) control group: cells were sham treated; (ii) H2O2 group: cells were treated with 200 lM H2O2; (iii) CSCP-treated groups: Cells pretreated with CSCP (gradient concentrations from 1 and 30 mg/ml) for 2 h before 200 lM H2O2 was added. The cells were continued to culture for 24 h before the cell viability assay. Cell viability was determined using methyl thiazoldiphenyl-tetrazolium bromide (MTT) assay. Briefly, cells were washed and incubated with 0.5 mg/ml MTT at 37 °C for 4 h following drug treatment. The formazan crystals were extracted and dissolved to dimethyl sulfoxide (DMSO) at room temperature. Absorbance was measured at a wavelength of 490 nm using a Molecular Devices VERSAmax microplate reader (Molecular devices, Sunnyvale, CA, USA). Animals Male Kunming mice (20 ± 2 g), SPF, were obtained from the Animal Department of Institute for drug control (Qingdao, China) and were allowed to quarantine and acclimate for a week prior to experimentation. The study protocol was approved by the ethical committee of our institution. All mice were housed under controlled conditions with temperature of 25 ± 2 °C, relative humidity of 60 ± 10 %, room air changes 12–18 times/h, and a 12-h light/dark cycle. Animals were allowed free access to food and water. Animal Grouping and Treatment
MTT Assay Firstly, cell proliferation was assessed on normal Chang liver cells by adding different concentrations of CSCP into the cells and cultured for 24 h. To assess the effect of CSCP on oxidative damaged cells, Chang liver cells were grouped as following: Table 1 Composition and molecular weight of CSCP Composition (%) Water
1.8
Ash
0.4
Protein
97.6
Hydroxyproline
6.1 Molecular weight (%)
[5,000 Da 3,000–5,000 Da
6.9 18.0
1,000–3,000 Da
40.6
500–1,000 Da
27.2
\500 Da
7.3
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Two acute hepatotoxic models were established by treating mice with CCl4 and acetaminophenum, respectively. For each model, the animals were divided into six groups with 10 mice in each group. For CCl4-induced acute hepatotoxicity model, animals were grouped and treated as follows: (i) control group: saline intragastrically for 10 days; (ii) CCl4-induced acute hepatotoxicity group: saline for 10 days ? CCl4 (100 mg/kg, ip. 0.2 % dissolved in peanut oil); (iii) DDB-treated group (positive control): DDB (200 mg/kg/day) for 10 days ? CCl4 (100 mg/kg, ip.); (iv) (v) (vi) low-, medium- and high-dosage CSCP-treated groups: CSCP (1 g, 2 g, 4 g/kg, respectively) for 10 days ? CCl4 (100 mg/kg, ip.). For acetaminophenum-induced acute hepatotoxicity model, animals were grouped and treated the same way as the CCl4 model except that they were treated with acetaminophenum (150 mg/kg) by oral administration. At the final stage of the experiment, the animals were fast overnight and sacrificed on the next day by cervical dislocation immediately after withdrawal of blood from the retrobulbar vessels. Afterward, the whole blood was centrifuged and serum samples were collected for liver
Cell Biochem Biophys
function tests. The whole liver was dissected out and washed immediately with ice-cold saline to remove as much blood as possible. After weighted, liver samples was immediately stored at -80 °C for future analysis. Calculation of Liver Index Livers were taken out right after the mice were scarified, and were gently cleaned off blood then weighted. The liver index was represented as mouse liver weight (g)/mouse weight (100 g). Liver Function Test Serum level of ALT and AST were measured using commercially available kits (Nanjing Jiancheng Bioengineering Research Institute, Nanjing, China) according to the manufacturer’s instructions. Estimation of Antioxidant Enzymes and Lipid Peroxidation Product Liver tissue samples were homogenized on ice in Tris–HCl (5 mmol/l containing 2 mmol/l EDTA, pH 7.4), and then centrifuged at 1,0009g for 15 min at 4 °C. The supernatants were used immediately for the assays of SOD, CAT, GSH, and MDA. All these enzymes were determined using commercially available kits (Nanjing Jiancheng Bioengineering Research Institute, Nanjing, China) according to the manufacturer’s instructions.
Following H2O2 treatment, cell viability showed a significant decrease compared with the control group and CSCP pretreatment effectively protected Chang liver cells against the H2O2-induced cell viability loss at the dosage from 100 to 1,000 lg/ml. The protective effect of CSCP was in a dose-dependent manner and after CSCP pretreatment the cell viabilities was increased to the level above the control group, as shown in Fig. 1b. Effects of CSCP on Liver Index in Mice As shown in Fig. 2a, liver index showed a significant decrease in the model group treated with CCL4 compared to the control group, indicating that the liver tissue was seriously damaged by CCL4 treatment. The CSCP pretreatment at the medium and high dosage, as well as DDB pretreatment, alleviated the CCL4-induced liver index loss. In contrast, as shown in Fig. 2b, liver index in the acetaminophenum-induced model had only a slight decrease (P [ 0.05) compared with the control group. Moreover, the liver index had no marked change following the CSCP and DDB treatment. Effects of CSCP on Serum ALT and AST in Mice
Statistical analysis was performed using SPSS13.0 software. One-way analysis of variance (ANOVA) was used to compare the means among different groups and p values of \0.05 were considered statistically significant.
The serum ALT and AST level were examined to further evaluate the liver injury of mice. In both model groups, the activities of ALT and AST were dramatically increased compared to the control group. When the two models were compared, CCL4 treatment resulted in a much higher level of ALT and AST than acetaminophenum. DDB pretreatment could pull down ALT and AST in both models. The medium and high dosage of CSCP pretreatment attenuated the acetaminophenum-induced increase in ALT and AST activities. Only the high dosage of CSCP pretreatment was effective to attenuate the CCL4-induced elevation in ALT and AST activities (Fig. 3a, b).
Results
Effects of CSCP on Antioxidant Enzyme and Lipid Peroxidation in Mice
Statistical Analysis
Effects of CSCP on Cell Proliferation in Normal and Oxidative Damaged Cell Effect of CSCP on normal Chang liver cell growth was shown in Fig. 1a. Cell growth was not affected by CSCP at dose lower than 10,000 lg/ml, indicating that CSCP was safe to Chang liver cells at dose lower than 10,000 lg/ml. Furthermore, following treatment with CSCP at 1,000 and 10,000 lg/ml, the cell viabilities was markedly increased compared with the non-CSCP-treated group. The result indicated that at the dose between 1,000 and 10,000 lg/ml CSCP could enhance the cell viability.
Liver injury induced by CCL4 and acetaminophenum provoked a significant reduction of SOD and CAT activity, as well as a decline of GSH content. However, MDA, one of the most important products of membrane lipid oxidation, was remarkably increased after CCL4 and acetaminophenum treatment (Figs. 4, 5). In the CCL4-induced liver injury, DDB and high dosage of CSCP pretreatment significantly increased the activities of SOD and CAT and decrease MDA. The medium dosage of CSCP was only effective on SOD and MDA, whereas the low dosage of CSCP had no influence to the antioxidants and MDA (Fig. 4). In contrast, in the acetaminophenum-induced liver
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Cell Biochem Biophys Fig. 1 CSCP increased cell viability in Chang liver cells. a Cell viability in Chang liver cells after concentration gradients of CSCP pretreatment. The cells were pretreated with CSCP and the cell viability was assessed by MTT 24 h later. Compared with the previous group, *P \ 0.05, **P \ 0.01. b Cell viability in H2O2-treated Chang liver cells after concentration gradients of CSCP pre-treatment. The cells were pre-treated with CSCP for 2 h before 200 lM H2O2 was administrated to cells. Cell viability was assessed by MTT 24 h later. Compared with the control group, *P \ 0.05; compared with the H2O2-treated group, ##P \ 0.01
injury model, both of DDB and CSCP at all experimental dosages enhanced SOD, CAT, and GSH, and reduced MDA (Fig. 5).
Discussion Marine species comprise approximately half of the total global biodiversity and the sea offers an enormous resource for nutritional or pharmaceutical applications. In recent years, marine originated collagen earned more and more attention due to biological safety, excellent biocompatibility, low antigenicity, high biodegradability, and cell growth potential [2, 3, 15]. However, few studies have investigated the biological activities of the collagen from marine animals. CSCP, compounds of hydroxyproline-rich collagen peptides (mainly 500–3,000 Da), were derived from cod skin which has been the wastage and pollution in the fish-processing industry. In the present study, the hepatoprotective effect of CSCP was examined in vitro and in vivo. We for the first time revealed the hepatoprotective effect of fish collagen peptides. Prior to the animal experiment, the Chang liver cell was chosen as a normal human hepatocyte line to assess whether CSCP could alleviate the H2O2-induced liver damage. Our results indicated that CSCP was safe to Chang liver
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cells at the dose lower than 10,000 lg/ml while CSCP at dosage higher than 10,000 lg/ml reduced the cell viability. CSCP was considered safe as the absorption dosage of collagen was far below 10,000 lg/ml. Furthermore, without H2O2 treatment, 1,000 to 10,000 lg/ml CSCP could effectively enhance the cell viability, which suggested that CSCP has the potential to improve liver function. Following H2O2 treatment, CSCP at the dose of 100 to 1,000 lg/ml protected Chang liver cells against the H2O2induced cell viability loss in a dose-dependent manner. Moreover, CSCP pretreatment restored the cell viabilities to the level higher than control. Taken together, these results demonstrated that CSCP could protect against the free radical-induced liver damage in vitro. In the animal experiment, two different liver damaged models were established in mice: the CCL4-induced model and the acetaminophenum-induced model. The hepatotoxic effect of CCL4 is caused by the conversion of CCL4 by the cytochrome-P450 mixed function enzyme in the smooth endoplasmic reticulum of the liver to the highly reactive CCL3, which yielded the free radical attack to liver to initiate peroxidate degradation of membrane lipids [16]. On the other hand, acetaminophenum is a well-known antipyretic and analgesic agent, which is harmless at therapeutic doses but can produce fatal hepatic necrosis at high doses. Acetaminophenum is also metabolized by
Cell Biochem Biophys
Fig. 2 CSCP enhanced liver index in acute liver injured mice. a Liver index in CCL4-induced acute liver damage in mice. The mice were oral administrated with 1, 2, 4 g CSCP, respectively, for 10-days. Two hours after CSCP pretreatment, animals received 100 mg/kg CCl4 by intraperitoneal injection. 16 h later, the mice were scarified and the livers were taken out to calculate the liver index. b Liver index in acetaminophenum-induced acute liver damage in mice. The mice were treated as above and the liver damage was induced by 150 mg/kg acetaminophenum oral administration at the same time point. Compared with the control group, *P \ 0.05; compared with the model group, #P \ 0.05
Fig. 3 CSCP elevated ALT and AST in acute liver injured mice. a The activities of ALT and AST in CCL4-induced acute liver damage in mice. The mice were scarified to take the eyeballs off for blood. The serum was separated and assessed. b The activities of ALT and AST in acetaminophenum-induced acute liver damage in mice. Compared with the control group, **P \ 0.01; compared with the model group, #P \ 0.05, ##P \ 0.01
cytochrome P450 and high dose of acetaminophenum forms the highly reactive N-acetyl-p-benzoquinone imine (NAPQI), which cannot be detoxified by enough GSH and harms the liver tissue [17]. Both of these two models represented free radical damage to liver, and the serum ALT and AST activities were remarkably increased and AST/ALT [1 in both models, indicating the severe impairment of liver function. Furthermore, the liver index was calculated to assess the liver injury. In the CCL4treated model, was significantly dropped, while in the acetaminophenum-treated model the liver index was not significantly changed. Therefore, we took the CCL4-treated model for severely damaged liversevere and the acetaminophenum-treated model as moderately damaged liver. Pretreated with gradient concentration of CSCP, the liver index of the CCL4-damaged groups was increased while that of the acetaminophenum-damaged groups remained unchanged. The results might be explained by the insensitivity of the acetaminophenum-induced moderate
damage to CSCP. In the CCL4-damaged groups, the medium- and high- dosage of CSCP treatment, as well as the DDB treatment, markedly enhanced the liver index compared to the model group, which indicated that the medium- and high- dosage of CSCP pretreatment could alleviate the CCL4-induced severe damage to the liver in mice. The serum ALT and AST activities were determined after the liver index assay. We found the ALT and AST activities in the CCL4-treated model mice were much higher than those in the acetaminophenum-treated model mice, which was in line with the results of liver index to further demonstrate that the CCL4-induced liver toxicity was more severe than the acetaminophenum-induced liver toxicity. Moreover, our result showed that in both models, CSCP pretreatment, as well as DDB treatment, could attenuate the elevated ALT and AST activities induced by CCL4 or acetaminophenum. We found that the high-dose of CSCP was effective in the CCL4-induced liver damage,
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Fig. 4 CSCP modulated the redox states in CCL4-induced acute liver damage in mice. a–d represented the activities of SOD, CAT, GSH and MDA in liver tissue in mice. Compared with the control group, **P \ 0.01; compared with the model group, #P \ 0.05, ##P \ 0.01
Fig. 5 CSCP modulated the redox states in acetaminophenum-induced acute liver damage in mice. a–d represented the activities of SOD, CAT, GSH and MDA in liver tissue in mice. Compared with the control group, **P \ 0.01; compared with the model group, #P \ 0.05, ##P \ 0.01
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while in acetaminophenum-induced liver damage, both of the medium- and high- dosage of CSCP could alleviate the liver injury. The effects of CSCP on antioxidant enzymes and lipid peroxidation products were further explored in liver tissue. After CCL4 or acetaminophenum treatment, the activities of antioxidant enzymes SOD, CAT, and GSH were remarkably suppressed while the lipid peroxidation product MDA was markedly enhanced, suggesting an increased oxidative stress in liver tissue. In the acetaminophenumtreated model, CSCP at all three doses as well as DDB were effective on restoring the redox balance by increasing the activities of antioxidant enzymes and decreasing MDA. In contrast, in the CCL4-induced model, only the high-dose of CSCP and DDB pretreatment partially restored the activities of antioxidant enzymes and MDA (medium dose of CSCP only effective in SOD and MDA). These results suggested that CSCP might yield its hepatoprotective effect through balancing the redox state in liver. In summary, CSCP pretreatment protected liver tissue against oxidative injury in vivo and in vitro. The mechanism might involve enhancing the activities of antioxidant enzymes and reducing the lipid peroxidation.
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12. Acknowledgments This work was supported by Agricultural Science and Technology Program of Qingdao (Grant No.14-2-3-50-nsh). Conflict of interest The authors declare that there is no conflict of interests regarding the publication of this paper.
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