1 INTRODUCTION

Alcoholism is a major global social and health problem，with amounts of deaths and disability every years due to the alcohol consumption (Rehm et al.，2009).

Liver is an important organ for detoxification，and deposition of endogenous and exogenous substances. Liver disease is considered to be a serious health problem (Diehl，2002; Mann et al.，2003; Ou et al，2010). Ethanol is a well-known hepatotoxin，and alcoholic liver disease (ALD) is caused by excessive ethanol consumption. The pathological mechanisms of ALD are complicated (Purohit et al.，2004; Fernando et al.，2010)，and predominantly result from alcohol metabolism，alcohol-induced oxidative stress，lipid peroxidation and free radicals，alcohol-mediated cytokines and inflammation (Arteel et al.，2003; Cohen et al.，2011). Previous studies have revealed that many antioxidants，especially those of natural origin，including polyphenol compounds (McKim et al.，2002; Hou et al.，2010)，amino acids and peptides (Li et al.，2012; Zhang et al.，2012) play a vital role in protecting liver tissues and hepatocytes from oxidative injury induced by acute or chronic alcohol consumption in rodents and humans (Lee and Kowdley，2012).

C-phycocyanin (C-PC) is a unique light-harvesting protein found in some algae. C-PC was shown to have several pharmacological activities，and its antioxidant activity is particularly prominent (Eriksen，2008; Fernández-Rojas et al.，2014). The reactive oxygen species (ROS) are known to play a crucial role in the pathology and progression of liver diseases (Vitaglione et al.，2004). C-PC was shown to have free radical scavenging properties and antioxidant activities (Bhat and Madyastha，2000).

In this study，we wanted to investigate whether C-PC has any protective effects against ethanolinduced hepatocyte damage，and its underlying mechanisms.

2 MATERIAL AND METHOD 2.1 Material

C-PC was extracted and purified from Spirulina . Briefly，the process of extraction and purification included immersion，precipitation with ammonium sulfate，centrifugation，dialysis and two rounds of chromatography with hydroxyapatites (Ou et al.，2010). The absorption spectra A620/A280=4.0，The purity ratio of C-PC is defined as the absorption ratio of A620/A280，which the purity of food grade is 0.7 and the 4.0 reach to Reagent level (Patil et al.，2006).

Aspartate transaminase (AST)，alanine aminotransferase (ALT)，low-density lipoprotein cholesterol (LDL)，triglyceride (TG)，and total cholesterol (CHOL) detection kits were purchased from Abbott Company (China).

Superoxide dismutase (SOD) and malondialdehyde (MDA) detection kits were purchased from Nanjing Jian Cheng Bioengineering Institute (China).

Absolute ethanol was purchased from Sinopharm Chemical Reagent Co. Ltd.，China.

2.2 Instrument

ABBOTT ARCHITECT c16000 automatic biochemical analyzer was used from Affiliated Hospital of Binzhou Medical College.

2.3 Animals and treatment

Female KM mice (18–22 g) were obtained from Shandong Lvye Pharmaceutical Co. Ltd.，(Yantai，China). Animal welfare and experimental procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals (Ministry of Science and Technology of China，2006)，and were approved by the animal ethics committee of Yantai University. The mice were quarantined and acclimated for one week prior to experimentation. The mice were maintained in room which has constant temperature and humidity. The method of making acute alcoholic liver injury model and administration according to the Chinese health food test and evaluation technical specification (Huang，2003). The animals were divided randomly into six groups，each containing 20 mice. The six groups were the control group，the model group and low-，middle-，high-dose C-phycocyanin groups and high-dose C-phycocyanin control group. Mice in the three doses of C-PC test groups and high-dose C-PC control group were treated with 100，200，and 400 mg/kg C-PC by gavaging every day，respectively，for 42 consecutive days，while mice in the control and model groups were treated with physiological saline. On the last day，the mice were fasted for 8h after the pretreatment and then gavaged with 50% absolute ethanol (0.14 mL/10 g body weight) once，except for those in the control group and high-dose C-phycocyanin control group，which were gavaged with physiological saline. The mice were sacrificed 4 hours later. Blood was collected into non-heparinized Eppendorf tubes and centrifuged (2 000 r/min，10 min，4℃). Serum was collected and assayed for biochemical markers. The liver was also removed and divided into two halves. One part was used for hepatic SOD and MDA assays，while the other half was excised and fixed in 10% formalin solution for histopathological analysis.

2.4 Biochemical assays 2.4.1 Assays for biochemical markers in serum samples

Serum AST，ALT，LDL，TG and CHOL levels were measured using an automatic biochemical analyzer.

2.4.2 Hepatic SOD and MDA assay

SOD and MDA in liver homogenates were measured by colorimetric enzymatic methods using commercial kits，according to the manufacturer’s protocols. The absorbance was read with a UV-1750 spectrophotometer (Shimadzu，Tokyo，Japan).

2.4.3 Histopathological analysis

After the mice were sacrificed，the livers were promptly and carefully extracted. The livers were examined for any abnormal color，exudate，edema，hyperplasia and atrophy. A square block of tissue from each liver was cut and fixed in 4% neutral formalin buffer overnight. The samples were repeatedly washed under running water to remove the fixing agent. Gradient ethanol (70%–100%) was used to dehydrate the samples，and xylene was used to make them transparent. The samples were embedded in paraffin，stained with hematoxylin and eosin (HE)，and observed under a microscope (400×magnification).

2.4.4 Statistical analysis

The data were analyzed using SPSS 22.0 software，and expressed as mean±standard deviation from replicate experiments. One-way analysis of variance was used to assess differences in biochemical indices. T -test variance analysis was also performed. P<0.05 was considered to be statistically significant.

3 RESULT 3.1 Protective effects of C-PC against acute ethanol liver injury

Pretreatment with C-PC for 42d effectively attenuated acute ethanol liver injury. As shown in Table 1，the TG，CHOL and LDL levels in the model group increased by 227.4%，64.6% and 39.1%(P<0.01)，respectively，as compared to the control group. Thus，the model of alcoholic liver injury in mice was successfully established. The TG levels in the three C-PC groups decreased by 53.29%，45.51%，and 20.96%(P<0.05 or P<0.01)，respectively，as compared to the model group. The CHOL levels decreased by 47.09%，33.72%，and 26.16%(P<0.01)，respectively，while the LDL levels decreased by 34.37%，25%，and 9.37%(P<0.05 or P<0.01)，respectively. As compared to the control group，serum ALT and AST levels in the model group increased by 150.5% and 102.6%(P<0.01)，respectively. As compared to the model group，the ALT levels in the three C-PC groups decreased by 49.68%，43.88%，and 42.09%(P<0.01)，respectively，while the AST levels decreased by 27.58%，34.55%，and 22.85%(P<0.01)，respectively. Compared to the control group，it’s no significant change in the level of ALT，AST，TG，CHO and LDL of high-dose C-phycocyanin control group mice.

C-PC effectively inhibited the increase in these indices. The 400 mg/kg C-PC treatment dosage was most effective in restoring the ALT，AST，TG，CHOL and LDL to their normal levels (Table 1). It’s no sideeffect of liver to use C-PC in a normal body.

3.2 Effects of C-PC on the hepatic antioxidant system

MDA and SOD levels in mice liver homogenates are shown in Table 2. As compared to the control group，the hepatic MDA level in the model group increased by 153%(P<0.01)，while the SOD content decreased by 38%(P<0.01). C-PC pretreatment for 42 d effectively suppressed the MDA increase and SOD depletion，and the MDA levels were restored to normal in C-PC (100 and 200 mg/kg) groups. C-PC pretreatment enhanced SOD levels. As compared to the model group，MDA levels decreased by 10.4%，32.9%，and 51.3%(P<0.05 or P<0.01)，respectively，and SOD levels increased by 19.3%，34.1%，and 49.3%(P<0.05 or ＜0.01)，respectively. Compared to the control group，it’s no significant change in the level of SOD and MDA of high-dose C-phycocyanin control group mice. Table 2 shows that C-PC could effectively inhibit the consumption of SOD caused by acute ethanol liver injury，and significantly improved the contents of SOD，thereby protecting the liver cells and reducing the extent of damage. It’s no side-effect of liver to use C-PC in normal mice.

3.3 Effects of alcohol and C-PC pretreatment on the hepatic pathology in mice

Pathological changes were observed by microscopy. Histopathological examination showed that the hepatic lobule structure was clear，hepatic cell cords were radially arranged in neat rows，liver sinus was normal and the nuclear structure was clear in the control group (Fig. 1a) mice and the high dose of C-PC control group (Fig. 1f) mice. Hepatic lobules in the model group (Fig. 1b) mice were not clear，hepatic cell cords were disorderly，liver sinus was narrowed，scattered and patchy hepatocyte ballooning，punctiform necrosis and infiltration of inflammatory cells were seen; hepatocytes were cloudy，cytoplasm and nucleus showed pale staining，diffuse cytoplasmic lipid droplets were seen，nuclei were ambiguous and necrosis of liver cells was observed. In C-PC high-dose group (Fig. 1e)，liver cells had recovered to normal，liver cell cords were arranged in neat rows，liver sinus had returned to normal; swelling of liver cells and infiltration of inflammatory cells had significantly reduced，spotty necrosis had disappeared; some hepatocytes around the central vein had returned to normal，while surrounding liver cells were still cloudy and swollen，indicating ballooning degeneration. The medium (Fig. 1d) and low dose groups (Fig. 1c) of C-PC showed clear liver lobule structure，liver sinus had returned to normal，liver cell cords were radially arranged; cloudiness and swelling of liver cells，inflammatory cell infiltration and punctate necrosis of liver cells had significantly decreased，scattered small lipid droplets were visible indicating the regeneration of liver cells，which was obviously inhibited by 42 d of C-PC pretreatment.

4 DISCUSSION

Ethanol liver injury includes ethanol-induced fatty liver，hepatitis，liver fibrosis and cirrhosis. At present，its pathogenesis is unclear，but lipid and free radicalmediated peroxidation are considered to be the most important pathogenic factors (Romero et al.，1988). The liver is the main organ for metabolism of alcohol. Excessive alcohol produces free radicals in liver cells that results in peroxidation of lecithin polyunsaturated fatty acids，which in turn produces more free radicals，resulting in the weakening and hyper-permeability of the cell membrane (Lakshman et al.，2004). Free radicals have high lipid affinity，and damage the cell and mitochondrial membranes，which releases transaminases into the bloodstream，causing inhibition of the mitochondrial three carboxylic acid cycle，fatty acid metabolism disorders (Halliwell，1990; Teli et al.，1995)，decrease in oxidation，and neutral fat accumulation in the surrounding liver cells (Kerial et al.，1999). Lipase activity in liver cells is inhibited，which decreases the ability to decompose fat in liver cells. In addition，excessive drinking stimulates the adrenal and pituitary adrenal axis，reduces mobility around the fat tissue，releases large amounts of fatty acids into the liver，thereby increasing hepatic TG synthesis and causing metabolite acetaldehyde damage of Golgi function. TG cannot be removed thereby aggravating liver cell damage，causing abnormal liver function，hepatic tissue fat metabolism，extensive vacuolar degeneration and necrosis of liver cells，which finally leads to extensive swelling and cell death. Therefore，the key to prevent ethanol liver injury occurrence and development is to remove the free radicals，inhibit lipid peroxidation，protect liver cells and restore normal liver function (Pascual and Romay，1992).

The present study showed that serum ALT，AST，TG，CHOL，LDL，TBIL，and MDA levels in the liver were significantly higher in mice in the model group that were gavaged with 50% absolute ethanol compared with the control group. Serum and liver SOD levels were significantly lower in the C-PC groups compared with the control and model groups. Liver biopsy tissue showed that the acute administration of alcohol caused fatty degeneration，thereby successfully establishing a model of acute ethanol liver damage. Treating mice with C-PC inhibited alcohol-induced acute liver cell damage effectively. Compared with the model group，serum ALT，AST TG，CHOL，LDL and TBIL levels were decreased significantly (P<0.05 and P<0.01) in C-PC group，suggesting that C-PC exerts good hepatoprotective effects. Mice treated with C-PC exhibited significantly reduced alcohol-induced lipid peroxidation，less of an increase in liver MDA levels. C-PC protected liver cells and increased SOD contents，thereby protecting liver cells. Although the high dose group shows a best effect on these liver markers，the effects between three dose groups have no significant difference and high dose of C-PC has no effect on normal mice. It indicated that C-PC maybe have an influence on body’s self-protective systems，and it not be improved significantly by using more C-PC. As shown in Table 2，C-PC inhibited alcohol-induced liver cell damage，and protected liver function in mice. C-PC could effectively prevent liver fatty degeneration，and focal necrosis of tissue.

5 CONCLUSION

The chemical medicine to protect alcohol-induced liver injury，as stimulant，vitamin and amino acid composition，can make the alcohol ingestion effects quickly，but it is difficult to protect liver function that the mechanism of the chemical medicine is only promoted the decomposition of alcohol in a temporary relief. The natural medicine often has obvious effects on the alcohol ingestion with a good curative effects on liver disease. Not only does it have the effects of systemic nursing，but also no side effects. As a natural marine active protein，C-phycocyanin showed obvious inhibitory effects on serum ALT，AST，TG，CHOL，LDL，and significantly improved the contents of SOD，thereby protecting the liver cells and reducing the extent of damage. It could effectively inhibit alcohol-induced acute liver cell injury in a dosedependent manner，thereby demonstrating its hepatoprotective effects in mice.

6 CONFLICT OF INTEREST

The authors declare that they have no conflict of interest. Animal welfare and experimental procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals (Ministry of Science and Technology of China，2006)，and were approved by the animal ethics committee of Yantai Institute of Coastal Zone Research，Chinese Academy of Science. Informed consent was obtained from all individual participants included in the study.