Introduction

Liver fibrosis, a pathological process common to chronic liver diseases, is caused by a range of actors, and has a highly deleterious impact on human health. It is well-established that liver fibrosis formation results from an excessive accumulation of extracellular matrix proteins including collagen, a process that occurs in most types of chronic liver disease1. Liver fibrosis is described in several stages, a normal liver is at stage 0 to 1, stage 2 represents light or minimal fibrosis, stage 3 is severe fibrosis, and stage 4 denotes advanced liver fibrosis resulting in cirrhosis, liver failure, and portal hypertension2,3. Any reversibility of advanced liver fibrosis prior to cirrhosis in patients would be of signal importance.

Because of the urgent clinical need, the development of an anti-fibrotic drug would be an auspicious moment in medical research. Up to now, several candidate treatments with anti-fibrotic properties have been developed, which variously act by blocking inflammatory pathways4,5, inhibiting profibrotic growth factors6,7,8, modulating epigenetic codes9,10, and interfering with morphogenic pathways11,12, and which have proven efficacy and tolerability in pre-clinical fibrosis models. However, they are not available to treat fibrotic diseases in clinical practice.

The Cygb protein was first discovered in 2001 by proteomic analysis in rat stellate cells, and was initially named stellate cell-activated protein (STAP)13. The protein sequence showed that Cygb was hexacoordinate hemoglobin, representing the fourth member of the globin superfamily in mammals14. Furthermore, immunohistochemical analysis revealed that Cygb was ubiquitously expressed in several organs, such as kidney and pancreas, and localized in fibroblast-like cells15. Cygb was a 21.4 kDa protein consisting of 190 amino acids and mapped at the 17q25.3 chromosomal segment. Exogenous over-expression of Cygb was soon demonstrated experimentally, both in vitro and in vivo, to protect hepatic stellate cells (HSC) from oxidative stress and suppress their differentiation to a myofibroblast-like phenotype16,17,18. Further, it was reported that Cygb had active peroxidase properties, which protected cells against oxidative damage by free radicals19,20,21. These findings prompted the hypothesis that Cygb could be effective in the prevention or treatment of liver fibrosis. In this study, therefore, we investigate the effect of rhCygb on CCl4-induced liver fibrosis in rats at various stages of the disease’s progression, from mild to severe fibrosis.

Materials and Methods

Preparation of rhCygb

Human cytoglobin cDNA was obtained from HepG2 cells by RT-PCR. A recombinant construct of pET28a-hCyt were transformed into E. coli BL21 (DE3). For expression, the bacteria cultures were induced for 4 h using 1 mM isopropyl β-d-thiogalactoside (IPTG) after the culture OD 600 has reached 0.6. The total bacteria cells were collected by centrifugation at 5,000 rpm for 10 min, resuspended in PBS, and broken down by sonication. The rhCygb was purified by sephadex G-25 and affinity chromatography which the monoclonal antibody was prepared by our laboratory. The purified protein was analyzed with SDS–PAGE and identified by mass spectrometry with ABI 4700 Proteomics Analyzer (Applied Biosystems, USA). The yields of rhCygb measured by the software BandScan 5.0 based on SDS-PAGE results. The total antioxidation capacity (T-AOC) of rhCygb was measured spectrophotometrically as described by Tang et al.22, using a T-AOC commercial kits (Jiancheng, Nanjing, China) according to the guidelines of the kit.

Ethics statement

All experimental procedures were conducted in conformity with institutional guidelines approved by the Chinese Association of Laboratory Animal Care. All experimental protocols were approved by the Institutional Animal Care and Use Committee in Southern Medical University, Guangzhou, China.

Animals

Sexually mature sprague–dawley (SD) rats (n = 150) weighing 180–220 g were obtained from the experimental animal center of Southern Medical University. Care of the animals used in this investigation was conducted according to the guidelines approved by the Chinese Association of Laboratory Animal Care. Rats were housed, five per cage, under constant temperature (20 ± 2 °C) and humidity (70%) with a 12 h light/dark cycle and with free access to chow and water.

Acute toxicity test

BALB/c mice of both sexes, weighing 18–24 g, were obtained from the experimental animal center of Southern Medical University. The acute toxicity of rhCygb was determined in vivo using the procedure approved by Chen et al.23. Uninfected mice were peritoneal injected rhCygb at doses of 100 and 500 mg/kg body weight. The animals were monitored for any toxic symptoms or mortality over a 14-day period.

Induction of fibrosis

Three degrees of liver fibrosis were induced by chronic carbon tetrachloride (CCl4) administration at three different dosage levels:

  1. a

    Mild model group: 2 ml/kg of 25% CCl4 in paraffin oil (Sigma Co., Milan, Italy) was administered by subcutaneous injection twice a week (Tuesday and Friday) for 10 weeks;

  2. b

    Medium model group: treatment in group (a) plus 50% CCl4 treatment for 3 more weeks;

  3. c

    Severe model group: treatment in group (b) plus 62.5% CCl4 for 2 more weeks.

Experiment design

To evaluate the effect of rhCygb on liver fibrosis, 30 rats were used in each of the groups (group a, b, c). A schedule of the treatment is shown in Fig. 1. In each group, followed by CCl4 treatment, rats were further treated with saline (vehicle group), CCl4 (model group) or rhCygb (rhCygb group, 2 mg/kg body weight/day, s.c. injection) in combination with CCl4 for 12 weeks. At the end of the experiments, all rats were sacrificed within 24 h after the last injection.

Figure 1: Schedule for the treatment and experimental tests.
figure 1

Rats were injected with CCl4 for three different doses to establish mild, medium and severe models at 10, 13 and 15 weeks, 2 times per week. After model was established, rats were treated daily with saline, CCl4 or rhCygb + CCl4 for 12 weeks. Rats were sacrificed within 24 h after the last treatment.

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Chemical analysis

Venous blood samples were collected for serum separation. Samples were stored at −20 °C until further analysis. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were measured spectrophotometrically by Olympus/AU 5200 (Konsesyum, Alternative Biomedical Services, Dallas, TX, USA).

Serum liver fibrosis markers hyaluronic acid (HA), laminin (LN), collagen III (Col III) and collagen IV (Col IV) were determined using enzyme-linked immunosorbent assay (ELISA) kits (SIGMA chemical, Corp.).

Pathological studies

In the end of the experiments, rats liver were removed and rinsed with cold saline water. Then liver lesions were fixed, embedded, and stained with Sirius red and hematoxylin-eosin (HE)24. Their evaluation was conducted using a light microscope (Axiphot2, Carl Zeiss) blindly. Fibrosis was scored according to the Metavir scoring system reported by Bedossa et al.25. Intralobular degeneration and focal necrosis, portal inflammation and fibrosis were compared across the groups. Three representative images of each histology sample section from each rat were selected randomly and all of the rats were scored.

Protein separation

Five liver samples (approximately 30–40 mg wet) were removed from vehicle group, CCl4 model group and rhCygb treated group (medium). The samples were then frozen in liquid nitrogen and homogenized with a mortar and pestle. Proteins were prepared in lysis buffer. A total of 120 μg of protein was separated by two-dimensional electrophoresis (2-DE) according to the product manual ‘2-DE for Proteomics’ (Bio-Rad, U.S). After silver staining, each spots were cut out from the gel and digested using trypsin according to the method described by Qualtieri et al.26.

MS data analysis

Peptide mixtures of each gel spot were dissolved in 0.1% TFA, desalted, and concentrated. The samples were then mixed with the same volume of matrix (CHCA in 30% ACN/0.1% TFA), spotted on a target disk, and allowed to air-dry. Samples were analyzed using a Bruker ultrafleXtreme MALDI-TOF mass spectrometer (Bruker Daltonics, Germany). The protein database search was performed using the MASCOT search engine (http://www.matrixscience.com/)27. Mass tolerance was allowed within 150 parts/million (ppm). Proteins matching more than five peptides and with a MASCOT score higher than 60 were considered significant (p < 0.05).

Bioinformatics analysis

To identify significantly represented biological themes and functional groups in the protein list (Table 1), the gene ontology (GO) and pathway analysis were performed using the database for annotation, visualization and integrated discovery (DAVID) program (https://david.ncifcrf.gov/)27. The GO analysis was used to identify enriched biological themes by the GeneCodis tool (http://genecodis.cnb.csic.es/)27. The list of identified proteins was used as the input data when the DAVID default population background was used; We used EASE scores, which modified Fisher’s exact test P values < 0.05 and corrected for multiple testing by the Benjamin–Hochberg method.

Table 1 Identification of 33 differentially expressed proteins among normal, CCl4 model, and rhCygb groups.
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Modified gene set enrichment analysis (GSEA) was used to assess functional significance at the level of sets of genes as previous described by Li et al.28. All of these potential targets were compared with the “c2_all” collection of curated gene sets from the Molecular Signatures Database (GSEA Version 2.0.14), consisting of 1077 gene sets corresponding to BIOCARTA, KEGG and REACTOME biological pathways. The False Discovery Rate (FDR) q value is deemed significant value at <0.05.

Statistical analysis

All data were expressed as the mean ± standard deviation from at least 5 independent experiments. Figures were obtained from at least 5 independent experiments with similar patterns. The biochemical experimental data were analyzed by two-way ANOVA. The Kruskal-Wallis was used for comparisons. Statistical analyses were performed using SPSS software version 19.0 (SPSS Inc, Chicago, USA).

Results

Purity and activity of rhCygb

The purity of rhCygb was 95% by BandScan 5.0 software. The antioxidant activity of rhCygb is 95.58 ± 2.67 U/mg (p < 0.01) (supplemental data).

Acute toxicity results

When given as a single dose of 100 and 500 mg/kg, rhCygb produced no signs of acute toxicity in the 14-day period of observation. No death was recorded in the 100- and 500-mg/kg-treated group. All groups of mice exhibited stable temperature values throughout the period of observation.

Developments of CCl4-induced hepatic fibrosis in rats

All experiments successfully induced fibrosis, as determined by the examination of liver histology and serum biochemistry (Fig. 2A–G). The mortality was 0 in all the experiment groups. According to the results of development of rat liver fibrosis model, rats treated with CCl4 were sacrificed at 3 different times, 10th week, 13th week and 15th week. At sacrifice, all rats presented marked fibrosis with significant differences in the amount of liver fibrotic tissue among groups. Hepatolobular injury was observed with centrilobular necrosis, balloon cells, and lipids accumulation in 15-weeks-model rats (Fig. 2A). The same results were obtained in an analysis of serum biochemistry (Fig. 2B–G). At the lowest dose (25% CCl4) for 10 weeks in SD rats, administration of CCl4 significantly (p < 0.01) increased the concentration of AST, ALT, LN, HA, Col III and Col IV in rat serum compared with the levels in the vehicle group (Table 2). Moreover, administration of a higher dose of CCl4 (50% CCl4) for a further 3 weeks in group b (medium) resulted in a marked prolongation of toxicity, increasing (p < 0.01) serum enzyme values about two-fold except for Col IV. However, there was no significant (p > 0.05) difference between the group b (medium) and group c (severe).

Figure 2: Examination of liver histology and serum biochemistry at different stages (10, 13, 15 weeks) in CCl4 model rats.
figure 2

(A) Representative photomicrographs of HE and Sirius red staining for observing the morphology of fibrosis of rat livers at 10, 13, 15 weeks (×200); Serum levels of (B) ALT, (C) AST, (D) LN, (E) HA, (F) Col III and (G) Col IV.

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Table 2 Effects of rhCygb on ALT, AST, LN, HA, Col III and Col IV of CCl4-induced liver injury SD rats ( ± SD, n = 10).
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Effects of rhCygb on hepatic histoarchitecture

We next evaluated the effect of rhCygb on liver fibrosis following chronic CCl4 administration. Rats were treated with rhCygb (2 mg/kg/day, s.c.) in combination with CCl4 (25%, 50% or 62.5%) for 12 weeks. All of the rats survived during the experimental period. The influence of CCl4 and rhCygb on liver was depicted in Fig. 3, in which liver fibrosis was advanced for 12 more weeks. In this group, fibrotic changes caused by CCl4 reversed dramatically by rhCygb. As shown in Fig. 3A, the liver appearance of CCl4-administered rats significantly changed, especially in the medium and severe groups. These groups, after treatment with rhCygb, exhibited a clear and marked improvement in their liver, when compared with those of the CCl4-administered groups. Photomicrographs of hepatic specimens stained with Sirius red were depicted in Fig. 3B, and the scores of fibrosis variables were presented as medians in Fig. 3C. As shown in the figures, no hepatic fibrosis occurred in the control group, which showed an integrated lobular structure with central venous and hepatic cord radiation. The staging score was 0. Extensive fibrosis and ballooning of hepatocytes were observed in all the CCl4 model groups. As expected, the high CCl4 treatment caused fibrous connective tissue proliferation as well as a good deal of collagen fiber formation. Moreover, increased dosages caused even more frequent pseudo lobules to appear in model control groups. The hepatic fibrosis score in the CCl4 groups increased to 3.40 ± 0.33, 3.80 ± 0.13 and 3.90 ± 0.1, respectively. In the rhCygb treated groups, liver had little collagen fiber formation in the peripheral area, and no pseudo lobules. The positive area of Sirius red staining was significantly lower than that of the CCl4 groups. The scores were 0.27 ± 0.58 (mild group), 0.8 ± 0.19 (medium group) and 0.57 ± 0.16 (severe group), respectively. Similar observations were made in rhCygb groups which evinced statistically significant lower fibrosis scores compared to CCl4 groups.

Figure 3: rhCygb treatment attenuates CCl4-induced rat liver fibrosis.
figure 3

(A) The liver appearance of vehicle control, CCl4 model and rhCygb groups. (B) Representative photomicrographs of Sirius red staining for observing the morphology of fibrosis of rat livers from different groups (×200). Black arrows indicate fibrosis. (C) Fibrosis scores (Y axis) of CCl4 model groups (n = 10) and rhCygb treatment groups (n = 30). Fibrosis scores are based on the percentage of liver area positively stained by Sirius red; ****p < 0.001.

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Indices of hepatotoxicity: liver marker enzymes

The levels of serum ALT and AST are markers of liver damage. As shown in Table 2, AST and ALT level significantly increased in mild, medium and severe CCl4 model groups compared with normal control groups, reflecting hepatocelluar damage in CCl4-induced liver fibrosis. Then rhCygb treatment significantly (p < 0.01) reversed serum ALT and AST levels in 10 + 12 weeks, 13 + 12 weeks and 15 + 12 weeks groups, to the levels almost equal to vehicle controls.

Effects of rhCygb on levels of LN, HA, Col III and Col IV

In this study, serum LN, HA, Col III and Col IV levels in CCl4 groups were higher (p < 0.01) than controls (Table 2). Compared with the CCl4 groups, rhCygb also markedly decreased (p < 0.01) liver fibrosis marker levels of LN, HA, Col III and Col IV in 10 + 12 weeks, 13 + 12 weeks and 15 + 12 weeks groups.

Differential 2-DE analysis of liver tissue proteins

To study the influence of rhCygb on the proteome of CCl4-induced fibrotic liver, liver tissues were harvested from the (13 + 12)-weeks-old control group, the CCl4 model group, and rhCygb treatment group of rats. When examined, the 2-DE gel showed an obvious separation of liver tissue proteins, with 1312 proteins in the vehicle group, 1325 proteins in the CCl4 model group and 1259 proteins in rhCygb group. Comparing the spots of vehicle to model group or model to rhCygb group by PDquest software for image analysis, a total of 75 protein spots exhibited significant (p < 0.05) differences in protein expression between vehicle and model or between model and rhCygb group (supplement data). These 75 protein spots were grouped into three major patterns (Fig. 4): pattern A: 53 spots differentially expressed between vehicle and CCl4 group; pattern C: 55 spots differentially expressed between CCl4 model and rhCygb group; pattern B: 33 spots which overlapped pattern A and pattern C. For example, the expression of cathepsin D in the vehicle group was lower compared to model, while that in rhCygb treated group was also lower. Among the 33 proteins, the expression of 21 proteins were three times higher and that of 12 proteins was three times lower in the vehicle and rhCygb treated groups than in the CCl4 model group significantly (p < 0.05). A total of 33 peptide mass fingerprints (PMFs) were matched and identified by MALDI-TOF-MS and protein database searching, as displayed in Table 1.

Figure 4: Grouping of differentially expressed protein spots.
figure 4

There were 53 differential spots between vehicle and CCl4 model groups (pattern A), 55 differential between model and rhCygb groups (pattern C), and 33 spots were overlapped between pattern A and pattern C, in which vehicle and rhCygb groups had the same tendency of differential expression compared with ones in model group (pattern B).

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Functional annotation of differentially expressed proteins

We analyzed the GO enrichment of these upregulated and downregulated proteins (Table 1). Relevant Protein functions were listed in Table 3. These dysregulated proteins were predicted to be involved in different metabolic processes including the response to stimulus, oxidation-reduction processes, heat-shock protein, the catabolic process and the cytoskeleton.

Table 3 Identification of the differential expressed proteins through the use of GO analysis.
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GSEA (Table 4) revealed that up-regulated genes in rhCygb treated group were significantly enriched in the down-regulated gene set in hepatocellular carcinoma (ranked 1st, p = 3.33 e−8, FDR = 2.27 e−4), or in the genes highly expressed in hepatocellular carcinoma with good survival (ranked 4th, p = 4.19 e−5, FDR = 2.77 e−2). FDR q-value was considered significant at < 0.05.

Table 4 GSEA result for the up-regulated genes in rhCygb treated groups vs CCl4 model group.
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Discussion

Much experimental evidence and clinical studies has shown that the physiological functions of Cygb are associated with various diseases, such as cancer29, gastroesophageal reflux disease30, psychomotor retardation epilepsy31, and certain neurodegenerative disorders32, especially liver fibrosis. Hence, the aim of the present research was to study the mechanism of liver fibrosis and rhCygb anti-fibrotic action.

Effects of rhCygb on serum markers of fibrosis

CCl4 proved to be highly useful as an experimental model for the study of liver damage in humans33,34,35. It is usually used through intraperitoneal injection or oral administration, and with a dosage of 0.2 to 5 ml/kg liver fibrosis is achieved between 4 and 18 weeks36,37. In this study, the mild, medium and severe liver fibrosis rat models were developed through differential doses (25–62.5%) and exposure duration (10–15 weeks) of CCl4. The results of the present study revealed that continuous and increasing doses of CCl4 resulted in advanced fibrosis which caused an elevation of levels of serum marker enzymes such as ALT, AST, HA, LN, Col III and Col IV from the mild to the severe model. The serum marker enzymes ALT and AST have previously been reported as important diagnostic factors for hepatic diseases, as they are cytosolic enzymes of the hepatocyte, and increased activity in their circulation reflects cell damage and leakage38. High ALT and AST levels are associated with a higher risk of fibrosis progression. The present study demonstrates that rhCygb can reduce heightened ALT and AST, indicating that they can promote the repair of injured liver cell.

In addition, HA, LN, Col III and Col IV have been found to be ideal serum markers of hepatic fibrosis, which play an important role in detecting the degree of hepatic fibrosis39. In the present study, CCl4 model groups increased markedly compared with normal groups. However, after the treatment of rhCygb (therapy but not removing CCl4), we detected that the serum contents of HA, LN, Col III and Col IVdecreased to the level of normal groups. This may be the basic mechanism of rhCygb reversing rat hepatic fibrosis.

Effects of rhCygb on stages of fibrosis

In response to liver injury induced by whatever cause, collagen and matrix proteins are produced in much higher quantities by activated hepatic stellate cells, which ultimately leads to liver fibrosis40. Fibrosis is evaluated by using Sirius red staining and scored using the fibrosis staging system (the Metavir score). The Metavir system scores necroinflammatory activity from 0 to 3 and fibrosis from 0 to 425. In this study, evaluation of staging revealed a score of 4 in different cases in CCl4 model groups: 70% in the mild group, 80% in the medium group and 90% in the severe group. However, score 4 didn’t show up in any of the rhCygb treated groups. In time, score 0 was present in 24 rats in the mild group, 18 rats in the medium group, and 19 rats in the severe group. These data are in solid agreement with the activities of serum biomarkers. Interestingly, the stages of fibrosis were previously believed to be progressive and largely irreversible. There is now mounting experimental and clinical evidence suggesting that liver fibrosis can regress in all chronic liver diseases by withdrawing the cause of liver injury or by treatment of the disease41,42. Our paper also obtains that CCl4-induced rat liver fibrosis was markedly reversed by administration of rhCygb. He et al. reported the similar findings in their use of rhCybg in the thioacetamide (TAA)-induced rat liver fibrosis model43.

Effects of rhCygb on protein species

We systematically analyzed the effect of rhCygb on liver proteome in the CCl4-induced rat model. Thirty-three differentially expressed proteins appeared among the three groups, with the same alteration profile in the vehicle and rhCygb treated groups compared with the CCl4 model group, identified through comparative proteomics. Functional annotation analysis showed several identified proteins involved in oxidative stress pathways, such as the response to chemical stimuli, oxidative-reduction, and xenobiotic metabolic processes. Oxidative stress is thought to be a critical factor in the development of CCl4-induced liver fibrosis44. Previous studies have reported the effects of rhCygb on the levels of oxidative stress biomarkers and anti-oxidative capacity45. The current results show that rhCygb regulates antioxidant enzymes such as peroxiredoxin-2, glutathione S-transferase, glutathione peroxidase 1, cytochrome P450 2C11, biliverdin reductase A, estrogen sulfotransferase, and taste receptor type 1 member 2. One of these proteins, P450 2C11, an enzyme that was increased by rhCygb, plays an important role in the deactivation of estrogens via 16a-hydroxylation and catechol formation. It is particularly active in relation to the development of fatty change, hepatic fibrosis, portal hypertension, and cirrhosis46. Earlier studies have shown that P450 2C11 activity in rat models of experimental liver disease including hepatic cirrhosis is produced by CCl447. The present study also demonstrated that the level of protein P450 2C11 in the liver decreased significantly in this model of liver fibrosis at the 25-week time point. In addition, we also found that enzymes involved in fatty acid β-oxidation (Long-chain-fatty-acid-CoA ligase 1, Enoyl-CoA hydratase, Carboxylesterase 1D and ATP synthase sub-unit alpha) were down-regulated in CCl4 model group, whereas in the rhCygb-treated group they were up-regulated (as they were in the vehicle group). These findings show that impairment of fatty acid oxidation may contribute to hepatic fibrosis.

In summary, the results of the present study demonstrate that in addition to the effect of reversing liver fibrosis, rhCygb could reduce the serum levels of ALT, AST, HA, LN, Col III and Col IV, indicating that it has definite effects in reversing the formation of liver fibrosis in the course of chronic hepatitis. The differentially expressed proteins have several groups of biological functions, especially relating to oxidative stress, which are helpful in revealing the underlying mechanisms of rhCygb action against liver fibrosis. rhCygb could be a novel candidate drug in combating liver fibrosis.

Additional Information

How to cite this article: Li, Z. et al. The Effect of rhCygb on CCl4-Induced Hepatic Fibrogenesis in Rat. Sci. Rep. 6, 23508; doi: 10.1038/srep23508 (2016).