Protective effect of Phellinus linteus polysaccharide extracts against thioacetamide-induced liver fibrosis in rats: a proteomics analysis
© Wang et al.; licensee BioMed Central Ltd. 2012
Received: 14 March 2012
Accepted: 9 October 2012
Published: 18 October 2012
The hepatoprotective potential of Phellinus linteus polysaccharide (PLP) extracts has been described. However, the molecular mechanism of PLP for the inhibition of liver fibrosis is unclear. This study aims to investigate the molecular protein signatures involved in the hepatoprotective mechanisms of PLP via a proteomics approach using a thioacetamide (TAA)-induced liver fibrosis rat model.
Male Sprague–Dawley rats were divided into three groups of six as follows: Normal group; TAA group, in which rats received TAA only; and PLP group, in which rats received PLP and TAA. Liver fibrosis was induced in the rats by repeated intraperitoneal injections of TAA at a dose of 200 mg/kg body weight twice a week for 4 weeks. PLP was given orally at a dose of 50 mg/kg body weight twice a day from the beginning of the TAA treatment until the end of the experiment. The development of liver cirrhosis was verified by histological examination. Liver proteomes were established by two-dimensional gel electrophoresis. Proteins with significantly altered expression levels were identified by matrix-assisted laser desorption/ionization-time of flight/time of flight mass spectrometry and the differentially expressed proteins were validated by immunohistochemical staining and reverse transcription polymerase chain reaction.
Histological staining showed a remarkable reduction in liver fibrosis in the rats with PLP treatment. A total of 13 differentially expressed proteins including actin, tubulin alpha-1C chain, preprohaptoglobin, hemopexin, galectin-5, glutathione S-transferase alpha-4 (GSTA4), branched chain keto acid dehydrogenase hterotetrameric E1 subunit alpha (BCKDHA), glutathione S-transferase mu (GSTmu); glyceraldehyde-3-phosphate dehydrogenase (GAPDH); thiosulfate sulfurtransferase (TFT); betaine-homocysteine S-methyltransferase 1 (BHMT1); quinoid dihydropteridine reductase (QDPR); ribonuclease UK114 were observed between the TAA and PLP groups. These proteins are involved in oxidative stress, heme and iron metabolism, cysteine metabolism, and branched-chain amino acid catabolism.
The proteomics data indicate that P. linteus may be protective against TAA-induced liver fibrosis via regulation of oxidative stress pathways, heat shock pathways, and metabolic pathways for amino acids and nucleic acids.
Most chronic liver diseases, including viral hepatitis (hepatitis B virus and hepatitis C virus), alcoholic liver disease, and biliary diseases, ultimately lead to liver fibrosis. Without effective treatments at an early stage, reversible liver fibrosis will lead to irreversible cirrhosis. Oxidative stress may cause liver damage[3, 4], and reducing oxidative stress by supplementation with antioxidants is effective for preventing liver fibrogenesis. However, evidence for the efficacy of antioxidants, such as vitamin E and superoxide dismutase, in the treatment of human liver fibrosis has not been established.
Phellinus linteus (Berk. et Curt.) Teng, an orange-colored mushroom, belongs to the Hymenochaetaceae Basidiomycetes and has been considered useful in preventing and treating liver fibrosis and liver cancers owing to its strong anti-inflammatory, antioxidative, antiangiogenic, and anticancer properties[7–10]. P. linteus has been used in Chinese medicine for the treatment of tumors, menstrual irregularities, and liver-related illnesses. Several reports from Korea and Japan have demonstrated that intake of P. linteus for a long time may induce spontaneous regression of hepatocellular carcinoma in patients with multiple metastases[12, 13]. Some in vivo and in vitro studies have also demonstrated that P. linteus exerts antitumor effects on hepatocellular carcinoma[14–16].
Over the last decade, accumulating evidence suggests that P. linteus may protect the liver against fibrosis via its antioxidative property. A study in 2002 demonstrated that an extract of P. linteus was able to suppress carbon tetrachloride-induced late liver fibrosis by reducing peroxidation products, restoring the activities of catalase and superoxide dismutase, and reviving the expression of aerobic respiration enzymes. Shon et al. demonstrated that a P. linteus polysaccharide (PLP) fraction was able to inhibit cytochrome P450 isozymes in the liver. Furthermore, a retinoic acid derivative isolated from P. linteus was reported to decrease transforming growth factor-beta-induced early liver fibrosis by downregulating reactive oxygen species generation and suppressing the expression of several proteins.
Although antioxidation is an important mechanism by which P. linteus suppresses liver fibrosis, the molecular mechanism of the antioxidative effect of P. linteus is still unclear. To date, studies on P. linteus-mediated protection of the liver against injury have only found a few target molecules. With the development of proteomics technology, it is possible to cover the expression of more proteins acting within a biological context to investigate the cellular processes involved in disease pathogenesis with high-throughput and in a quantitative manner[18, 19].
In the present study, we aim to assess the hepatoprotective effects of P. linteus against thioacetamide (TAA)-induced liver fibrosis by high-resolution two-dimensional polyacrylamide gel electrophoresis (2-DE) coupled with mass spectrometry technology.
Preparation of PLP
Sang Hwang 125 capsules containing a lyophilized hot water extract of wild-type P. linteus were donated by Dr. Frankie Chan (Amazing Grace Health Products Limited Partnership, Thailand). Each Sang Hwang capsule contained 400 mg of pure extracts from natural P. linteus. The polysaccharides and glucan contents of the P. linteus natural compound are 53–63% and 24%, respectively, as previously reported by us. This natural compound has been shown to possess strong antioxidative and immunomodulatory properties. PLP was prepared by dissolving 100 g of freeze-dried powder from Sang Hwang 125 capsules in 1 L of distilled water, followed by the addition of 2 L of ethanol (Merck, Germany) at −20°C. The precipitated polysaccharides were collected by centrifugation at 3000 × g for 1 h, dissolved in a small volume of distilled water, and lyophilized. The resulting powder was stored at −20°C until use.
Eight-week-old male Sprague–Dawley rats (weighing approximately 200 g) were obtained from Hallym University (Korea). All rats were kept in an animal house under a 12-h/12-h light/dark cycle, with controlled temperature and humidity and free access to food and water. After 1 week of acclimatization, the rats were arbitrarily divided into three groups: Normal group; TAA group, in which rats received TAA only; and PLP group, in which rats received PLP and TAA. TAA (Sigma-Aldrich, USA) was intraperitoneally injected at a dose of 200 mg/kg body weight twice a week for 4 weeks. PLP was given orally at a dose of 50 mg/kg body weight twice a day from the beginning of the TAA treatment until the end of the experiment. All rats were euthanized after 4 weeks by intraperitoneal injection of 200 mg/kg sodium pentobarbital (Sigma-Aldrich, USA). Dissection was carried out, and liver samples were fixed in 10% buffered formalin solution (Surgipath, Germany) for histological staining. Tissues from the same portion of the liver were collected from the TAA and PLP groups for the proteomics analysis.
The study protocol was approved by the Hallym University, South Korea. Animal care complied with institutional guidelines.
Histological examination of the liver
The fixed liver tissues were embedded in paraffin and sectioned at 5-μm thickness. For each liver sample, the stage of hepatic fibrosis was established. The liver sections were stained with Masson’s trichrome (Sigma-Aldrich, USA) and observed under NIKON model SE microscope (NIKON, Japan) to evaluate the degree of fibrosis.
Sample preparation for proteomics analysis
Liver samples were snap-frozen in liquid nitrogen and stored at −80°C for the proteomics analysis. The frozen liver tissue samples from the TAA and PLP groups were disrupted with a tissue teaser (Biospec Products, USA) in a lysis buffer containing 25 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA disodium salt, 1 mM dithiothreitol (DTT) (USB, USA), 1% (v/v) Triton X-100 (USB, USA), and 1% (v/v) Protease Inhibitor Cocktail Set III (Bio-Rad, USA). The superfluous salt in the extract was removed by incubation with 20% (w/v) trichloroacetic acid (TCA)-acetone solution and 20 mM DTT in acetone (Merck, Germany) for 4 hours at −40°C. The protein pellet was obtained by centrifugation at 15,800 × g for 30 min at 4°C. Excess TCA was removed by three washes with acetone containing 20 mM DTT. After air-drying, the protein pellet was resuspended in buffer comprising 7 M urea, 2 M thiourea, 100 mM DTT, 5% (v/v) glycerol, and 4% (w/v) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) (USB, USA), and the resulting protein solution was stored at −80°C until 2-DE analysis. The protein concentration was determined by the Bradford assay (Bio-Rad, USA).
Two-dimensional gel electrophoresis
The 2-DE procedures were performed according to our previous study with some modifications. The tissue samples were processed in duplicate and a total of 12 gels (six for the TAA group and six for the PLP group) were used. For the first-dimension electrophoresis, 100-μg protein samples were mixed with 350 μL of rehydration buffer comprising 9.5 M urea, 2% (w/v) CHAPS, 0.28% (w/v) DTT, 0.002% (w/v) bromophenol blue (USB, USA) and 1% (v/v) immobilized pH gradient buffer (pH 3–10) (Bio-Rad, USA), and then applied to an Ettan IPGphor 3 isoelectric focusing electrophoresis system (GE healthcare, USA). The samples were rehydrated for 7 h before isoelectric focusing with the following programs: (a) linear increase up to 500 V over 1 h; (b) holding at 500 V for 2 h; (c) linear increase up to 10,000 V over 4 h; (d) linear increase up to 10,000 V over 3 h; and (e) final hold at 10,000 V to reach a total of 120,000 V × h. The focused immobilized pH gradient gel strips were equilibrated for 15 min in a solution comprising 50 mM Tris–HCl, pH 8.8, 6 M urea, 30% (v/v) glycerol, 2% (w/v) sodium dodecyl sulfate (SDS) and 20 mM DTT, followed by incubation with the same buffer containing 20 mM iodoacetamide (Sigma-Aldrich, USA) for another 15 min. The second-dimension separation was performed by 12.5% SDS polyacrylamide gel electrophoresis (PAGE) at a constant current of 30 mA for 30 min, followed by a 60-mA current for the rest of the analysis until the bromophenol blue line reach the bottom of the gels.
Image acquisition and analysis
After the 2-DE, the gels were stained with SYPRO® Ruby Protein Stain (Bio-Rad, USA) according to the manufacturer’s protocol. The stained gels were scanned with a Molecular Imager PharosFX Plus System (Bio-Rad, USA) and analyzed by PDQuest 8.0 software (Bio-Rad, USA). Each expression level was calculated as the percentage volume (% vol), and exported for statistical analysis. The relative intensities of spots were used for comparison between the two groups, and only those spots with significant differences (≥ 1.5-fold increase or decrease; P < 0.05) were selected for protein identification.
Spots showing differential expression (P < 0.05) between the TAA and PLP groups were sent to the Genome Research Centre (The University of Hong Kong, Hong Kong) for protein identification. The proteins were digested with sequencing grade modified trypsin (Promega, USA) and applied to matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometer analysis using a 4800 MALDI TOF/TOF Analyzer (Applied Biosystems, USA). Matches between the experimental data and mass values calculated from a candidate protein were carried out by Mascot search engine (Matrix Science, UK) that uses MS data to identify proteins from the NCBInr database with taxonomy limited to Rattus norvegicus. The database allowed up to one missed cleavage and the mass tolerance was set as 75 ppm peptide limited by fixed modification of carbamidomethyl and variable modification of oxidation, with monoisotopic values. Mascot reported the molecular weight search (MOWSE) score, which is calculated by −10 × log10 (P), where P is the probability that the observed match is a random event. The P value is limited by the size of the sequence database being searched (limited by taxonomy), the conditions, and the settings of trypsin digestion. Each calculated value that falls within a given mass tolerance of an experimental value counts as a match. The accepted threshold is that an event is significant if it would be expected to occur at random with a frequency of < 5%. In this study, a protein match with a score of > 71 was regarded as significant.
Western blot analysis for validation of differentially expressed proteins
Western blot analysis was employed to validate the proteomic data. Liver protein extracts were mixed with sample buffer (62.5 mM Tris–HCl, pH 6.8, 25% (v/v) glycerol, 2% (w/v) SDS, 350 mM DTT, and 0.01% (w/v) bromophenol blue) at a ratio of 1:1 and incubated in boiling water for 5 min. Aliquots of the samples (30 mg of protein) were separated by electrophoresis in 12.5% SDS-PAGE gels at constant voltage (120 V) and then transferred to polyvinylidene difluoride membranes (GE Healthcare, USA) using a TE77 PWR Semi-dry Transfer Unit (GE Healthcare, USA). The membranes were blocked with 5% (w/v) non-fat dry milk in phosphate buffer saline overnight at 4°C. The membrane was incubated with primary antibodies: anti-haptoglobin (1:1000), anti-hemopexin (1:1000;), anti-hemoglobin (1:1000), anti-GSTA4 (1:500), and anti-GSTmu (1:1000) (Abcam, USA) for one hour and then incubated with their corresponding secondary horseradish peroxidase-conjugated antibodies (Bio-Rad, USA) for another one hour. The blots were washed five times with 0.05% Tween-20 in phosphate buffer saline between steps. Proteins were detected with an enhanced chemiluminescence system (GE Healthcare, USA) and the band intensity was measured with the Quantity One software (Bio-Rad, USA).
Reverse transcription polymerase chain reaction
Primers used for the PCR analyses
Primer sequence (5’-3’)
Fragment size (bp)
Annealing temperature (°C)
All data are presented as the mean ± standard deviation (SD). The significance of differences in data between the groups was determined by one-way analysis of variance followed by the Tukey test for equality of variances using SPSS 17.0 (IBM, USA). Differences were considered statistically significant at P < 0.05.
Histological assessment of liver fibrosis
Identification of protein spots on 2-DE gels
Differentially expressed liver proteins between the TAA-induced liver fibrosis rats in the PLP and TAA groups
Expression quantity (×102) TAA
Expression quantity (×102) PLP
Expression change (PLP/TAA)
actin, cytoplasmic 2
62.6 ± 24.5
97.1 ± 13.0
tubulin alpha-1C chain
29.4 ± 13.0
55.4 ± 14.0
100.5 ± 34.1
61.4 ± 15.4
11.5 ± 9.5
75.3 ± 44.9
41.3 ± 9.1
61.0 ± 11.5
49.3 ± 14.7
73.8 ± 14.0
Glutathione S-transferase alpha-4
15.1 ± 7.6
28.9 ± 11.3
54.8 ± 20.6
92.5 ± 16.2
172.2 ± 69.2
78.9 ± 45.1
quinoid dihydropteridine reductase, isoform CRA_c
83.7 ± 22.3
128.5 ± 25.5
betaine-homocysteine S-methyltransferase 1
128.6 ± 42.6
65.1 ± 12
141.6 ± 69.1
225.8 ± 44.3
63.8 ± 15.7
99.7 ± 22.8
Western blot analysis for validation of differentially expressed proteins in the proteomics analysis
Quantitative RT-PCR for gene expression analysis
The present study demonstrates that a natural product derived from P. linteus was able to protect against liver fibrosis induced in rats by chronic insult with TAA. The histopathological data clearly showed a reduction in collagen accumulation in the liver with PLP treatment. The present study thus supports the earlier findings that P. linteus possesses the capability to suppress liver injury[17, 23] and exhibits strong and specific inhibitory activities to reduce peroxidation products and increase antioxidant enzymes in the liver[17, 23].
Major biofunctions of the identified proteins
The highest binding affinity for heme, iron metabolism
The highest binding affinity for hemoglobin
glutathione S-transferase alpha-4 (GSTA4)
betaine-homocysteine S-methyltransferase 1 (BHMT1)
cysteine metabolism and GSH synthesis regulation
Branched-chain amino acids catabolism
dihydrofolate reductase (DHFR)
Synthesis of nucleic acid precursors
quinoid dihydropteridine reductase (QDPR)
Tetrahydrobiopterin recycle, amino acid metabolism
glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
Glucose metabolism, initiation of apoptosis
Mitochondrion. Cytoplasm. Nucleus. Peroxisome
Cytoplasm Cell surface of rat reticulocytes and erythrocytes
Erythrocyte differentiation and reticulocyte maturation
The proteomic data showed that the expression of preprohaptoglobin was 1.7-fold higher while that of hemopexin was 1.6-fold lower in the PLP group compared with the TAA group. The expression changes of these two proteins were validated by western blot analysis (Figure3). The increase in hemopexin and decrease in haptoglobin are potential markers for fibrosis because of their involvement in the regulation of liver iron homeostasis. The aspect of whether the protective effect of PLP against the TAA-induced liver fibrosis occurred via the regulation of iron homeostasis cannot be concluded in the present study, because the liver and serum iron concentrations were not determined. In a previous study, chelation of ferrous ions by P. linteus was described, and PLP was able to protect hepatocytes against iron overload-mediated oxidative stress. Iron homeostasis regulation has been suggested as a potential PLP treatment target in liver fibrosis.
Glutathione (GSH) plays an important role in cellular detoxification, because it effectively scavenges free radicals and other reactive oxygen species. In GSH-related antioxidative detoxification, glutathione S-transferases (GSTs) play central role; GSTA4 plays a role in the cellular defense against oxidative stress and lipid oxidation during liver injury. Dwivedi et al. demonstrated that mGSTA4 null (−/−) mice showed much quicker and greater carbon tetrachloride-induced hepatotoxicity than wild-type (+/+) mice. In the present study, the expression of GSTA4 was 1.9-fold higher in the PLP group than in the TAA group, and the change was confirmed by western blot analysis. The upregulated expression of GSTA4 might protect the liver against the injury and oxidative stress induced by TAA. However, the western blot analysis did not show a significant change in GSTmu between the PLP and TAA groups. These results could arise through non-specificity of the antibody for GSTmu or because GSTA4 was likely to be regulated by P. linteus.
The expression of BHMT was 2-fold lower in the PLP group than in the TAA group, suggesting that homocysteine was inclined to be converted to cysteine in the transsulfuration reaction, generating more cysteine for GSH synthesis. P. linteus may promote the accumulation of substrates for GSH synthesis, cysteine and glutamate, by regulating the expression levels of BHMT and BCKDHA. BHMT reduces the conversion of homocysteine to cysteine by catalyzing the remethylation of homocysteine back to methionine.
Branched-chain amino acid (BCAA) catabolism is an important intercellular source of glutamate. The branched-chain α-keto acid dehydrogenase (BCKD) complex is the rate-limiting enzyme for the whole BCAA catabolism. The Bckdha gene encodes the E1 α subunit of the BCKD. The expression of BCKDHA was 1.5-fold higher in the PLP group than in the TAA group, suggesting that more glutamate was generated for GSH synthesis in the PLP group.
Several proteins that showed higher expression in the PLP group are involved in amino acid metabolism and nucleic acid metabolism. These include BCKDHA (1.5-fold), QDPR (1.6-fold), and DHFR (1.5-fold). In clinical treatment of liver diseases, supplementation with BCAAs is considered useful to relieve protein malnutrition[33, 34]. QDPR is an enzyme that takes part in the tetrahydrobiopterin recycling pathway, and tetrahydrobiopterin is the precursor of phenylalanine and tyrosine. The higher expression of QDPR in the PLP group suggests that PLP may expedite protein and nucleic acid synthesis in the fibrotic liver. DHFR is important for regulating the cellular amount of tetrahydrofolate, which is essential for purine and thymidylate synthesis[36, 37]. The higher expression of DHFR in the PLP group indicates that PLP may aid in the regeneration of liver injury. The expression of ribonuclease UK114, a translational inhibitor mostly present in the liver and kidney, was 2.2-fold lower in the PLP group, meeting the requirement for protein synthesis for liver regeneration. In a clinical study, downregulation of ribonuclease UK114 was observed in human hepatocellular carcinoma.
GAPDH catalyzes a step of glycolysis. The expression of GAPDH was 1.6-fold higher in the PLP group, suggesting a higher energy requirement for liver amelioration. Several studies have illustrated that GAPDH may work in non-metabolic processes, such as transcription regulation and apoptosis initiation[40, 41]. This may be another reason for the upregulation of GAPDH in the PLP group.
Galectins comprise a family of evolutionarily conserved glycan-binding proteins that take part in acute and chronic inflammation[42, 43]. Galectin-5 contributes to erythrocyte differentiation and reticulocyte maturation, but its function in liver injury remains unclear[44, 45]. The much higher expression of galectin-5 in the PLP group suggests that PLP may promote erythropoiesis, inflammation regulation, and liver regeneration.
The present study has demonstrated that PLP can protect rats against TAA-induced liver fibrosis in at least two possible ways: 1) protection of the liver against oxidative stress, especially by scavenging of iron-related free radicals; and 2) regulation of the metabolism of amino acids and nucleic acids for liver amelioration. Our findings provide novel molecular mechanisms for the protective effects of P. linteus against liver fibrosis.
Phellinus linteus polysaccharide
Two-dimensional polyacrylamide gel electrophoresis
- MALDI-TOF/TOF MS:
Matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight mass spectrometry
Reverse transcription polymerase chain reaction
Glutathione S-transferase alpha-4
Branched chain keto acid dehydrogenase heterotetrameric E1 subunit alpha
Glutathione S-transferase mu
Betaine-homocysteine S-methyltransferase 1
Quinoid dihydropteridine reductase
Sodium dodecyl sulfate
Polyacrylamide gel electrophoresis
Molecular weight search
Heat shock protein 70
Branched-chain amino acid
Branched-chain α-keto acid dehydrogenase.
This work was supported by grants from the Chinese Medicine fund and the general research funding of the University of Hong Kong. We acknowledge Dr. Frankie Chan of The Amazing Grace Health Products Limited Partnership of Thailand for providing the P. linteus sample.
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