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[20]. This natural compound has been shown to possess strong antioxidative and immunomodulatory properties[21]. 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.

Animal experiments

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[22] 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.

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

Statistical analysis

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

TAA treatment of rats for 4 weeks resulted in liver fibrosis, which was characterized by alterations in the quality of the hepatic extracellular matrix (Figure1B&C), compared with the livers of rats in the Normal group (Figure1A). Extended collagen deposition and large septa of the hepatic lobules were observed after 4 weeks of TAA treatment (Figure1B). In addition, lymphoid infiltration was observed around the central and portal veins in the TAA-treated livers. PLP treatment markedly reduced the severity of the fibrosis and inflammation induced by TAA (Figure1C).

Identification of protein spots on 2-DE gels

On each 2-DE gel, nearly 1000 individual protein spots were detected, and 13 spots with notable changes found by the PDQuest software between the PLP and TAA groups were identified by MS (Figure2, Table2). The proteins with increased expression levels in the PLP group compared with the TAA group included actin cytoplasmic 2, tubulin alpha-1C chain, galectin-5, BCKDHA, DHFR, preprohaptoglobin, GSTA4, QDPR, GAPDH, and TFT. The proteins with decreased expression levels in the PLP group compared with the TAA group were hemopexin, ribonuclease UK114, and BHMT1.

Western blot analysis for validation of differentially expressed proteins in the proteomics analysis

Owning to the limitations of anti-rat protein antibodies, many of the identified differentially expressed proteins could not be measured by western blot analysis. Haptoglobin, hemopexin, heat-shock protein 70 (HSP70), and GSTA4 were successfully measured and used to validate the results obtained in the proteomic analysis. The western blot results were in general agreement with the differentially expressed proteins obtained in the proteomic analysis. As shown in Figure3, the level of hemopexin (P = 0.049) was lower and the levels of haptoglobin (P = 0.042) and GSTA4 (P = 0.040) were much higher in the PLP group compared with the TAA group. The levels of hemoglobin (P = 0.047) and HSP70 (P = 0.041) were higher in the PLP group than in the TAA group. GSTmu did not show a significant difference in the western blot analysis.

Quantitative RT-PCR for gene expression analysis

To investigate whether the expression changes of the identified proteins occurred at the transcriptional level, we determined the mRNA expression changes of these proteins by semiquantitative RT-PCR. As shown in Figure4, the mRNA expression of many of the identified genes changed in a similar tendency as their protein expression change showed in 2D proteomic results, suggesting that the effects of P. linteus were exerted at the protein expression level, i.e. focused on the translation and post-translation steps. Haptoglobin, BCKDHA, and BHMT showed significant differences between the TAA and PLP groups.