Genistein, taurine, 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), Tris, SDS, DL-dithiothreitol (DTT), trichloroacetic acid (TCA), Dulbecco's modified Eagle's medium (DMEM), sodium dihydrogen phosphate (NaH₂PO₄), potassium chloride (KCl), acetone and acetonitrile (ACN) were all purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). EGCG was acquired from Sichuan Yuga Tea Development Co., Ltd. (Sichuan, China). Reagents for iTRAQ were obtained from AB Sciex Pte., Ltd. (Concord, ON, Canada). Phosphate buffered saline (PBS; pH 7.4), ampholine and apotinin were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Furthermore, radioimmunoprecipitation assay lysis buffer was purchased from Beyotime Institute of Biotechnology (Shanghai, China) and Trypsin Gold was acquired from Promega Corporation (Madison, WI, USA).

The HSC-T6 cell line, an immortalized rat HSC line exhibiting a stable phenotype and biochemical characteristics of liver fibrosis, was purchased from the Xiangya School of Medicine, Central South University (Hunan, China). HSC-T6 cells were cultured in DMEM supplemented with heat-inactivated 10% fetal bovine serum (both Gibco; Thermo Fisher Scientific, Inc.) at 37°C in a humidified atmosphere containing 5% CO₂. Cells were passaged via trypsinization every 3 days.

HSC-T6 cells were seeded into 50-ml culture flasks at a density of 5×10⁴ cells/ml. Following 24 h culture, cells were treated with or without combined treatment with 0.03 mg/ml taurine, 0.035 mg/ml EGCG and 0.007 mg/ml genistein for 24 h, as previously reported (15). Subsequently cells were trypsinized, and centrifuged at 12,000 × g and 4°C for 20 min to obtain cell pellets. Pellets were washed three times with ice-cold PBS, resuspended in 1 ml lysis buffer (containing 9 mol/l urea, 4% CHAPS, 40 mmol/l Tris, 1% DTT, 0.8% ampholine and 0.002% apotinin), placed on ice and vortexed every 5 min for a total of 20 min. Cell supernatants were collected following centrifugation at 12,000 × g for 20 min at 4°C. Supernatants were then precipitated with pre-chilled acetone (containing 10% v/v TCA) for 2 h and centrifuged at 12,000 × g for 15 min at 4°C. The resulting pellets were washed with pre-chilled acetone, incubated at 4°C for 2 h and centrifuged again at 12,000 g for 15 min at 4°C. Subsequently, cells were washed with pre-chilled acetone a further three times. The final pellets were lyophilized and protein concentration was determined using bovine serum albumin (Wuhan Boster Biological Technology, Ltd., Wuhan, China) as the standard (24).

A total of 100 µg processed protein was reduced, blocked with cysteine (25) and digested with Trypsin Gold at a ratio of protein to trypsin of 20:1 at 37°C for 12 h. Following trypsin digestion, peptides were dried using vacuum centrifugation (250 × g, −20°C, 1 h). Subsequently, peptides were reconstituted in 0.5 mol/l tetraethylammonium bromide and processed using 4-plex iTRAQ (both AB Sciex Pte., Ltd.) following the manufacturer's protocol. One unit of iTRAQ reagent was thawed and reconstituted in 150 µl 100% isopropanol. Peptides were individually labeled with iTRAQ tags as follows: Control group, 114; and the combination treatment group, 115. Following incubation for 2 h at room temperature with the iTRAQ reagent, labeled samples were mixed equally prior to further analysis (25).

SCX chromatography was performed using a LC-20AB high performance liquid chromatography pump system (Shimadzu Corporation, Kyoto, Japan). iTRAQ-labeled peptide mixtures were reconstituted with 4 ml buffer A (25 mmol/l NaH₂PO₄ in 25% ACN, pH 2.7) and loaded onto a Ultremex SCX column (4.6×250 mm, 5 µm; Phenomenex, Torrance, CA, USA). Peptides were eluted with a gradient of buffer A for 10 min, 5–35% buffer B (25 mmol/l NaH₂PO₄, 1 mol/l KCl in 25% ACN, pH 2.7) for 11 min and 35–80% buffer B for 1 min at a flow rate of 1 ml/min. The system was maintained in 80% buffer B for 3 min prior to equilibrating with buffer A for 10 min before the next injection. Elution was measured at 214 nm and fractions were collected every min. Eluted peptides were pooled into 20 fractions, desalted with a Strata X C18 column (Phenomenex) and vacuum-dried.

Each fraction was resuspended in buffer A, which consisted of 2% ACN, 0.1% formic acid (FA), and centrifuged at 20,000 rpm for 10 min. In each fraction, the final concentration of peptide was ~0.25 µg/µl. Using an auto sampler, 9 µl supernatant was loaded onto a Symmetry C18 column (180 µm ×20 mm, 5 µm). LC-ESI-MS/MS was performed using a nanoACQuity UPLC system (Waters Corporation, Milford, MA, USA). The sample was eluted with buffer A at 2 µl/min for 15 min. Peptides were eluted onto a BEH130 C18 column (100 µm ×10 mm, 1.7 µm; Waters Corporation) for online trapping, desalting and analytical separation. Samples were loaded at a flow rate of 300 nl/min with 5% buffer B (98% ACN and 0.1% FA) for 1 min, eluted with a 40-min gradient from 5–35% buffer B, followed by a 5-min linear gradient to 80% buffer B and maintenance with 80% buffer B for 5 min. Initial chromatographic conditions were restored following 2 min.

A TripleTOF 5600 system fitted with a Nanospray III source (both AB Sciex Pte., Ltd.) and a pulled quartz tip as the emitter (New Objective, Inc., Woburn, MA, USA) was used for data acquisition. Data were acquired using an ion spray voltage of 2.5 kV, curtain gas of 30 PSI, nebulizer gas of 5 PSI and an interface heater (temperature, 150°C). Survey scans were acquired in 250 msec and the top-30 product ion scans were collected if a threshold of 120 counts per sec was exceeded and a +2 to +5 charge state was exhibited. Four time bins were summed for each scan at a pulse frequency value of 11 kHz by monitoring the 40 GHz multichannel TDC detector with 4-anode channel detection. A sweeping collision energy setting of 35±5 eV was applied to all precursor ions for collision-induced dissociation. Dynamic exclusion was set for the ½ peak width (~18 sec) and the precursor was refreshed off of the exclusion list. All iTRAQ experiments were performed in triplicate.

Changes in the expression of hexokinase-2 (HK2) and lysosome-associated membrane glycoprotein 1 (LAMP 1) were determined by western blot analysis following iTRAQ analysis. Proteins were extracted in the iTRAQ experiment and quantified with a bicinchonic acid protein assay kit (Wuhan Boster Biological Technology, Ltd.). Subsequently, 50 µg total protein were separated in each lane via a 10% SDS-PAGE gels (Wuhan Boster Biological Technology, Ltd.) and transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). Membranes were blocked for 1 h at room temperature with 5% non-fat dried milk in Tris-buffered saline with Tween 20 (TBST). Subsequently, proteins were washed and incubated with anti-HK2 (cat. no. ab227198), anti-LAMP 1 (cat. no. ab62562; both 1:1,000; Abcam, Cambridge, MA, USA) and anti-GAPDH (cat. no. A00227-1; 1:500; Wuhan Boster Biological Technology, Ltd.) antibodies at room temperature for 1 h. Membranes were washed three times with TBST and incubated with secondary antibodies (IRDye^® 680LT Goat anti-Rabbit IgG; cat. no. 926-68021; 1:10,000; LI-COR Biosciences; Lincoln, NE, USA) for 1 h at room temperature. Blots were washed three times with TBST and detected using an LI-COR Odyssey^® Infrared Imaging system (Odyssey System, Version 2.0.25, LI-COR Biosciences). GAPDH was used as the loading control and total protein content in each lane was quantified using densitometry. Experiments were performed in triplicate.

Data analyses were performed using Protein-Pilot software 4.0 (AB Sciex Pte., Ltd.) and the search parameter for cysteine was determined to be the carbamidomethylation of cysteine. Resulting MS/MS spectra were searched against the International Protein Index (IPI) rat sequence databases (version 3.87; 39,925 sequences; http://www.ebi.ac.uk/IPI/IPIhelp.html) using MASCOT software (version 2.3.02; Matrix Science, London, UK). For protein identification and quantification, a peptide mass tolerance of 8.6 ppm was used for intact peptide masses and 0.05 Da for fragmented ions. One missed cleavage was accepted in the trypsin digests, carbamidomethylation of cysteine was considered to be a fixed modification and the conversion of N-terminal glutamine to pyro-glutamic acid and methionine oxidation were considered variable modifications. All identified peptides had an ion score above the Mascot peptide identity threshold and a protein was considered identified if at least one such unique peptide match was apparent for the protein. For protein-abundance ratios measured using iTRAQ, fold-changes >1.3 or <0.7 were set as the threshold and P<0.05 was considered to indicate a statistically significant difference. The accession numbers of proteins that were significantly differentially expressed were converted to a gene list using the Database for Annotation Visualization and Integrated Discovery (DAVID) functional annotation tool (http://david.abcc.ncifcrf.gov/summary.jsp) and biological processes, cell components and molecular functions were analyzed using the Gene Ontology terms (26). The protein interaction network mode was created using the Search Tool for the Retrieval of Interacting Genes (STRING) database (http://string-db.org/), which quantitatively integrated interaction data for a large number of organisms and transferred information between these organisms where applicable.

To determine the proteins that were differentially expressed, another two separate experiments were performed as aforementioned. Statistical analysis was performed using SPSS 16.0 for Windows (SPSS, Inc., Chicago, IL, USA). Data were presented as the mean ± standard deviation from three independent experiments. Quantitative variables were analyzed using Student's t-test. Spearman's rank was used to determine whether there was a correlation between parameters. P<0.05 was determined to indicate a statistical significance.

Of the 166 differentially expressed proteins, the expression of 76 were increased and 90 were decreased following combination treatment. Details of the proteins associated with cell signaling pathways associated with fibrosis are listed in Table I. Peptide Mass (https://web.expasy.org/peptide_mass/) and Prot Param (https://web.expasy.org/protparam/) tools in ExPASy were used to predict peptides produced by trypsin digestion of heme oxygenase 1 and analyze physical and chemical properties, including peptides molecular weight, isoelectric point, amino acid, atom, molar absorptivity, half-life, instability coefficient, aliphatic amino acid coefficient and average hydrophilicity coefficient. Peptides with good solubility and stability, and satisfying mass spectrometry detectable mass-to-charge ratio were selected, and analyzed for peptide homology using online software BlastP (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome) in NBCI database. Skyline (version 4.1; MacCoss Lab Software, Washington, USA) was used to predict specific peptide scanning mass spectrometry fragment information. Therefore, a representative mass spectrum and quantitative information of the heme oxygenase 1 peptide (sequence: PSLFPAASGAFSSFR) is indicated in Fig. 1. First order mass and tandem mass spectra of heme oxygenase 1 were presented in Fig. 1 (the control group, n=114 and the combination treatment group, n=115).

The 166 differentially expressed proteins were analyzed using the DAVID functional annotation tool to determine their cellular components, molecular functions and participation in biological processes (data not shown). The top three biological processes included cellular processes (11.86%), single-organism processes (10.09%) and metabolic processes (9.85%). The top three molecular function categories included binding (49.82%), catalytic activity (24.21%) and structural molecule activity (6.67%). The top three associated cellular components included the cell (37.60%), organelle (27.15%) and membrane (11.55%).

In order to characterize the protein-protein interactions of the identified differentially expressed proteins, the STRING database was assessed to establish a protein-protein interaction network (Fig. 2). Proteins that responded significantly to combination treatment and interacted with each other included Stat1, Icaml, Timp1, Cfd, Got2, Gpi, Pmpcb, Cox7a2, Phf5a, Nop58, Rps17, Rpl18, Rp18 and Gfm1 (Fig. 2). These seed proteins serve important roles in catalytic activity, biological regulation and coagulation systems (27–33). Furthermore, pathway annotation indicated that certain differentially expressed proteins, including glucose-6-phosphate isomerase (GPI) and ribosomal protein L8 (RPL8), were involved in eight different Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways (glycolysis and gluconeogenesis; lysosome metabolism; ribosome metabolism; spliceosome metabolism; butanoate metabolism; arginine and proline metabolism; cardiac muscle contraction; and pyruvate metabolism signaling pathways; Table II). Notably, GPI is a crucial enzyme that catalyzes the interconversion of glucose-6-phosphate and fructose-6-phosphate in glycolysis and gluconeogenesis (34). Rp18 is a component of the 60S subunit of the ribosome that is involved in the protein synthesis pathway. Depletion of Rp18 impairs drosophila development and is associated with apoptosis (35). The results suggest that the interactions and signaling pathways associated with the differentially expressed proteins may serve important roles in the pathogenesis of liver fibrosis.

To further investigate the expression of the differentially expressed proteins obtained from the iTRAQ-based quantitative proteomics study, western blot analysis was performed to assess the expression of HK2 and LAMP 1. Levels of HK2 and LAMP 1 were significantly downregulated and upregulated in HSC-T6 cells following combination therapy, respectively (each, P<0.05; Fig. 3). This pattern was similar to the changes in the expression of these proteins obtained following iTRAQ (Table I), supporting the validity of the proteomic analysis performed in the present study.