Introduction
Hepatic fibrosis refers to the excessive accumulation of extracellular matrix (ECM) components in the liver. It occurs in most types of chronic liver diseases, including liver cirrhosis, liver failure, and portal hypertension, and often requires liver transplantation (1). The activation of hepatic stellate cells (HSCs) is an important event by which this cell type, which is otherwise quiescent, expresses α-smooth muscle actin (α-SMA), assumes a myofibroblastic phenotype and synthesizes fibrillar collagens (2,3). Therefore, the inhibition of HSC proliferation, the regulation of the HSC cell cycle, and the facilitation of HSC apoptosis are important therapeutic approaches for hepatic fibrosis-related liver diseases.
Taurine (2-aminoethanesulfonic acid) is an organic acid which is abundant in the human body. Natural taurine (NTau) has emerged as an alternative candidate for therapeutic intervention since it is effective in preventing hepatic fibrosis and reducing cirrhosis (4,5). Supplementation with exogenous taurine is able to extensively inhibit the deposition of ECM and mitigate the degree of hepatic fibrosis (2). Several studies have focused on the specific gene regulation associated with the protective effect of taurine against hepatic damage (6–8), but the genome-wide genes, proteins and functional pathways underlying the hepatic protection have yet to be fully elucidated.
Gene-expression profiling through microarray analysis may shed light on useful clues to the taurine-mediated gene regulation. Furthermore, a proteomics approach may also be used to elucidate global protein expression and facilitate the discovery of potential drug targets. However, studies using microarray or proteomic technologies to investigate the molecular mechanism of taurine treatment for liver diseases have not been previously been conducted. Therefore, an integrative analysis of transcriptome and proteome levels was designed to illuminate the changes of gene and protein expression in human HSCs treated with NTau.
Materials and methods
NTau extraction
NTau (2-aminoethanesulfonic acid) was extracted from black clams (Meretrix meretrix L.). Briefly, the clam meats were weighed and minced in an electrical blender (4000 rpm), for ~10 sec. The mince was further homogenized for 30 min after adding distilled water (1 liter). The mixture was boiled in water for 30 min, followed by filtering through 4 layers of gauze. The residue on the top of the gauze was discarded, and the filtrate was then centrifuged (3000 rpm) to obtain the supernatant, which was then de-acidified with HCl (HCl:H2O=3). After centrifuging, the proteins were adjusted to a pH of 10 with a NaOH (20%) aqueous solution to yield the de-alkalinated protein. Following adjustment of the pH value to 5, the supernatant was further condensed. The other unwanted amino acids and pigments were removed by column chromatography using strong-acid cation-exchange resin as the solid phase and eluting with distilled water. The resultant NTau was quantitatively measured by high-performance liquid chromatography (HPLC), and the purity of the NTau was determined to be 98.8%.
Cell culture
LX-2 human HSCs (purchased from the Cell Bank at Xiangya Central Experiment Laboratory of Central South University, Changsha, China) were cultivated at 37°C in Dulbecco’s modified Eagle’s medium (DMEM; Gibco-BRL, Carlsbad, CA, USA), and were supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin in a 5% CO2 humidified incubator. Cells (1×104 or 5×105) were seeded in 96-well plates or 35-mm dishes. The cells were cultured in serum-free medium for 24 h and then treated with 40 mM NTau for 48 h.
Cell proliferation analysis
The proliferation activity of the LX-2 cells was measured by a microculture tetrazolium (MTT) colorimetric assay. LX-2 cells (1×104 cells/ml) were cultured in 96-well plates in DMEM with 10% FBS medium and then tranferred to serum-free medium for an additional 24 h, and triplicate wells of cells were incubated for 48 h in the presence 0–50 mM NTau. Cells in the various treatment groups were then incubated with MTT [5 mg/ml in phosphate-buffered solution (PBS)] for 4 h before harvesting. The optical density was measured using an ELISA reader at 570 nm with a reference wavelength of 630 nm.
Cell cycle analysis
The LX-2 cells were incubated for 24 h in a 5-ml cell culture flask in DMEM with 10% FBS medium and then cultured in serum-free medium for an additional 24 h. The cells were treated with 40 mM NTau for 48 h and then fixed with 70% ice-cold ethanol. The fixed cells were permeabilized with PBST (137 mM NaCl, 3 mM KCl, 8 mM NA2HPO4 and 0.1% Tween-20; pH 7.4) and then stained with 100 mg/l RNase A and 50 mg/l propidium iodide (PI) in the dark for cell cycle analysis. Cell cycle analysis was performed on a Coulter ELITE flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) through a 488-nm (LP) filter. The data were analyzed using the MultiCycle AV software (Phoenix Flow Systems, San Diego, CA, USA) for cell cycle distribution.
Apoptosis detection
LX-2 cells were incubated for 48 h in the presence 40 mM NTau and then re-suspended in 100 μl buffer containing calcium ions. The cells were treated with 5 μl Annexin V-FITC dye for 20–30 min and then with 5 μl PI dye for 5 min. The cell concentration was adjusted to ~1×105/ml by adding the appropriate amount of calcium ion-containing buffer. The cells were loaded on a flow cytometer (Coulter® Elite) within 1 h. Apoptotic analysis was performed using a Coulter® Elite flow cytometer (Beckman Coulter, Miami, FL, USA) with excitation and emission wavelengths of 488 and 530 nm, respectively.
Two-dimensional electrophoresis (2DE)
LX-2 cell lysates were collected and centrifuged at 14,000 rpm for 10 min at 4°C. The supernatants were analyzed by 2DE, and isoelectric focusing (IEF) was performed using an IPGphor IEF system (Bio-Rad, Hercules, CA, USA). The protein extract (200 μg) was mixed with rehydration buffer to 350 μl and loaded onto 17-cm, immobilized, nonlinear pH gradient (IPG) dry strips (pH 4–7; Bio-Rad). The IPG strips were equilibrated for 15 min in an equilibration buffer (6 M urea, 30% glycerol, 2% SDS and 50 mM Tris-HCl) containing 10 mg/ml dithiothreitol (DTT), followed by 15 min in an equilibration buffer containing 40 mg/ml iodoacetamide. Following equilibration, strips were applied to 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and sealed with agarose sealing solution. Following electrophoresis, the SDS-PAGE gels were silver stained. Stained gels were scanned using an image scanner (Amersham Biosciences, Piscataway, NJ, USA) in transmission mode. Analysis of the gels was accomplished using the PDQuest analysis software (Bio-Rad) including background subtraction, spots detection and the establishment of a reference gel. Protein spots were selected based on the criterion of >2-fold variation of expression between NTau-treated and control samples.
Mass spectrometry (MS) analysis
MS was performed on a UPLC-ESI-MS/MS (Waters/Micromass, Manchester, UK) equipped with an electrospray ionization (ESI) source. Data-dependent analysis was employed (the 3 most abundant ions in each cycle): 1 sec MS m/z 350-1,600, and max 5 sec MS/MS m/z 50-2,000 (continuum mode), with 50 sec dynamic exclusion. The positive-ion mode was employed, and the capillary voltage was set at 3.0 kV. The cone voltage was set at 35 V to investigate the intensities and distribution of ions in the mass spectra of samples. The MS/MS spectra were processed, searched using ProteinLynx Global SERVER™ (PLGS) v2.3 (Waters/Micromass), and searched against the NCBInr database by MASCOT (http://www.matrixscience.co.uk) using the following constraints: only tryptic peptides with up to 2 missed cleavage sites were allowed and 0.3-Da mass tolerances for MS and MS/MS fragment ions. The results were filtered by a peptide score of ≥30.
Western blot analysis
Total proteins in LX-2 cell lysates were quantified by the Bradford method (9,10) and analyzed on 12% SDS-PAGE gels. The gels were transferred onto a nitrocellulose membrane using a Trans-Blot SD apparatus (Bio-Rad). The membrane was incubated with anti-IgY [dilution 1:1,000 in Tris-buffered saline (TBS)] followed by incubation with secondary antibody (dilution 1:5,000 in TBS). Visualization of the protein bands was achieved by the chemiluminescence method, and the films were developed and fixed. Glyeraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal reference.
Gene ontology (GO) analysis
GO is a stratified tree structure for the analysis of the functions of genes and proteins (11). The 3 hierarchical principles of GO are ‘Biological Process’ (BP), ‘Cellular Component’ (CC) and ‘Molecular Function’ (MF). We analyzed the functional distribution of differential gene expression and protein production over the 3 principles. To accurately detect significantly over-represented GO terms, the Database for Annotation, Visualization, and Integrated Discovery (DAVID) tool (http://david.abcc.ncifcrf.gov) was used by analyzing into the fourth layers (12). The threshold value of group membership counts was set at 3, and the EASE score was set at 0.1. Then, the functional annotation clustering tool in DAVID was used to cluster functionally related annotations into groups for a 2D view of the related gene-term relationship (12). We ranked the importance of annotation groups with an enrichment score. In addition, we also used the DAVID tool to map differential gene expression and protein production into the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways database (13) to facilitate the biological interpretation in a network context. Fisher’s exact test was used and P<0.05 was considered to indicate a statistically significant difference.
Microarray analysis
A genome-wide 70-mer oligonucleotide microarray including 22,000 well-characterized human genes (14) was obtained from CapitalBio Corporation (Beijing, China) to investigate the expression profiling of NTau-treated LX-2 cells. The cDNA targets were prepared from 5 μg total RNA and were labeled with fluorescent dyes (Cy5 and Cy3-dCTP) by the Eberwine linear RNA amplification method and a subsequent enzymatic reaction (14). The RNA samples from the NTau-treated LX-2 cells were labeled with Cy3-dUTP and named as ‘1.’ The RNA samples from the blank control cells were labeled with Cy5-dUTP and named as ‘2.’ We then prepared the hybridization solution in hybridization buffer (25% formamide, 3X SSC, 0.2% SDS and 5X Denhardt’s solution) and hybridized it with sample at 42°C overnight in a humid environment. The hybridized microarrays were scanned with a confocal LuxScan™ scanner (CapitalBio Corporation, Beijing, China) at 2 wavelengths to detect emission from both Cy3 and Cy5. The images obtained were then analyzed using LuxScan™ 3.0 software (CapitalBio Corporation). Then, an intensity-dependent locally weighted scatterplot-smoothing regression (LOWESS) algorithm was used to normalize the 2-channel ratio values by an R package (15).
Gene set enrichment analysis (GSEA)
GSEA is a software (https://www.broad.harvard.edu/gsea/) for searching in predefined gene sets (ex. pathways) and ranking genes to identify significant biological changes in microarray data sets (16). First, ratios of filtered genes were operated by logarithm function to the base 1.5, and the input ‘rnk’ file was made based on the ranked value of log1.5(ratio). These expression matrices were exported to GSEA software and searched against the background dataset of ‘c4: computational gene sets’ with ‘GSEA Pre-ranked’ option. All the default settings except ‘gene set permutation’ with 1,000 iterations were used for the analysis.
RNA extraction and real-time polymerase chain reaction (PCR)
Total RNA was extracted using TRIzol (Invitrogen, Gaithersburg, MD, USA) reagent according to the manufacturer’s instructions. RNA was purified using the NucleoSpin RNA Clean-up kit (Macherey-Nagel, Düren, Germany). RNA quality from each sample was assessed by visualization of the 28S/18S ribosomal RNA ratio on 1% agarose gels. First-strand cDNA was synthesized using 1 μg total RNA in a 20-μl final volume by reverse transcription utilizing ReverTra Ace® reverse transcriptase (Toyobo, Co., Ltd., Osaka, Japan) with random hexamer primers or oligo(dT)18 primers (Invitrogen). PCR was performed using 0.5 μl cDNA, with specific primers and Ex Taq™ Polymerase (Takara Bio, Inc., Otsu, Japan) in a volume of 12.5 μl. The PCR products were then separated on 1.5% agarose gels. The real-time PCR reactions were performed using iQ™ SYBR®-Green Supermix kit according to the manufacturer’s instructions (Bio-Rad). RNA was amplified using the ABI Prism 7500 Sequence Detection system (Applied Biosystems, Carlsbad, CA, USA). The primers (Invitrogen) are shown in Table I. For all the real-time PCR experiments, negative controls were a non-reverse transcriptase reaction, and a non-sample reaction (data not shown). GAPDH was amplified as an internal standard.
Statistical analysis
All experiments were performed ≥3 times with triplicate measurements, and data are expressed as the mean ± standard error of the mean (SEM). Statistical analysis was performed using the R software (version 2.9.2, http://www.r-project.org/). Student’s t-test was performed to evaluate the differences of cell proliferation rate among the groups treated with different concentrations of NTau. The Student’s t-test was also used to evaluate the gene expression changes in real-time PCR results between 2 phenotypes. The Chi-square test was used to assess the effect of NTau on the cell cycle of HSCs. Fisher’s exact test was used to identify the significant GO terms of target genes relative to genome backgrounds. P<0.05 was considered to indicate a statistically significant difference.
Results
Effect of NTau on cell proliferation and cell cycle of HSCs
As shown in Fig. 1, an inverse correlation between the proliferation of LX-2 cells and the concentration of NTau was detected. Treatment with different concentrations of NTau significantly inhibited the growth of LX-2 cells when compared with that of the non-treated control cells (one-way ANOVA, P<0.005). The growth rate of LX-2 cells in the presence of 30–40 mmol/l NTau was reduced by 43% compared with that of the non-treated cells. As assessed by cell cycle analysis, the percentage of LX-2 cells in the G0/G1 phase increased from 43.9 to 50.9% in response to 40 mmol/l NTau, while exposure of the LX-2 cells to 40 mmol/l NTau for 48 h caused a 1- to 3-fold reduction (P<0.04) of the S- and G2/M-phase cell populations compared with those in the non-treated control cells.
Detection of NTau-induced apoptosis in HSCs
To investigate the mechanism underlying the growth-inhibitory effects of NTau on HSCs, the cell-apoptosis analysis of NTau-treated LX-2 cells was performed. As shown in Fig. 2, the increase in apoptotic cells in the NTau-treated LX-2 cells was comparable to that in the non-treated control cells (13.6±3.3 vs. 4.65±1.1%, P<0.05). These findings suggest that the induction of cellular apoptosis contributed, at least in part, to the HSC growth-inhibiting effects of NTau.
Identification of differentially expressed proteins in HSCs following NTau treatment
The protein profiles of HSCs were analyzed by 2DE and visualized using the PDQuest image analysis software. By comparing the protein profiles of the NTau-treated and non-treated control HSCs, 15 differentially expressed proteins were successfully identified. We picked up protein spots in the 2D gel image with identified changes and prepared them for MS analysis. Table II summarizes the code name, relative molecular weight, isoelectric point, and peptide fragment coverage for the differentially expressed proteins. The most significantly upregulated expression was of CAA32649, MYL9, PSMB6, ANXA1, MDH1, HSPB1, LASP1, LOC100134370, and SOD1, while ATP5H, BAF82933, ECHS1, PRDX2, HNRNPA2B1, and BAG36698 showed the most markedly downregulated protein expression.
Functional analysis of differentially expressed proteins in HSCs following NTau treatment and validation of proteomic data
We analyzed the functional enrichment of the differentially expressed proteins using the DAVID tool based on their annotation keywords from the UniProt database (17). Significant protein functions terms and corresponding proteins were identified (Fisher’s exact test, P<0.05). As shown in Table III, proteins that corresponded to the category of ‘acetylation,’ ‘direct protein sequencing,’ ‘antioxidant,’ ‘cytoplasm’ and ‘oxidoreductase’ were significantly affected in the NTau-treated HSCs.
Among the differentially expressed proteins identified successfully by MS, the upregulated proteins ANXA1 and PSMB6 and the downregulated proteins ECHS1 and PRDX2 were selected and subjected to western blot analysis. As shown in Fig. 3, NTau treatment significantly upregulated the expression of ANXA1 and downregulated the expression of ECHS1 and PRDX2 in HSCs. The expression of PSMB6 was not significantly different following NTau treatment. Therefore, western blot analysis of the differentially expressed proteins confirmed the reliability and validity of the proteomic high throughput experiments.
Differential gene expression in HSCs following NTau treatment
According to the filtering principles described above, 6,109 normally expressed genes (28.38%) with high confidence were screened among 21,522 genes. By applying the threshold of 1.5-fold change for an intensity ratio of NTau-treated vs. control HSCs, 658 genes (3.06%) were shown to be differentially expressed in NTau-treated HSCs. Among the differentially expressed genes, 241 were upregulated (1.12%) and 417 were downregulated (1.94%). The top 9 upregulated and the top 10 downregulated genes are presented in Table IV.
Functional categorization and clustering for the differentially expressed genes
The differentially expressed genes were then categorized according to their GO function using the DAVID tool. Six function categories of ‘Molecular Function’ (MF), 51 function categories of ‘Cellular Component’ (CC), and 42 function categories of ‘Biological Process’ (BP) were significantly enriched in the differentially expressed genes (Fisher’s exact test, P<0.05). Fig. 4 lists the top 5 significantly enriched GO terms identified after screening with a threshold of false discovery rate (FDR) of <0.01. Functional categorization of the GO terms demonstrated that these differentially expressed genes were strongly associated with biological processes of ‘NADH reduction and oxidation reaction’ and ‘RNA processing’. Based on the analysis above, the heuristic fuzzy clustering was used to classify the groups of similar annotations by the κ statistic values (18). Table V provides a visualized network of the 9 significantly enriched GO terms (geometric mean of member’s P-value, P<0.05). Clusters 1, 2, 3, 4, 7 and 9 correspond to the categories of ‘cellular transport and translation,’ ‘oxidant reaction’ and ‘mitosis process,’ respectively. This suggests a crucial role of these biological processes in NTau-treated HSCs.
Pathway-based (GSEA) microarray analysis of NTau-treated HSCs and validation of microarray analysis
mRNA expression profiling using GSEA microarrays and quantitative PCR (qPCR) was performed in NTau-treated HSCs. Analysis of the expression of individual mRNAs demonstrated 2 different patterns of expression. A number of genes including nucb2, adam9, tpp2, mtif2, abce1, pkn2, ndufb1, brcc3, adamtsl3 and gbp1 correlated with reduction and oxidation of NADH showed a reduced expression in the NTau-treated HSCs. The second group, consisting of genes related to cell proliferation and cell cycle regulation including hdac3, tgfβ1i1 and hrh1, showed an increased expression in NTau-treated HSCs. We selected 2 of the gene sets ‘Module 62’ and ‘MORF_RAD21’ to illustrate the enrichment scores as shown in Fig. 5, which was driven by the group of genes within a gene set that showed the highest correlation with NTau treatment.
Five selected candidate genes, snrpe, hnrph3, eif1ay, nucb2 and vim, were used as reference genes were assayed by qPCR in order to confirm the expression profiles found using microarray analysis (Fig. 6). Another 5 selected genes, cox5a, cox6c, ndufb1, ndufc1 and tgfβ1i1, were assayed to identify the difference between NTau-treated and control HSCs. The primers designed for real-time PCR are listed in Table I. As shown in Fig. 7, tgfβ1i1 mRNA showed a significantly increased expression level (2.26±0.41 vs. 1, P=0.01), which coincided with pathway-based (GSEA) microarray analysis progression in the regulation of cell proliferation group. However, cox5a mRNA (0.62±0.03 vs. 1, P=2×10−4), cox6c mRNA(0.51±0.07 vs. 1, P=1.3×10−3), ndufb1 mRNA(0.53±0.07 vs. 1, P=2.7×10−5), and ndufc1 mRNA (0.45±0.06 vs. 1, P=1.1×10−3) showed a reduced expression in the NTau-treated HSCs, which are known to play a role in the regulation of NADH dehydrogenase (ubiquinone) activity, which is involved in NADH oxidation.
Discussion
Taurine is a sulfur-containing β-amino acid with several potential therapeutic applications 19,20). Substantial progress has been made over the last 10–20 years in elucidating the bio-physiological function of taurine in the treatment of liver fibrosis. It has been reported that taurine is able to protect hepatocytes from chemically induced injury and mitigate the fibrosis of the liver (6,7,21). Although recent findings suggest that taurine is able to inhibit the proliferation of HSCs and cause a G0/G1 phase arrest (2), the exact mechanism has not yet been fully elucidated. During chronic liver injury, HSCs undergo a phenotypic transformation with the acquisition of myofibroblast-like features by increased proliferation and synthesis of ECM components and play a pivotal role in the formation of fibrosis (22). Therefore, the inhibition of HSC proliferation, the regulation of the cell cycle of HSCs, and the facilitation of HSC apoptosis are important therapeutic approaches for hepatic fibrosis-related liver diseases. The present study is congruent with the study by Chen (21), according to which taurine not only inhibits the proliferation of HSCs, but it is also able to promote HSC apoptosis.
The process of apoptosis is controlled by a diverse range of cell signals, which may originate either extracellularly or intracellularly. A cell initiates intracellular apoptotic signaling in response to stress, which may cause cell suicide. The binding of nuclear receptors by glucocorticoids, heat, radiation increase and intracellular calcium concentration, for example, by causing damage to the membrane, are all able to trigger the release of intracellular apoptotic signals by a damaged cell (23,24).
The proteins ANXA1, ECHS1 and PRDX2 were selected for validation by western blot analysis. Functional analysis showed that these proteins were related to the biological processes of ‘cellular apoptosis’ and ‘oxidation reaction.’ Since flow cytometric analysis has shown that taurine-treated HSCs had a significantly increased apoptosis rate compared to the control group, the proteomic analysis may reveal the relevant mechanism. Upregulated protein Annexin I belongs to a family of Ca2+-dependent phospholipid-binding proteins, which are able to change the intracellular calcium concentration and cause apoptosis. Reactive oxygen species (ROS) are closely associated with apoptotic induction (25) and downregulate PRDX2 protein, a kind of antioxidant enzyme. Since altered cellular oxidation-reduction one of the key events in apoptosis that affects the mitochondria (26), it may be involved in oxidation reaction and HSC apoptosis.
GO analysis indicated that reduction and oxidation of NADH had significant functional enrichment. Fuzzy heuristic clustering of GO categories suggested that intracellular components of HSCs were influenced by NTau treatment. Furthermore, many differentially expressed genes were classified into function clusters relating to reduction-oxidation of mitochondrial NADH and mitosis. A variety of key events in apoptosis focus on the mitochondria, including the release of caspase activators, such as cytochrome c, changes in electron transport and altered cellular oxidation-reduction (26,27).
Among the validated genes, upregulated tgfβ1i1, which is transforming growth factor β1-induced transcript 1, is involved in the negative regulation of cell proliferation (28). This means that the higher its expression level, the slower the cell proliferation. Its upregulated level may indicate taurine’s function in inhibiting the proliferation of LX-2 cells. The downregulated genes cox6c, cox5a, ndufb1, and ndufc1 are components of the electron transport chain in the mitochondrion. Therefore, we hypothesize that NTau may regulate the reduction-oxidation of NADH and thereby lead to the inhibition of HSC proliferation.
The pathogenesis of hepatic fibrosis involves the activation of HSCs. This procedure is accelerated by HSC proliferation and the progression of the cell cycle (29). NTau has been demonstrated to inhibit HSC proliferation and prevent HSCs in the G0/G1 phase from entering the S and G2/M phases by flow cytometric analysis, which suggests that NTau is able to modulate hepatic fibrosis. Functional clustering and GSEA analyses also showed that mitosis, and especially the M phase of mitotic cell cycle, was regulated by NTau treatment. Therefore, it was not only demonstrated that NTau was beneficial to hepatic fibrosis therapy, but valuable evidence to elucidate the underlying molecular mechanism by investigating responded genes and proteins was also provided.
It is worth noting that reduction-oxidation of NADH may play an important role in the protective effect of NTau against hepatic fibrosis, since GO functional clustering and GSEA analysis consistently came to the same results. The reduction-oxidation state is often used to describe the balance of NAD+/NADH and NADP+/NADPH in a biological system such as a cell (30). Oxidative stress is important in the pathogenesis of hepatic fibrosis, which is the result of deposition of excess ECM proteins produced by activated HSCs (6). NTau may be able to attenuate ROS production by modulating the balance of reduction-oxidation of NADH. Furthermore, the relationship between the reduction-oxidation of NADH and HSC proliferation or cell cycle regulation has been previously investigated. Zou et al(31) have demonstrated that ROS derived from NAD(P)H oxidases activated the phosphotidylinositol-3-kinase (PI3K)/Akt pathway, thus promoting cellular proliferation in HSCs.
Taurine may be synthesized chemically or extracted from natural sources. However, NTau is superior to synthetic taurine in promoting HSC apoptosis (32). The advantage of the present study is reflected in the selection of NTau for investigation of its mechanism in regulating HSC apoptosis. To the best of our knowledge, the present study provided for the first time evidence concerning taurine-mediated transcriptional changes in HSCs by microarray analysis. Additionally, proteomic approaches were used to delineate protein expression changes in NTau-treated HSCs. These variations correspond to biological processes such as ‘oxidant reaction’ and ‘mitosis process,’ which promote HSC apoptosis. While these observations systematically investigated the underlying mechanism of NTau in inhibiting the activation of HSCs, our data provide strong support for the use of NTau as a potential therapy for hepatic fibrosis.