Female C57BL/6J mice (8–9 weeks old, weighting 21–23 g, specific pathogen-free) were purchased from the Animal Center of the Chinese Academy of Medical Sciences. Animal care was carried out according to the guidelines of the Principles of Laboratory Animal Care (18), all experimental protocols were approved by the Institutional Animal Care and Use Committee at the Shanghai Institute of Materia Medica (approval no. 2018-12-LC-11; Shanghai, China). The animals were allowed free access to a standard laboratory diet and tap water. All mice were kept in standard laboratory conditions (21±2°C, 12-h light/dark cycle), and were fed adaptively for 1 week before starting the experiments. A total of 24 mice were randomly divided into three groups with 8 mice per group: i) The normal control group; ii) the solvent group of CCl4-challenged mice; and iii) the givinostat treatment group of CCl4-challenged mice. In the givinostat treatment group, mice were stimulated with CCl4 for 8 weeks, and in the last 6 weeks, the animals received an intraperitoneal (i.p.) injection of givinostat (10 mg/kg, formulated in PBS) daily (19). Mice were i.p. injected with 10% CCl4 dissolved in olive oil at a dose of 1 ml/kg body weight twice a week for 8 weeks to trigger liver fibrosis (20). Givinostat or PBS solvent was i.p. injected after CCl4 treatment for 2 weeks, when mild fibrosis was shown. At the end of the experiment, the mice were sacrificed, and blood as well as liver samples were harvested. Although there was a total of 24 mice used overall, too little blood was collected during blood collection to be used for experiments so the number of experimental results displayed was n=8 in normal control group, n=6 in CCl4 group and n=7 in the CCl4 + givinostat group. All surgeries (blood and liver samples were harvested) were performed under sodium pentobarbital anesthesia (50 mg/kg), and then all mice were euthanized by 5% isoflurane (cat. no. HR135327; Hairui Chemical). Death of the mice was confirmed by checking whether their heartbeat had completely stopped and whether their pupils were dilated.

Liver tissues were fixed in 4% paraformaldehyde for 24 h at 37°C, dehydrated and paraffin embedded. The 3–4-mm thick liver sections were stained with hematoxylin and eosin (cat. no. G1005; Wuhan Servicebio Technology Co., Ltd.) at 37°C for 5 min and 15 sec, respectively and Sirius Red (cat. no. G1018; Wuhan Servicebio Technology Co., Ltd.). The liver tissue sections were deparaffinized using xylene (Wuhan Servicebio Technology Co., Ltd.), rehydrated with graded alcohol, treated with 0.3% endogenous peroxidase blocking solution (Sigma-Aldrich; Merck KGaA) for 10 min. Following high pressure heating retrieval (125°C and 103 kPa) and blocking with 10% normal goat serum (Wuhan Servicebio Technology Co., Ltd.) at 37°C for 30 min, the sections were incubated overnight at 4°C with the following primary antibodies (Wuhan Servicebio Technology Co., Ltd.): anti-α-SMA (cat. no. GB13044; 1:100) and anti-Col1α1 (cat. no. GB11022-1; 1:100). Following washing with PBS, goat anti-rabbit non-biotinylated reagents (cat. no. G1213; 1:1,000; Wuhan Servicebio Technology Co., Ltd.) were used to react with the primary antibody for 2 h at 37°C. Images were captured by observers who were blinded to the experimental conditions at 6–8 non-consecutive random fields under a light microscope (magnification, ×100), and were used to assess the histological changes using Image-Pro Plus 6.0 software (Media Cybernetics, Inc.). Representative views were displayed.

The human HSC LX-2 cell line and the rat HSC-T6 cell line were obtained from the FuHeng Cell Center, and were cultured in Dulbecco's modified Eagle's medium (DMEM cat. no. L110KJ; Shanghai BasalMedia Technologies Co., Ltd.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin and streptomycin (Gibco; Thermo Fisher Scientific, Inc.), at 37°C in a 95% air humidified atmosphere containing 5% CO₂. For stimulation, the cells were starved in serum-free DMEM for 24 h before being treated with recombinant human TGF-β1 (10 ng/ml; cat. no. 100-21C; PeproTech, Inc.) (21) and/or givinostat (900, 300 or 100 nM; cat. no. CSN16577; CSNpharm) for 24 h.

HSC LX-2 cells (5×10⁵ cells/well) were seeded in 96-well cell culture plates (Cellvis) overnight and sequentially stimulated with TGF-β1 and compounds (2 µM) or with TGF-β1 solely. Cells were harvested 24 h after TGF-β1 stimulation with Total RNA extraction reagent (Vazyme Biotech Co., Ltd.). Cytolysis was then subjected to RT-qPCR using TransScript^® Green One-Step qRT-PCR SuperMix (cat. no. AQ211; TransGen Biotech Co., Ltd.) (14). The compounds library containing 46 molecule probes targeting epigenetic proteins was screened to find small molecule compounds able to inhibit α-SMA expression.

Total RNA was isolated from flash-frozen mice liver tissues. Total RNA was isolated and purified using DNase I (Takara Bio, Inc.) and Dynabeads Oligo (dT) 25 (Thermo Fisher Scientific, Inc.). Subsequently, purified RNA (100 ng) was used for cDNA library construction, using the NEBNext Ultra™ RNA Library Prep kit for Illumina^® (cat. no. E7530L; New England BioLabs, Inc.). Sequencing data was collected on an Illumina HiSeq 2500 instrument. The RNA integrity number (RIN) value was used to assess the quality of the isolated RNAs. Only RNAs with RIN ≥7.0 were used for sequencing. The sequencing reads were located to mm10 by STAR 2.5 (22), and gene counting was quantified using featureCounts (Subread package 2.0.0) (23). The edgeR R package (24) was used for differential gene expression analysis. The P-value was adjusted using the Benjamini and Hochberg method (25), and a 5% FDR cutoff value and fold-change >1.5 were set as the threshold of the significant gene. The differentially expressed genes were further analyzed by gene-annotation enrichment analysis using The Database for Annotation, Visualization and Integrated Discovery 6.8 bioinformatics platform (26). Cytoscape was used for network analysis (27). The original data generated using high-throughput sequencing methodologies has been submitted to the GEO database (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE161981).

Msln siRNA (sense, 5′-GCCUUGCUUUCCAGAACAU-3′ and antisense, 5′-AUGUUCUGGAAAGCAAGGC-3′; and sense, 5′-GGACGUCCUAAAGCAUAAA-3′ and antisense, 5′-UUUAUGCUUUAGGACGUCC-3′), Dmkn siRNA (sense, 5′-GCAGAGACGAUCAGAACUA-3′ and antisense, 5′-UAGUUCUGAUCGUCUCUGC-3′; and sense, 5′-GCCUAUGGUGGGAAGUACU-3′ and antisense, 5′-AGUACUUCCCACCAUAGGC-3′) and Upk3b siRNA (sense, 5′-GCCCUACACACCACAGAUA-3′ and antisense, 5′-UAUCUGUGGUGUGUAGGGC-3′; and sense, 5′-GCUACAUGACCCACCACAU-3′ and antisense, 5′-AUGUGGUGGGUCAUGUAGC-3′) for human cells were synthesized by Shanghai GenePharma Co., Ltd. Transfection with siRNA against Msln, Upk3b or Dmkn, or with control siRNA (sense, 5′-UUCUCCGAACGUGUCACGU-3′ and antisense, 5′-ACGUGACACGUUCGGAGAA-3′) was performed according to the manufacturer's protocol. LX-2 cells were seeded in a 6-well plate at 60–80% confluence. Briefly, siRNA (20 µM, 1.5 µl) and 9 µl Lipofectamine^® RNAiMAX transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.) were mixed with 150 µl Opti MEM (cat. no. 31985070; Gibco; Thermo Fisher Scientific, Inc.). Next, diluted siRNA was added to diluted Lipofectamine RNAiMAX reagent and cultured for 5 min at room temperature. siRNA-lipid complex was added to cells for 6–8 h at 37°C. Subsequent experiments were performed 24 h after transfection.

Total RNA was extracted from HSC LX-2 cells, HSC-T6 cells or liver tissues using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocols. Total RNA (1,000 ng) was reverse transcribed into cDNA using a cDNA synthesis kit (Vazyme Biotech Co., Ltd.). The reverse transcription temperature protocol was as follows: 50°C for 15 min, followed by 80°C for 5 sec. RT-qPCR was performed using SYBR-Green (Vazyme Biotech Co., Ltd.). The thermocycling conditions were as follows: Pre-denaturation at 95°C for 10 min, 40 cycles of denaturation at 95°C for 15 sec, and annealing at 60°C for 30 sec, followed by extension at 72°C for 1 min. Subsequently, the expression values of mRNA were calculated using the 2^−ΔΔCq method (28) The expression of target genes was normalized to GAPDH expression. The primer sequences are shown in Table I.

The levels of serum aspartate aminotransferase (AST; cat. no. C010-3-1) and alanine aminotransferase (ALT; cat. no. C009-3-1) were measured using the corresponding kits (Nanjing Jiancheng Bioengineering Institute) and were assessed using a Hitachi 7020 automatic analyzer (Hitachi, Ltd.).

Total protein was extracted from HSC LX-2 cells, HSC-T6 cells or liver tissues with 1X SDS sample loading buffer (250 mM Tris HCL pH 6.8, 10% SDS, 30% glycerol, 5% β-mercaptoethanol and 0.02% bromophenol blue). Protein concentration was determined using a BCA kit (Thermo Fisher Scientific, Inc.). The lysates (25 µg/lane) were separated via SDS-PAGE on 6, 10 or 12% gels, and subsequently transferred to a nitrocellulose membrane (EMD Millipore). Following blocking with 5% milk at room temperature for 1 h, membranes were incubated with primary antibodies at 4°C overnight. Subsequently, the membranes were incubated with a HRP-conjugated goat anti-Rabbit IgG secondary antibody (1:10,000; cat. no. D110058; Sangon Biotech Co., Ltd.) for 1 h at room temperature. The bands were visualized using an enhanced chemiluminescence kit (Thermo Fisher Scientific, Inc.) with a ChemiScope 3400 mini imaging system (Clinx Science Instruments Co., Ltd.). Densitometry was performed for each group using ImageJ software (v1.50b; National Institutes of Health). The following primary antibodies were used: Anti-α-SMA (1:1,000; cat. no. 19245; Cell Signaling Technology, Inc.), anti-Col1α1 (1:1,000; cat. no. 72026; Cell Signaling Technology, Inc.) and anti-GAPDH (1:5,000; cat. no. 8884; Cell Signaling Technology, Inc.), which was used as the loading control.

All numerical results are expressed as the mean ± standard deviation, and represent data from a minimum of three independent experiments. Two-tailed unpaired t-test was used to analyze differences between two groups. One-way ANOVA or two-way ANOVA were used to compare the means of multiple groups followed by LSD post hoc test. All analyses were performed using GraphPad Prism 7.0 statistical software (GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.

Aiming to find potential candidate compounds that can inhibit HSC activation for the treatment of liver fibrosis, the present study established a high-throughput screening assay based on one-step RT-qPCR for detecting α-SMA mRNA expression as a readout for HSC activation, since activated HSCs are characterized by the expression of α-SMA (21). A library of small molecule inhibitors targeting epigenetic enzymes was screened, and givinostat was identified as the most potent compound that inhibited TGF-β1-induced α-SMA expression (Fig. 1A). Givinostat, a pan-HDACi that belongs to the hydroxamic acids family with HDAC type I and type II inhibitory activity, has been used in phase I/II clinical trials for the treatment of Duchenne muscular dystrophy. To validate the inhibitory effect of givinostat on HSC activation, the mRNA and protein expression levels of Col1α1 and α-SMA after givinostat treatment were examined. Givinostat dose-dependently inhibited Col1α1 and α-SMA mRNA expression in the presence of TGF-β1 (Fig. 1B). Consistently, western blotting also confirmed that givinostat inhibited Col1α1 and α-SMA protein expression levels in a dose-dependent manner (Fig. 1C). Based on these data, givinostat was identified as a potent inhibitor of HSC activation in vitro.

Whether givinostat could inhibit HSC activation in vivo and alleviate liver fibrosis, which has not been extensively studied, was further examined in the present study, which investigated its potential role in the treatment of chronic liver fibrotic diseases. A widely used mouse liver fibrosis model with i.p. repeated injection of CCl4 was used to evaluate the efficacy of givinostat for the treatment of hepatic injury and liver fibrosis (29). C57BL/6J mice were i.p. injected with CCl4 for 2 weeks; at that time there was already mild liver fibrosis (30). Mice were then treated with givinostat for the following 6 weeks after CCl4 treatment (Fig. 2A). As shown in Fig. 2B, persistent i.p. injection of CCl4 for 8 weeks caused hepatocyte steatosis (Fig. 2B middle panel), varying degrees of central venous wall thickening and collagen deposition (Fig. 2C middle panel), which are consistent with previous reports (31,32). The results indicated that givinostat treatment markedly ameliorated the extent of hepatocyte steatosis (Fig. 2B right panel). Since the deposition of ECM (primarily collagens) is the major characteristic of liver fibrosis, the collagen contents were evaluated based on Sirius Red staining. Images (n=6–8 images per group) showed that hepatic lobules maintained a normal physiological structure in the liver of control mice, while higher collagen accumulation was observed in the liver of CCl4-treated mice (P<0.0001; Fig. 2C). In the givinostat treatment group, the hepatic structure was improved and collagen was decreased (P<0.0001; Fig. 2C). There was a significant increase in the levels of serum ALT (n=6; 469±41.65; P<0.0001) and AST (n=6; 536.8±95.53; P<0.01) in CCl4-treated mice, whereas treatment with givinostat significantly decreased these serum markers of liver injury, ALT (n=6; 137.7±71.72; P<0.01) and AST (n=6; 156.5±71.44; P<0.01) (Fig. 2D). Furthermore, no systemic toxicity was observed at the dose of givinostat used in this experiment (data not shown). Thus, givinostat treatment significantly alleviated liver fibrosis and injury in vivo.

Quiescent HSCs are activated on account of liver injury and considered to be the primary source of ECM yielding during hepatic fibrosis. Since givinostat significantly inhibited HSC activation in vitro and alleviated liver fibrosis in vivo, the present study assessed whether it inhibited HSC activation in vivo. α-SMA and Col1α1 are the most abundant ECM proteins in liver tissue, and are markers of HSC activation (33,34). Thus, α-SMA or Col1α1-positive cells were detected by morphometric quantification to evaluate the accumulation of activated HSCs in mouse liver tissues. Immunohistochemical staining for α-SMA (n=6; 65.800±14.861; P<0.01) or Col1α1 (n=6; 8.215±1.069; P<0.0001) showed that positively stained brown-colored cells were notably increased in the liver tissues of mice treated with CCl4 (Fig. 3A middle panel). By contrast, immunohistochemical staining for α-SMA (n=6; 12.886±5.603; P<0.01) or Col1α1 (n=6; 3.140±859.9; P<0.01) in the liver tissue of givinostat-treated mice was much weaker compared with that of solvent-treated mice, almost at a level similar to that of the normal control group (Fig. 3A right panel). RT-qPCR analysis confirmed that the increase in mRNA expression of α-SMA and Col1α1 in the liver tissues of CCl4-challenged mice was significantly reduced by givinostat treatment (P<0.01; Fig. 3B). Consistently, western blot analysis further confirmed that the protein expression levels of α-SMA and Col1α1 in mouse liver tissues were increased in the CCl4-chanlleged group, and were reduced by givinostat treatment (P<0.05; Fig. 3C). Taken together, these results demonstrated that givinostat alleviated liver fibrosis and inhibited HSC activation in vivo.

To explore the mechanism underlying the improvement of hepatic fibrosis by givinostat treatment in CCl4-challenged mice, RNA-seq analysis was performed to compare the gene expression profile of liver tissues from CCl4-challenged mice with or without givinostat treatment. Differential gene expression analysis identified genes upregulated or downregulated in givinostat-treated group compared with their expression in the solvent group in CCl4-challenged mice. The most significantly regulated genes by givinostat treatment are shown in Fig. 4A. RT-qPCR analysis confirmed that givinostat treatment inhibited or upregulated the mRNA expression of these genes in liver tissues (Fig. 4B), which was consistent with the RNA-seq results.

The present study next examined whether givinostat regulated the transcription of these genes in vitro in HSCs. The mRNA expression of these genes was analyzed via RT-qPCR in HSC LX-2 cells stimulated by TGF-β1 in the absence or presence of givinostat treatment. The mRNA expression levels of Msln, Dmkn and Upk3b were also reduced in HSC LX-2 cells (Fig. 4C), which was consistent with the findings in liver tissues of mice treated with givinostat. As for the genes that were most notably upregulated by givinostat treatment in vivo, their mRNA expression was increased in vitro to a much lower extent (Fig. 4C). As shown in Fig. 4D, givinostat dose-dependently inhibited Msln, Dmkn and Upk3b mRNA expression in the presence of TGF-β1. These results confirmed that givinostat inhibited Msln, Dmkn and Upk3b gene expression in vivo and in vitro.

To further validate whether these givinostat-regulated genes play crucial roles during HSC activation, the effect of Msln, Dmkn and Upk3b depletion on TGF-β1-induced HSC activation was examined. RT-qPCR analysis confirmed the knockdown efficiency of Msln, Dmkn and Upk3b by siRNA, which resulted in >70% reduction in mRNA levels in HSC LX-2 cells compared with that of the siControl group (P<0.0001; Fig. 5A). RT-qPCR analysis showed that the knockdown of Msln, Dmkn and Upk3b inhibited the mRNA levels of α-SMA and Col1α1 in HSC-LX2 cells stimulated with TGF-β1 compared with the siControl + TGF-β1 group (P<0.0001; Fig. 5B and C). These results demonstrated that givinostat inhibited Msln, Dmkn and Upk3b expression, and these genes were shown to be crucial for HSC activation.