All animal procedures conducted in this study were approved by the Committee for Care and Use of Laboratory Animals at Seoul National University, according to the Guide for Animal Experiments edited by the Korean Academy for Medical Sciences. Male 8-week-old C57BL/6 mice were purchased from Orient Bio (Seoul, Korea). The mice were randomly assigned to four groups. The vehicle group (n=4) received saline, and the TAA group (n=4) received thioacetamide intraperitoneally three times a week for a total duration of 6 weeks. The injection doses of TAA (Sigma-Aldrich, St. Louis, MO, USA) were 50 mg/kg for the first injection, 100 mg/kg for the second injection and 150 mg/kg for third to sixth injections and 300 mg/kg for the rest of the injections. The TAA and HL156A co-treatment groups (n=5 each) received the same TAA as the TAA group and HL156A was intraperitoneally injected on the alternative days of TAA injection at a dose of either 2 mg/kg or 10 mg/kg. After 6 weeks of treatment, mice were euthanized via deep anesthesia followed by cardiac perfusion.

The paraffin sections were de-paraffinized and stained with hematoxyline and eosin (H&E) or Picro-Sirius Red (Abcam, Cambridge, MA, USA) according to manufacturer’s instructions. Immunohistochemistry was performed as previously described (19). Briefly, paraffin sections were subjected to antigen retrieval by incubating in Tris-EDTA buffer (10 mM Tris Base, 1 mM EDTA, 0.05% Tween-20, pH 9.0) for 40 min at 95°C. After blocking with 5% normal donkey serum (Sigma-Aldrich)/PBS solution, the sections were incubated overnight at 4°C with primary antibodies for α-smooth muscle actin (α-SMA) (1:200, Dako, San Diego, CA, USA), Desmin (1:200, Abcam), and Laminin (1:200, Sigma). After extensive washing in PBS/0.1% Tween-20 solution, the sections were treated with Alexa-488 or 546-conjugated secondary antibodies (1:750, Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature followed by counter staining with Hoechst (Sigma). Fluorescent images were taken under confocal microscope (Carl Zeiss AG, Oberkochen, Germany), and immuno-positive areas were quantified by ImageJ software.

HSC-T6 cells were kindly provided by Dr Scott L. Friedman of Icahn School of Medicine at Mount Sinai. HSC-T6 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; GenDEPOT, Barker, TX, USA) containing 10% fetal bovine serum (FBS, GenDEPOT), 100 U/ml penicillin and 100 μg/ml streptomycin (GenDEPOT). To investigate the effect of HL156A on HSC activation, cells were pre-treated with HL156A at indicated doses for 2 h and then treated with TGF-β1 (Peprotech, Rocky Hill, NJ, USA) for 16 h in serum-free condition. Raw264.7 cells were purchased from Korean Cell Line Bank (KCLB, Seoul, Korea) and maintained in DMEM containing 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. The cells were pre-treated with HL156A for 2 h at indicated doses and with LPS (Sigma) for another 4 h. Primary macrophages were obtained from cultures of bone marrow cells from femora and tibiae of 6 to 8-week-old male C57BL/6 mice. In brief, mouse total bone marrow cells were isolated by flushing the diaphysis of femora and tibiae with cold PBS and incubating overnight in cell culture dishes in α-modified Eagle medium (α-MEM; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS, to remove non-hematopoietic lineage cells. After discarding adherent cells, floating cells were further incubated in α-MEM supplemented with 10% FBS and macrophage colony-stimulating factor (M-CSF) (30 ng/ml, BioLegend, San Diego, CA, USA) in Petri dishes. After 6 days, non-adherent cells were removed and bone marrow macrophages (BMMs) were re-plated and used in the experiments.

Liver tissues were incubated in Trizol Reagent (Invitrogen) for 5 min followed by homogenization using Tissue Lyser II (Qiagen, Hilden, Germany). Total RNA was isolated according to the manufacturer’s instructions. Total RNA (2 μg) from each sample was reverse-transcribed with Moloney murine leukemia virus (MMLV) reverse transcriptase (Promega Corp., Madison, WI, USA). Quantitative real-time PCR was then performed using StepOnePlus real-time PCR system (Applied Biosystems, Foster city, CA, USA) with RealHelix qPCR kit (NanoHelix, Seoul, Korea). The relative mRNA levels were normalized using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal control. Primer sequences used for PCR are summarized in Table I.

Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer containing 25 mM Tris pH 7.4, 150 mM NaCl, 5 mM MgCl₂, 0.5% NP-40, phosphatase inhibitor cocktail (Sigma) and proteinase inhibitor cocktail (Calbiochem, Billerica, MA, USA). Protein concentrations were determined using a bicinchoninic acid (BCA) Assay kit (Thermo). Lysates (20 to 30 μg) were resolved on poly acrylamide gel and then immunoblotted as previously described (20). The following antibodies were used; mouse anti-α-SMA (Dako, Glostrup, Denmark), rabbit anti-vinculin (Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-phosphoAMPK Thr172 (Cell Signaling Technology, Beverly, MA, USA), and rabbit anti-phosphoSmad2 (Cell Signaling Technology).

NO assay was carried out in Raw264.7 cells as previously described (20). In brief, sub-confluent cells were first treated with HL156A for 2 h and then incubated in the presence or absence of lipopolysaccharide (LPS) (100 ng/ml) for 24 h. The conditioned medium was then collected and mixed with the same volume of Griess reagent (1:1 mixture of 1% sulfanilamide in 30% acetate and 0.1% N-1-aphthylethylenediamine dihydrochloride in 60% acetate) at room temperature for 10 min. The absorbance of the incubated samples was measured by using microplate reader at 540 nm. The concentration of nitrite in each sample was calculated based on a standard curve built with known concentrations of sodium nitrite.

Cells were plated on 96-well plates and treated with serial doses of HL156A for 24 h to investigate its effect on cell viability. Relative cell viability was measured using CellTiter 96^® AQueous One Solution Cell Proliferation Assay System (Promega Corp.) according to manufacturer’s protocol.

HL156A is a derivative of phenyl biguanide, which is designed and synthesized by Hanall Biopharma Inc. (Seoul, Korea). The detailed procedure of HL156A synthesis was described in a previous study (18).

Data were expressed as mean ± SEM. One-way ANOVA followed by Tukey’s test or two-tailed Student’s t-test was used for statistical analysis. Differences were considered significant at a P-value <0.05.

TAA is one of the common hepatotoxins used in experimental liver fibrosis that causes centrilobular fibrosis (21). Since TAA-induced liver fibrosis is reversible by the withdrawal of TAA exposure, mice were treated with HL156A and TAA together to investigate the anti-fibrotic effect of HL156A in liver. Low (2 mg/kg) or high (10 mg/kg) dose of treatment was decided based on a previous study done in rodents (18). Repeated TAA injection for a total duration of 6 weeks resulted in inflammation and alteration of liver histology (Fig. 1B, arrows in upper row). Massive ECM deposition was also obvious in TAA group as revealed by Sirius Red staining (Fig. 1B, lower row). Co-treatment with low dose HL156A slightly reduced ECM deposition but the difference relative to TAA alone group was not statistically significant. High dose of HL156A, however, significantly reduced TAA-induced ECM deposition by approximately 40% when compared to TAA group (Fig. 1B and C).

To analyze the alterations in gene expression caused by HL156A, RNA was extracted from liver tissues of each experimental group and subjected to RT-qPCR. Among the several ECM components, the mRNA expressions of col1a1 and col3a1 were drastically increased by up to 10-fold by TAA. Co-treatment with HL156A reduced col3a1 levels by up to 60% and those of col1a1 by up to 30% at either 2 mg/kg or 10 mg/kg dose (Fig. 2A and B). Of note, the mRNA level of tgf-β1, a potent profibrogenic factor was also significantly decreased by HL156A co-treatment (Fig. 2C).

One major feature of hepatic fibrosis is the activation of hepatic stellate cells, which is characterized by the expression of myofibroblast markers such as α-SMA and desmin. To address cellular events regulated by HL156A, we analyzed liver histology using myofibroblast markers. As shown earlier, TAA administration led to upregulation of α-SMA- as well as desmin-positive cell populations (Fig. 3A). HL156A significantly and dose-dependently diminished both α-SMA and desmin-positive immunoreactivity (Fig. 3A–C). These results indicate that HL156A inhibits HSC activation, which is a critical step in hepatic fibrogenesis.

Several lines of evidence revealed that liver sinusoidal endothelium underwent vascular remodeling upon fibrogenesis (6,22). The loss of liver sinusoid characteristics, referred to as capillarization, is known to accelerate HSC activation (7). As previously reported (23), we observed an increase in laminin-positive area in close proximity to fibrosis area in the liver sections of TAA treated animals (Fig. 3A). High dose of HL156A (10 mg/kg) significantly reduced laminin area while there was no significant reduction in the laminin immunoreactivity at low doses of HL156A (2 mg/kg).

To further study the anti-fibrotic effect of HL156A in vitro, we took advantage of the rat HSC cell line HSC-T6. MTS assay was carried out to determine proper doses of HL156A without cytotoxicity. HSC-T6 cells were treated with serial doses of HL156A ranging from 1 to 100 μM for 24 h and relative cell viabilities were measured. As shown in Fig. 4A, no significant cytotoxicity was observed up to 20 μM while HL156A concentrations over 50 μM showed some cytotoxicity. Preliminary experiments revealed that there is no or limited effect of HL156A on TGF-β1-induced HSC activation at concentrations <10 μM (data not shown). The treatment of HSC-T6 cells with TGF-β1 resulted in induction of α-SMA; simultaneous treatment with HL156A attenuated TGF-β1-mediated α-SMA induction at doses of 10 to 20 μM (Fig. 4B). Since HL156A is a new AMPK activator, we next investigated whether or not the inhibitory effect of HL156A on HSC activation involved AMPK activation. Treatment with HL156A promoted AMPK phosphorylation in a dose-dependent manner, whereas Smad phosphorylation by TGF-β1 was not affected by HL156A (Fig. 4C). Moreover, the inhibitory effect of HL156A on α-SMA expression was reversed by co-treatment with the AMPK inhibitor, compound C (Fig. D). These data revealed that HL156A has an inhibitory effect on HSC activation, probably via the stimulation of AMPK signaling.

Inflammation is closely associated with liver fibrosis (3) and there are several studies reporting protective effects of AMPK signaling in inflammation (11,16). To examine the anti-inflammatory effect of HL156A, LPS, a potent proinflammatory agent was added to Raw264.7 cell culture, which is a widely used mouse macrophage cell line. As expected, LPS treatment of Raw264.7 cells increased the production and release of NO by up to 60-fold. This LPS-induced NO release was diminished by HL156A in a dose-dependent manner (Fig. 5A). We next examined if HL156A was capable of regulating proinflammatory cytokines. Raw264.7 cells were treated with LPS for 4 h with or without HL156A, and the relative mRNA levels of proinflammatory cytokines were analyzed. A single treatment of inactivated Raw264.7 cells with LPS resulted in up to 350-fold increase in IL-6 and 25,000-fold increase in IL-1β transcripts. However, HL156A decreased the levels of IL-6 and IL-1β transcripts by approximately 30 and 20%, respectively (Fig. 5B and C). The inhibitory effect of HL156A on LPS-induced inflammation was further confirmed in primary macrophages, which were isolated and differentiated from mouse bone marrow. HL156A reduced the levels of both IL-6 and IL-1β mRNA in primary macrophages also and more efficiently than in Raw264.7 cells (Fig. 5D and E). Together, these results demonstrate that HL156A inhibits inflammation, which is probably responsible for its anti-fibrotic activity, at least in part.