The A3AR agonist, Namodenoson, is a compound known generically as 2-chloro-N6-(3-iodobenzyl)-adenos ine-5′-N-methyl-uronamide, was synthesized for Can-Fite BioPharma by Albany Molecular Research Inc. A stock solution of 5.44 mg/ml was prepared in DMSO and further diluted in PBS for the in vivo and in vitro experiments.

C57BL/6 mice (14-day-pregnant, female) were obtained from Japan SLC, Inc. All animals used in this study were housed and cared for in accordance with the Japanese Pharmacological Society Guidelines for Animal Use. The animals were maintained in a SPF facility under controlled conditions of temperature (23±2°C), humidity (45±10%), lighting (12-h artificial light and dark cycle; light from 08:00 to 20:00) and air exchange. A high pressure was maintained in the experimental room to prevent the contamination of the facility. The animals were housed in TPX cages (CLEA Japan, Inc.) with a maximum of 4 mice per cage. Sterilized Paper-Clean (Japan SLC) was used for bedding and was replaced once a week. A sterilized solid high-fat diet (HFD) was provided ad libitum. NASH was induced in 24 male mice by a single subcutaneous injection of 200 µg streptozotocin (STZ, Sigma-Aldrich Co. LLC.) solution 2 days after birth. At 4 weeks of age, feeding with a HFD (57 kcal% fat, cat. no. HFD32, CLEA Japan, Inc.) was initiated till sacrifice. Namodenoson at 200 µg/kg (n=8) or equal amounts of DMSO (n=16) treatments were administered p.o., thrice daily between weeks 6-9.

Eleven-week-old male C57bl/6J male mice (n=21), from Harlan Laboratories, and housed in a barrier facility and received care according to the NIH guidelines. All procedures involving laboratory animals were evaluated and approved by The Hebrew University Institutional Animal Care and Use Committee and followed the guidelines for laboratory animal welfare.

The mice (n=16) received an intraperitoneal (i.p.) injection of CCl4 at 0.5 ml/kg body weight (1:10 v/v in corn oil) (Sigma). Namodenoson at the dose of 100 µg/kg body was administered by i.p. injection twice a week, commencing one day after the first dose of CCl4 (n=8) or the vehicle (n=8) (corn oil) twice a week for 6 weeks, as previously described (18). In addition, 5 naïve mice with no treatments have been included as well. The mice were sacrificed at 72 h after the final CCl4 injection. Whole livers and serum were collected for histological, cytological, biochemical and molecular analyses.

The animals of both models were sacrificed by exsangui-nation through direct cardiac puncture under ether anesthesia.

Blood samples were collected from C57bl/6J male mouse hearts at a volume of 0.5 ml. The samples were centrifuged for 5 min at 3,584 × g, at 4°C, and the supernatants (volume of 600 µl) were transferred to Eppendorf tubes. Serum samples of 32 µl were dropped onto the strip of the ALT (Reflotron) and analyzed by Reflovet^® plus (Roche).

Livers from the STAM and CCl4 model mice were harvested and fixed in 4% formaldehyde for 48 h. Paraffin slides of 3-5 µ thickness were stained as described below. The evaluation of the slides was performed using a microscope (Olympus BX60, serial no. 7D04032 at a magnification of ×10) and microscope Camera (Olympus DP73, serial no. OH05504). Ten random fields were observed for each slide. For the STAM model slides, hematoxylin and eosin (H&E) staining was used to analyze inflammation, steatosis and ballooning, combined as the NAFLD activity score (NAS) and calculated according to the Kleiner criteria (19) (Table I). To assess the adiponectin quantity, immunofluorescence mouse anti-adiponectin (1:50, cat. no. ab22554, Abcam) was used. The stained sections were examined under a fluorescence microscope (E600, Nikon) equipped with a Plan Fluor objective connected to a CCD camera (DMX1200F, Nikon). Digital images were collected and analyzed using Image-Pro Plus software 6.3. Images were assembled using Adobe Photoshop (Adobe Systems). In addition, immunohistological analysis utilizing rabbit anti-adiponectin/Acrp30 antibody (cat. no. NB100-6581; Novus Biologicals) at a dilution of 1:10 was performed to reproduce images. For the CCl4 model slides, the following analyses were performed: i) Sirius Red staining for fibrosis assessment; and ii) immunohistological analysis utilizing rabbit anti-adiponectin/Acrp30 antibody and rabbit anti leptin/OB antibody (cat. no. NBP1-59324; Novus Biologicals) at a 1:10 dilution, respectively, for the assessment of adiponectin and leptin.

For the assessment of inflammation, H&E staining was carried out. The sections were graded as follows: Grade 0, no inflammation; grade 1, mild/few inflammatory cells, 10-20 per X20 high-power field (HPF); grade 2, moderate/more inflammatory cells, 20-50 per X20 HPF; and grade 3, severe/many inflammatory cells, >50 per X20 HPF.

The liver sections were stained with Picro-Sirius Red solution (Waldeck). Morphometric analysis was performed using 'Image Pro Plus; version 6.3 (Media Cybernetics)'.

For adiponectin and leptin assessment, the sections were graded as follows: Grade 0, no positive reaction of IHC at all; grade 1, only few cells are immunopositive to the stain (<5 cells per a ×10 field); grade 2, very mild immune reaction (>5 and <15 cells per a ×10 field); grade 3, mild immune reaction (>15 and <25 cells per a ×10 field); grade 4, moderate immune reaction (>25 and <50 cells per a ×10 field); and grade 5, high immune reaction (>50 cells per a ×10 field).

RT-qPCR. Total cellular RNA was isolated from liver tissue, using 2 ml TRI Reagent (Bio LAB; Jerusalem, Israel) for each 3 cm of tissue. The samples were homogenized for 5 min at room temperature of 25°C. 0.2 ml of Chloroform (Bio LAB; Jerusalem, Israel) were added to each sample, incubated for 15 min at room temperature and centrifuged (1,400 rpm) for 15 min at 4°C. The supernatant in each sample was transferred to new Eppendorf, 0.5 ml of isopro-panol (Bio-Lab Ltd.) were added to the sample for 10 min at 25°C and centrifuged (32,256 × g) for 10 min at 4°C. The supernatants were removed and 1 ml of 75% ethanol was added to the pellet and centrifuged (12,600 × g) for 5 min at 4°C. The pellets were dried in air at room temperature for 15 min, 50 µl of DEPC were added, and the samples were heated for 10 min at 55°C. The preparation of cDNA was performed using High Capacity cDNA Isolation kit (R&D Systems). RT-qPCR was performed for the quantification of the expression of the gene that encoded α-SMA (Rhenium Assay ID: Mm00725412s1), compared to GAPDH (Rhenium Assay ID: Mm99999915g1) as a housekeeping gene using TaqMan Fast Advanced Master Mix (Applied Biosystems). Relative gene expression data were analyzed using the 2^−ΔΔCq method (20).

For the LX2 cells, total RNA was extracted using TRI-Reagent (Sigma). RNA (500 ng) was reverse transcribed using the high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific) according to the manufacturer's instructions. RT-qPCR was performed for the quantification gene expression of the encoded α-SMA, compared to β-actin as a housekeeping gene using TaqMan Fast Advanced Master Mix (Applied Biosystems). For α-SMA, the primers forward. 5′-ACT GGG ACG ACA TGG AAA AG-3′ and reverse. 5′-TAC ATG GCT GGG ACA TTG AA-3′ were added. The PCR reaction was performed as follows: Heating to 94°C for 2 min, 25 cycles of 94°C for 15 sec, 56°C for 45 sec, and 68°C for 2 min. For the amplification of human β-actin, the primers forward. 5′-TGG GAA TGG GTC AGA AGG ACT-3′ and reverse. 5′-TTT CAC GGT TGG CCT TAG GGT-3′ were used. The PCR condition included heating to 94°C for 2 min, 25 cycles of 94°C for 30 sec, 56°C for 1 min and 30 sec, and 72°C for 45 sec. The PCR products were electrophoresed on 2% agarose gels, stained with ethidium bromide and visualized with UV illumination. Total Lab v1.11 software was used for quantification.

LX2 human hepatic stellate cells (HSCs; ATCC) were grown in DMEM containing penicillin (10 units/ml), streptomycin (10 µg/ml), L-glutamine (2 mM) and 10% fetal bovine serum (FBS). The cells were maintained in T-75 flasks at 37°C in a 5% CO₂ incubator and transferred to a freshly prepared medium twice weekly. For all experiments, serum-starved cells were used. FBS was omitted from the cultures for 18 h and the experiment was carried out on monolayers of cells in DMEM supplemented with 1% FBS in a 37°C, 5% in a CO₂ incubator.

³H-thymidine incorporation assay was used to evaluate cell growth. LX2 cells (5,000 cells/well) were incubated (37°C) with 10 nM Namodenoson in the absence or presence of the antagonist, MRS 1523 (5 nM) (cat. no. M1809; Sigma-Aldrich), in a 96-well plate for 48 h. Each well was pulsed with 1 µ Ci ³H-thymidine for the last 24 h. The cells were harvested and the ³H-thymidine uptake was determined in an LKB liquid scintillation counter (LKB). These experiments were repeated at least 4 times.

Protein extracts from liver tissue or the LX-2 cell line were utilized. The liver tissue was homogenized with an ice-cold RIPA lysis buffer (Sigma) with protease phosphatase inhibitor cocktail (Roche). The LX2 cells were rinsed with ice-cold PBS and transferred to ice-cold lysis buffer (TNN buffer, 50 mM Tris buffer pH 7.5, 150 mM NaCl, NP 40 0.5% for 20 min). Cell debris was removed by centrifugation for 10 min, at 7,500 × g, 4°C. The supernatant was utilized for western blot analysis. Protein concentrations were determined using the NanoDrop spectrophotometer (Thermo Fisher Scientific). Equal amounts of the sample (50 µg) were separated by SDS-PAGE, using 4-12% polyacrylamide gels. The resolved proteins were then electroblotted onto nitrocellulose membranes (Pall Corp.). The membranes were then blocked with 5% bovine serum albumin and incubated with the desired primary antibody [A3AR, sc-13938; PI3K, sc-1637; glycogen synthase kinase 3β (GSK-3β), sc-9166; β-catenin, sc-7963; cyclin D1, sc-8396; NF-κB, sc-372; lymphoid enhancer-binding factor 1 (LEF-1), sc-374522; α-SMA, sc-32251; and β-actin, sc-47778; dilution 1:1,000; all from Santa Cruz Biotechnology] for 24 h at 4°C. The blots were then washed and incubated with a secondary antibody (Abcam; mouse ab97020, rabbit ab97048) for 1 h at room temperature. Bands were recorded using the BCIP/NBT color development kit (Promega). Densitometry of protein expression was normalized against β-actin and expressed as a percentage of the control.

Statistical analyses for the comparison between 2 groups were performed using the Student's t-test. Significant differences between multiple groups were determined using one-way ANOVA followed by Tukey's post-hoc analysis. P-values <0.05 were considered to indicate statistically significant differences. Data are presented as the means ± SD. The Chi-squared test was used for the assessment of ascites. For both analyses, P-values <0.05 were considered indicate statistically significant differences.

A representative photomicrograph of H&E-stained liver sections is depicted in Fig. 1A, demonstrating that in the vehicle-treated group, inflammatory cell infiltration, severe micro- and macrovesicular fat deposition and hepatocellular ballooning had occurred. Namodenoson treatment between weeks 6 to 9 resulted in a significant reduction in steatosis and an improvement in inflammation and ballooning, as demonstrated by morphometric analysis (P<0.05, P<0.05 and P<0.01, respectively), summing up to a significant decrease in the NAS score compared to the vehicle-treated group (Fig. 1B). Moreover, intense adiponectin staining was observed in liver sections derived from the Namodenoson-treated group compared to the vehicle treated one (Fig. 1C, left panel) and quantification is depicted in the right panel of Fig. 1C, utilizing immunofluorescence antibody (P<0.01).

In the CCl4 model, the serum ALT levels increased from 63.65±2.36 to 294.75±69.24 (P<0.01). Namodenoson significantly decreased the serum ALT levels, returning them to near normal levels (69.35±17.64; P<0.01; Fig. 2A). Namodenoson also reversed the ascites induced by CCl4 compared to the vehicle-treated group (P<0.05; Fig. 2B). Morphometric analysis of the liver sections derived from the CCL4-treated mice revealed an upregulation of inflammation, fibrosis, adiponectin and leptin, whereas a significant reduction was noted in the samples derived from the Namodenoson-treated mice (P<0.01; Fig. 2C).

Western blot analysis of the liver extracts derived from the animals in the CCl4 model revealed that Namodenoson treatment induced a decrease in the A3AR and PI3K expression levels followed by an increase in the GSK-3β expression level, with a subsequent reduction in the levels of β-catenin and cyclin D1, key proteins of the Wnt signaling pathway (Fig. 2D). The mRNA expression level of α-SMA was markedly decreased in the Namodenoson-treated group compared with the vehicle-treated group (P<0.01; Fig. 2E).

Namodenoson inhibited LX2 cell proliferation as was measured by ³H-thymidine incorporation. The highly specific A3AR antagonist, MRS 1523, counteracted the inhibitory effect of Namodenoson (P<0.01; Fig. 3A). Western blot analysis revealed that upstream, Namodenoson treatment induced the downregulation of A3AR, PI3K, NF-κB and α-SMA expression; these effects were reversed by MRS 1523, demonstrating the specificity of the response to the agonist (Fig. 3B). Downstream, GSK-3β was up-regulated whereas β-catenin, Lef-1 and cyclin D1 were down-regulated (Fig. 3C). Moreover, a decrease in α-SMA mRNA expression was noted (Fig. 3D). As can been seen, the protein profile was very similar to that of the liver extracts derived from the Namodenoson-treated CCL4 animals, supporting the notion that Namodenoson deregulates the Wnt/β-catenin pathway.