Experiments were performed according to the guidelines of the Animal Care and Use Committee of the Hebrew University (NIH approval number OPRR-A01-5011). Male C57BL/6JOlaHsd mice, aged 4 weeks for NASH experiments and 6 weeks for the CCl₄ fibrosis model (Harlan; Ein Kerem, Israel), were housed (five per cage), in a pathogen-free unit under controlled 12-h light/12-h dark cycle and an ambient temperature of 21 ± 1°C and humidity 40%–50%. The cages contained Teklad Sani-chips (ENVIGO) bedding and two 2" small play tunnels for environmental enrichment. Male mice were selected because they develop a more severe form of the disease than females and have lower antioxidant enzymes (23).

The normal diet (ND) consisted of Teklad 2918SC radiated pellets (ENVIGO) containing 13.9% kcal from fat, 62.9% from carbohydrates, and 23.1% from protein. The modified WD (Envigo-Teklad TD.150235) had 50.5% kcal fat [trans-fat (12% of fatty acids) and saturated fat (50% of fatty acids)], 38% carbohydrates and 11.5% protein, 20% sucrose, 10% fructose, and 1.25% cholesterol with reduced choline (900 mg/kg). This diet, modified from the WD described in Farrell et al. (24), caused hepatic steatosis in mice after 1 month ( Figure S1B ) and inflammation and fibrosis within 4 months.

The mice were maintained for 2 months on WD (n = 30) or ND (n = 15) and weighed twice weekly. Then, under ketamine 100 mg/kg/xylazine 10 mg/kg anesthesia, they were implanted with mps delivering saline, or AN1284 (1 or 5 mg/kg/day)/month (ND n = 5/dose) (WD n = 10/dose) for the next 2 months ( Figure S1A ). A new pump was implanted under anesthesia in the second month. In a previous study, there were no significant differences in the effects of 2.5 and 5 mg/kg/day of AN1284 on the parameters measured in diabetic mice (22). Therefore, in the current study, we administered 1 and 5 mg/kg/day. At the end of the experiment, blood was collected by cardiac puncture under ketamine/xylazine anesthesia, and the livers were excised, weighed, and prepared for histological, cytological, biochemical, and molecular analyses.

Mice (n = 25) that were fed with ND were injected i.p. with carbon tetrachloride (CCl₄) (0.5 mg/kg in corn oil) (Sigma), twice weekly for 7 weeks. Four controls were injected with saline (1 ml/kg). Four weeks later, five mice injected with CCl₄ were sacrificed and the livers were examined to confirm the presence of fibrosis. The remaining eight mice were given saline by s.c. injection and seven others were implanted with mps delivering AN1284 (1 mg/kg/day) for 3 weeks.

While the current study was in progress, we completed an examination of the pharmacokinetics and metabolism of AN1284 in mice. Peak drug concentrations were similar in plasma and liver after s.c. injection, but nearly 50-fold higher in the liver when the compound was given orally (25). This suggested that oral administration should enable AN1284 to reduce hepatic damage. Therefore, AN1284 (1 mg/kg/day) was given to eight mice (four per cage) for 3 weeks via the drinking fluid, 4 weeks after they had developed fibrosis induced by CCl₄ injections. Ten others received normal drinking fluid. They were weighed once weekly, their fluid intake was measured twice weekly, and the concentration of AN1284 in the fluid was adjusted accordingly. Seven weeks after commencement of the CCl₄ injections, the mice were processed for histological and biochemical analyses as described below.

The livers were extracted as described in Ref (26). and their triglyceride content was determined using the Cobas C-111 bio-analyzer (Roche, Switzerland), normalized to wet tissue weight. Plasma ALT was measured by Reflotron chemical blood analyzer (Roche Diagnostics, Mannheim, Germany). Frozen liver was placed in an embedding medium and used for the measurement of hepatic fat content by Oil Red O (ORO) staining. The rest of the liver was fixed for 24 h in 4% formaldehyde solution (Bio-Heart Ltd., Jerusalem, Israel), induced in 70% ethanol and embedded in paraffin, cut into 5-µm slices, and stained with hematoxylin and eosin (H&E) for general damage. Fibrosis was assessed with Sirius Red (SR) (Sigma, 365548), collagen 4 (Col4) (Abcam, ab236640), and immunohistochemical staining with primary antibodies against α-SMA (Sigma, A2547). Antibodies against Ly6B (Bio-Rad, MCA771) were used for neutrophils and natural killer cells, F4/80 for macrophages (Bio-Rad, MCA497), CD3 for T cells (Bio-Rad, MCA1477), CD45R for B cells (Santa Cruz, sc-19597), CD36 (Abcam, ab133625), and iNOS (Abcam, ab3523). Histopathological analysis was performed by a light microscope using the program Cellsens Entry (Olympus, Japan). Macrophages, ORO, α-SMA, and SR were quantified in 12 random images at ×40 magnification. Using the ImageJ software, the colored area was calculated, normalized, and expressed as a percentage of the whole picture.

RNA was extracted from snap-frozen liver tissues (miRNeasy Micro Kit, Qiagen), from six samples/group. Its quantity and integrity were checked (Nanodrop, spectrophotometer) and reverse-transcribed into complementary DNA (qScript cDNA Synthesis Kit, QuantaBio). Genes were determined by PCR with an SYBR Green Kit and (QuantaBio) on the CFX384 Touch Real-Time PCR Detection System (Bio-Rad). The relative expression of target genes was normalized by hydroxyl methyl bilane synthase expression as an internal control. The primer sequences used are listed in Table 1 .

For RNA-Seq analysis, an Illumina Hi-seq sequencer was used to measure the differences in global gene expression between the experimental groups. Each sample generated approximately 70 × 10⁶ reads at the length of 86 bases. Differential expression data of the whole transcriptome was subjected to Gene Set Enrichment Analysis (GSEA) with the corresponding human ortholog gene symbols. GSEA uses all differential expression data (cutoff independent) to determine whether a priori-defined set of genes show statistically significant, concordant differences between two biological states. The hallmark gene set collection from MSigDB (molecular signature database) was used for the analysis. For each comparison, all statistically significant, differentially expressed genes were subjected to pathway enrichment analysis using QIAGEN’s ingenuity pathway analysis (IPA, QIAGEN Redwood City, www.qiagen.com/ingenuity), GeneAnalytics and EnrichR, and functions/diseases enrichment analysis by IPA.

HUH7 human hepatoma cells were incubated for 24 h in medium containing BSA. To see whether AN1284 can reduce steatosis from an FFA by a direct action on liver cells, PA was added together with different concentrations of AN1284 for 24 h. Lipid content was quantified by ORO staining. Since RNA-seq analysis suggested that AN1284 could act via the aryl hydrocarbon receptor (AhR), we measured the effect of AN1284 on the nuclear translocation of AhR, by immunofluorescence intensity, 15 min after its addition to the cells. We used a specific antibody (Abcam, ab190797), analyzed its intensity with ImageJ, and normalized it to the control group. RT-qPCR was used to measure the target genes of AhR after 24 h: fatty acid synthase (FASN), SREBP-1c, acyl-CoA oxidase 1 (ACOX1), and cytochrome P450-1 (CYP1a1). siRNA for human AhR from TriFECTa Kit DsiRNA Duplex purchased from ITD was used to silence AhR. The reverse transfection of these siRNAs onto HUH7 cells was performed by means of TransIT-X2 Dynamic Delivery System (MC-MIR-6000, Mirus) according to the manufacturer’s instructions. To examine the effect of siRNA on AhR expression, total protein was extracted from cells 48–72 h after transfection, and AhR protein levels were measured using Western blot (WB) with primary AhR antibody (Abcam, ab190797). The siRNA-transfected cells were treated with AN1284 and analyzed.

Liver samples were homogenized (100 mg/ml) in phosphate buffered saline. Twenty microliters of internal standard (rivastigmine 750 ng/ml) and 20 µl of ultra-pure water were added. AN1284 and its oxidized metabolite, AN1422, were extracted and measured as described in Weitman et al. (25).

Studies were designed to generate groups of equal size whenever possible, and any variation in group size within an experiment was due to unexpected loss of an animal or sample for measurement. All statistical analyses were performed using GraphPad Prism 9.50 (GraphPad Software Inc., San Diego, CA, USA). Data were compared by the Kruskal–Wallis non-parametric method, followed by the Mann–Whitney post-hoc test if F achieved P < 0.05. Body weight changes over time were compared by a two-way repeated measures ANOVA using SPSS version 28. Data are expressed as the mean ± SD. A p-value of <0.05 was considered to be significant.

There were no significant differences in the hepatic concentrations of AN1284 after administration of 1 or 5 mg/kg/day (37.9 ± 9.7 and 51.4 ± 12.0 ng/g), respectively, but those of the indole metabolite, AN1422, were significantly higher after the 5 mg/kg/day dose (3.4 ± 1.1 vs. 9.4 ± 4.9 mg/kg).

During 4 months of feeding, mice on WD gained significantly more weight than those on the normal diet (p < 0.0001; Figure 1B ). There were no significant differences in the weight gain between the three groups of mice on the WD. At this time, livers of mice on the WD showed significant hepatic fat content as showed with ORO staining ( Figure S1B ). During the last 2 months after implantation of the mps, there was still a significant difference in weight gain between saline-treated mice on a WD and those on an ND. AN1284 only significantly reduced weight gain at a dose 5 mg/kg/day ( Figure 1B ). After 4 months, the livers of saline-treated mice fed a WD showed extensive cell ballooning, inflammation (H&E), and fat accumulation (ORO) ( Figure 1A ). AN1284 (1 and 5 mg/kg/day) significantly decreased liver weight ( Figures 1C, D ), lipid content ( Figure 1E ), triglycerides ( Figure 1F ), and serum ALT ( Figure 1G ), despite the fact that they remained on the WD throughout the entire period. AN1284 also reduced hepatic cell ballooning and inflammation ( Figure 1A ). Additionally, we checked whether AN1284 has any effect in mice fed a ND. Neither dose of AN1284 had any significant effect on body weight, liver weight, ALT, and oil red content.

Moderate pericellular fibrosis in the livers of mice on the WD was demonstrated by an increase in staining with SR and Col4 and by TGF-β1 mRNA levels ( Figures 2A-D ). AN1284 (1 and 5 mg/kg/day) decreased SR. TGF-β1 mRNA was significantly reduced by 1 and 5 mg/kg/day but Col4 was significantly reduced only by a dose of 1 mg/kg/day. Since the degree of fibrosis was only moderate in the mice on WD, we performed additional experiments to assess the effect of AN1284 in mice on ND in which liver fibrosis was induced by injections of CCl₄ during a 7-week period. Fibrosis, assessed by SR and α-SMA staining, was already present at 4 weeks ( Figure 2E ). SR intensity increased significantly by 7 weeks. AN1284 (1 mg/kg/day) given by mps or orally, started after 4 weeks of CCl₄ injections when fibrosis was clearly present, decreased the levels of SR, but α-SMA was only reduced significantly after oral administration. The results indicate that AN1284 is able to halt the progression of liver fibrosis ( Figures 2E-G ).

We used RNA-Seq analysis to elucidate the influence of AN1284 on WD-induced hepatic gene expression profile. This enabled us to identify the canonical pathways altered by both the WD and drug treatment and to assess the differences in global gene expression between groups. Principal component analysis (PCA) showed that the six experimental groups could clearly be separated by the first two principal components (PC1 and PC2; Figure 3A ). Compared to a ND, the WD significantly changed the expression of 4,600 genes [with a Base Mean (BM) >150]. Those most changed by the WD and reversed by AN1284 are shown in Figure 3B . IPA and GSEA also revealed the top 20 pathways significantly altered by the diet that are associated with liver diseases ( Figure 3C ). The WD strongly activated pathways of hepatic steatosis and fibrosis and those encoding inflammation, oxidative stress, liver damage, and liver necrosis. All were significantly altered by AN1284 treatment, together with liver metabolism and elevation of the (FXR)/retinoid X receptor (RXR) and liver X (LXR) receptors ( Figures 3D, E ). The xenobiotic metabolism and AhR pathways were also significantly altered by AN1284. IPA prediction of the up- or downstream regulators by AN1284 ( Supplementary Table 1 ) indicated a role of several nuclear receptors (AhR, RXR, LXR, CAR, and FXR) and the inhibition of several cytokines and growth factors (i.e., TGF-β, TNF-α, IL-1β, and FGF). IPA pathways analysis suggested a decrease for AhR and an elevation of RXR and LXR ( Figures S2–S4 ).

In recent years, the role of nuclear receptors in liver steatosis and NASH has been investigated. While some of them were initially characterized as xenobiotic receptors, subsequent observations have pointed to their equally important metabolic functions (27, 28). FXR and LXR control metabolic processes abundantly expressed in the liver. IPA and GSEAs indicated that AN1284 treatment activated the FXR/RXR pathway with a p-value of 20.7 and the LXR/RXR pathway with a p-value of 15.6 ( Figure 3D , S2 , S3 ). This was verified by WB analysis, which showed that levels of hepatic RXRα protein increased by the diet were further elevated by AN1284 ( Figures 4A, B ). Although RXRα protein levels did not change in the liver of mice, 7 weeks after CCl₄ injections, they were greatly increased by both routes of AN1284 administration ( Figures 4C, D ). The WD also increased the percent area of fatty acid translocase (CD36)-positive cells ( Figures 4E, F ) and hepatic mRNA levels of FASN ( Figure 4G ) as suggested from RNA-Seq results ( Figure S2 ). AN1284 (1 and 5 mg/kg/day) significantly decreased gene expression of CD36, ACC, and FASN.

Hepatic inflammation plays an important role in the progression of NASH. Since the AhR is involved in many inflammatory responses, including suppression of cytokine release in LPS activated macrophages (28), we examined whether AN1284 also influences hepatic inflammation. In the livers of saline-treated mice, the WD significantly increased the number of hepatic T cells (CD3), macrophages (F4/80), and B cells (CD45R), but not neutrophils (Ly6B; Figures 5A-E ). It also increased hepatic gene expression of CCL2 ( Figure 5F ), a marker of immune activation. AN1284 (1 mg/kg/day), but not 5 mg, increased the number of neutrophils and further increased that of macrophages, B cells, and T cells ( Figures 5A-E ). AN1284 depressed CCL2 gene expression ( Figure 5F ) and increased that of IL-10 ( Figure 5G ).

Previous studies indicated that AhR acts as a “double-edged sword” in the progression of NAFLD, depending on the specific ligand (29). In order to determine whether AN1284 can have a direct effect on liver cells, we used a HUH7- human hepatoma cell line. The addition of PA/BSA complex to HUH7 cells for 48 h increased fat content (p < 0.0001). This was significantly reduced by AN1284 (0.21, 2.1, and 6.3 ng/ml) ( Figures 6A, B ). The concentrations were in the range of those found in the liver of mice treated with 1 and 5 mg/kg/day. BSA alone had no effect on the measurements. Since the RNA-Seq results suggested AhR as an upstream regulator, we analyzed its nuclear translocation in the HUH7 cells incubated with PA, 15 min after the addition of AN1284, and found this to be increased by AN1284 (6.3 ng/ml) ( Figures 6C, D ), together with upregulation in the expression of AhR target gene CYP1a1 and also ACOX1 ( Figures 6E, F ) 24 h later. AN1284 also decreased SREBP-1c mRNA, the principal transcriptional regulator of FASN that was elevated by PA (p < 0.001, Figure 7E ). Similarly, FASN mRNA was decreased by AN1284 (2.1 and 6.3 ng/ml), opposing the increase caused by PA addition (p < 0.001; Figure 7F ). In order to confirm that AN1284 suppresses fat accumulation in HUH7 cells through AhR, we silenced the receptor by using siRNA. AN1284 no longer reduced lipid in cells pre-treated with siRNA ( Figures 7A, B ). AhR protein levels were substantially reduced in HUH7 cells treated with AhR siRNA ( Figures 7C, D ). When these cells were incubated with PA and pre-treated with AN1284 siRNA, the levels of SREBP-1 and FASN genes remained elevated ( Figures 7E, F ). We also checked if AN1284 directly elevated RXR-α in the hepatoma cells. No change was observed in its protein levels in the WB analysis ( Figures 7C, D ).