Materials and Methods

Chemicals and reagents

Dios (Fig. S1) was from the College of Pharmaceutical Sciences, Southwest University, P. R. China. The standard diet (SD, HD005) and high-fat diet (HFD, HD001+2% cholesterol, containing 45% fat and 2% cholesterol) were purchased from BiotechHD Co.Ltd. (Beijing, China). The aspartate aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) commercial kits were obtained from Nanjing Jiancheng Bioengineering Institute (Jiangsu, China) and the triglyceride (TG) commercial kit was purchased from Applygen Technologies Inc. (Beijing, China). The Oil Red O and Nile Red staining kits were obtained slarbio (Beijing, China). Dulbecco’s modified Eagle medium (DMEM) was purchased from KeyGen Biotech (Jiangsu, China), and fetal bovine serum (FBS) was obtained from Tianhang (Zhejiang, China). Palmitic acid (PA) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Fludarabine (HY-B0069, STAT1 inhibitor) was purchased from MedChemExpress (NJ, USA). siRNA for CXCL10 (human) and the control siRNA (human) were purchased from Tsingke Biological Technology (Beijing, China). The STAT1 overexpression plasmid STAT1-pcDNA3.1 and the control plasmid pcDNA3.1 were obtained from Pulateze (Hunan, China). Lipo6000™ transfection reagent was obtained from Beyotime Biotechnology (Shanghai, China). Antibodies against STAT1 (1:5000), CXCL10 (1:500), sterol regulatory element-binding proteins-1c (SREBP-1c) (1:1000), carbohydrate response element-binding protein (CHREBP) (1:1000), liver X receptor-alpha (LXRα) (1:1000), liver X receptor-beta (LXRβ) (1:1000), nuclear factor-κB p65 (p65) (1:1000), phosphorylated-p65 (p-p65) (1:1000), tumor necrosis factor-α (TNF-α) (1:1000), interleukin-6 (IL-6) (1:1000), β-actin (1:2000) and Lamin B (1:2500) were obtained from Proteintech Group, Inc. (Hubei, China). Antibody against STAT1Y701 (1:1000) was purchased from Affinity Biosciences (Jiangsu, China). Antibody against STAT1S727 (1:1000) was obtained from Beyotime Biotechnology (Shanghai, China).

Animals experiments

Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny, Browne, Cuthill, Emerson & Altman, 2010) and with the recommendations made by the British Journal of Pharmacology (Curtis et al., 2018). All animal experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The animal studies were performed after receiving approval of the Institutional Animal Care and Use Committee (IACUC) in Southwest University, P.R. China (IACUC approval No. yxy202007). All the animals were housed under specific pathogen-free (SPF) conditions at a controlled temperature (22-25 °C) and humidity (50 ± 5%), and alternating 12-hour light / dark cycles. The animals used in this study were housed in individually ventilated cages with natural soft sawdust as bedding up to five per cage and maintained on a normal chow diet with clean water ad libitum.
Eight-week-old male C57BL/6J mice (18-22g) were obtained from Beijing Huafukang Biotechnology Co., Ltd. (SCXK-Jing-2019-0008, Beijing, China). Mice were fed with either SD or HFD for 14 weeks to induce NASH model. Thereafter, the HFD-fed NASH mice were randomly divided into HFD group and HFD + Dios group (n = 5 per group) and administered intragastrically either Dios of 60 mg·kg-1·day or saline for four weeks (Yang et al., 2017). Mice were euthanized by cervical dislocation under anesthesia, and the livers were removed by two independent individuals who were blinded to the treatments and either snap-frozen or fixed in buffered 4% formalin for RNA-Seq and followed experiments.
To explore the potential mechanism of STAT1 in the beneficial effect of Dios in NASH, another animal experiment was carried out ( Eight-week-old Male C57BL/6J, SCXK-Jing-2019-0010) that mice were injected with fludarabine (Flu, a STAT1 inhibitor, 0.8 mg·kg-1·day) by the abdominal cavity two cycles for 5 days every 2 weeks in the whole experiment, and mice were fed with either SD or HFD for 14 weeks to induce NASH. Thereafter, the HFD-fed mice were randomly divided into different groups (n = 5 per group) including HFD, HFD + Dios, Flu-HFD, and Flu-HFD + Dios, and those mice were either administered intragastrically Dios (60 mg·kg-1·day) or saline for four weeks for four weeks. The diet intake of the mouse was monitored per day and the body weight was recorded weekly. After the last administration, all the mice euthanized by cervical dislocation under anesthesia, and the livers, kidneys, spleens, and abdominal fat pads were removed and weighed by two independent individuals who were blinded to the treatments. Serum was prepared by solidification and centrifugation (4 °C, 12000 × g, 10 min) and stored at -80 °C until the analysis of biochemical parameters. Liver samples were either snap-frozen or fixed in buffered 4% formalin for histological staining, hepatic triglyceride content measurement, quantitative real-time PCR, and western blot assay.

Cell culture and treatment

HepG2 cells were obtained from the Cell Bank at the Chinese Academy of Sciences (Shanghai, China) and cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin in a 5 % CO2humidified incubator at 37 °C. HepG2 cells were treated with different doses (0.10, 0.15, 0.20, 0.25, 0.30, 0.35 and 0.40 mM) of PA for 24 h to induce NASH cell model . To detect the effect of Dios, HepG2 cells were treated with or without Dios under the indicated concentrations combined with PA treatment for 24 h. To study the importance of STAT1 in Dios-mediated hepatoprotective effect, the STAT1 inhibitor fludarabine (10 μM) was used to pretreat HepG2 cells for 1 h before Dios and PA.

Transfection of overexpression plasmid and siRNA

HepG2 cells (3× 105 cells per well) were seeded in 6-well plates and incubated until the cells reached 70-80% confluence. Cells were then transfected with the STAT1 reporter vector STAT1-pcDNA3.1 (Fig. S5) , the control vector pcDNA3.1 and siCXCL10 (5’-GGUCUUUAGAAAAACUUGATT-3’, 3’-UCAAGUUUUUUCUAA-AGACCTT-5’) mixed in Lipo6000™ reagent according to the manufacturer’s guidelines. Then, the medium was exchanged with fresh complete medium after 6 h. After 24 h transfection, cells were incubated with 0.2 mM PA in the presence or absence of Dios (80 μM) for 24 h. After treatment, cells were harvested for further experiments.

RNA Sequencing (RNA-Seq)

Total RNA isolated from the livers of C57BL/6J mice was used to construct high throughput sequencing libraries using NEBNext® UltraTM RNA Library Prep Kit. High throughput RNA-sequencing was performed using a HiSeq 4000 instrument (Illumina) at Novogene (Beijing, China). Adaptor sequences and low-quality reads were initially filtered from the raw data. Then the remaining ones, called clean reads, were aligned to the reference genome of a mouse, using the HISAT2 v2.0.5 program. Subsequently, unigene expression was calculated as the FPKM (fragments per kilobases of exons for per million mapped reads) with featureCounts v1.5.0-p3. Differential expression genes (DEGs) of two groups were performed using the DESeq2 R package, and when the P-value was less than 0.05 and the log2 ratio was greater than 1 (two-fold change), the unigenes were considered to be differentially expressed.

GO and pathway enrichment analyses of DEGs

The enrichment analysis of GO (Gene Ontology) and KEGG (Kyoto encyclopedia of genes and genomes) pathway was performed for DEGs using David which is an online biological information database for annotation, visualization, and integrated discovery (Huang, Sherman & Lempicki, 2009). GO consists of biological processes (BP), cellular components (CC), and molecular functions (MF). P-value < 0.05 was chosen as the cut-off point for GO and KEGG analyses.

PPI network, module analysis, and hub genes identification

Protein-protein interaction (PPI) analysis of differentially expressed genes was based on the STRING database. The significant module in the PPI network was identified by molecular complex detection (MCODE). The parameters of DEGs clustering and scoring were set as follows: MCODE score ≥ 4, degree cut-off = 2, node score cut-off = 0.2, max depth = 100, and k-score = 2. Hub genes were identified using Cytohubba (a plug-in of Cytoscape software) filtered with the criterion of degrees > 10 criteria (each node had more than 10 interactions).

Biochemical assessment of serum and liver

Serum triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), alanine transaminase (ALT) and aspartate aminotransferase (AST) were quantified using enzymatic assays. The content of TC and TG of liver tissues and HepG2 cells were determined with an enzymatic assay kit and the protein concentrations were determined with a BCA protein assay kit. The results were expressed as mmol per g protein (mmol·g-1). All procedures were carried out according to the manufacturer’s instructions.

Histological assessment

Histologic examination of the liver was performed by hematoxylin-eosin staining. Liver samples were fixed briefly in 4% paraformaldehyde and embedded with paraffin. The samples were cut into 5 μm sections, deparaffinized, rehydrated, then stained with hematoxylin-eosin or mason trichrome. Images of liver sections were captured with light microscope. The amounts of steatosis, activity, and fibrosis were scored using the Fatty Liver Inhibition of Progression (FLIP) Algorithm (Bedossa & Consortium, 2014). The SAF score was defined as the total of scores for steatosis (S0-S3), activity including lobular inflammation (0-2) and hepatocellular ballooning (0-2) scores, and the fibrosis (F0-F4) score.

Immunofluorescence analysis

Liver samples embedded with paraffin were dewaxed, blocked, and then incubated with phosphorylated STAT1Tyr701 and phosphorylated STAT1Ser727 antibodies overnight at 4 °C. Slides were washed three times with PBS and then incubated with secondary antibody labeled fluorescence for 30 min at 37 °C. The nuclei were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI) for 5 min at room temperature. Images were captured using fluorescent microscopy.

Lipid content analysis

Hepatic lipid accumulation was detected by Oil Red O staining. Liver sample cryosections and hepatocytes were prepared for staining with pre-warmed Oil Red O working solution for 20 min, being rinsed with 60% isopropanol 3 times. And then counterstained with hematoxylin for 3 min, gently washed with 60% isopropanol. The images were captured using light microscopy. HepG2 cells were seeded in a 24-well plate and treated with 0.2 mM PA and indicated concentrations of Dios for 24 h. The cells were washed twice with PBS and fixed with 4% formaldehyde for 20 min and then stained with 1 μg·mL-1 Nile red for 30 min at room temperature. Lipid-bound Nile red fluorescence was detected using fluorescence microscopy.

Quantitative real-time polymerase chain reaction(qRT-PCR)

To verify the quantification of gene expression levels, qRT-PCR was performed as previously described (Zhou et al., 2018). Total RNA was extracted from livers and HepG2 cells using Trizol reagent (Sangon Biotech Co., Ltd., Shanghai, China), and RNA was reverse-transcribed using the Fastingking RT kit (TiangenBiotech CO. LTD., Beijing, China). Quantitative real-time PCR was carried out with SYBR Premix Plus (TiangenBiotech CO. LTD., Beijing, China) according to the manufacturer’s instructions, and the gene primers listed in Tab. 1 . Relative mRNA expression was determined by a comparative method (2-ΔΔCt) using GAPDH as a reference gene.

Western blotting analysis

The antibody-based procedures used in this study comply with the recommendations made by the British Journal of Pharmacology (Alexander et al., 2018). The proteins were extracted from mouse liver and HepG2 cells, then equal amounts of protein extract were denatured. After separated by electrophoresis in 12% SDS–PAGE, the protein samples were transferred onto Polyvinylidene Fluoride (PVDF) membranes. The membranes were blocked with 5% skimmed milk for 2 h, subsequently incubated with primary antibodies against STAT1 (1:5000), p-STAT1Y701(1:1000), p-STAT1S727 (1:1000), CXCL10 (1:500), p65 (1:1000), p-p65 (1:1000), IL-6 (1:1000), TNFα (1:1000), LXRα (1:1000), LXRβ (1:1000), CHREBP (1:1000), SREBP-1c (1:1000), β-actin (1:2000) and Lamin B (1:2500) overnight at 4 °C. After three times washing, the membranes were incubated with an HRP-conjugated secondary antibody at room temperature for 2 h. Antibody binding was detected by enhanced chemiluminescence detection kit (Affinity Biosciences, Jiangsu, China) and the digital images were analyzed by Image J. The relative protein levels were normalized to β-actin or Lamin B.

Data and statistical analysis

The data and statistical analysis comply with the recommendations on experimental design and analysis in British Journal of Pharmacology (Curtis et al., 2018). Randomization was used to assign samples to the experimental groups and treatment conditions for all in vivo studies. Data collection and acquisition of all in vivo and in vitro experimental paradigms were performed in a blinded manner. All results are expressed as the mean ± SEM (n = 5). Difference among groups was analyzed with one-way analysis of variance (ANOVA) followed by a Bonferroni post hoc analysis using GraphPad Prism 5 software. Post hoc tests were conducted only if F was significant, and there was no variance inhomogeneity. A value of p < 0.05 was considered statistically significant.