Abstract

   Peroxisome proliferator-activated receptor α/δ (PPAR α/δ), regulating
   glucolipid metabolism and immune inflammation, has been identified as
   an effective therapeutic target in non-alcoholic steatohepatitis
   (NASH). Dual PPAR α/δ agonist, such as GFT505 (also known as
   elafibranor), demonstrated potential therapeutic effect for NASH in
   clinical trials. To profile the regulatory network of PPAR α/δ agonist
   in NASH, the choline-deficient, L-amino acid-defined, high-fat diet
   (CDAHFD) induced NASH model was used to test the pharmacodynamics and
   transcriptome regulation of GFT505 in this study. The results showed
   that GFT505 ameliorated hepatic steatosis, inflammation and fibrosis in
   CDAHFD mice model. RNA-sequencing yielded 3995 up-regulated and 3576
   down-regulated genes with GFT505 treatment. And the most significant
   differentialy expressed genes involved in glucolipid metabolism (Pparα,
   Acox1, Cpt1b, Fabp4, Ehhadh, Fabp3), inflammation (Ccl6, Ccl9, Cxcl14)
   and fibrosis (Timp1, Lamc3, Timp2, Col3a1, Col1a2, Col1a1, Hapln4,
   Timp3, Pik3r5, Pdgfα, Pdgfβ, Tgfβ1, Tgfβ2) were confirmed by RT-qPCR.
   The down-regulated genes were enriched in cytokine-cytokine receptor
   interaction pathway and ECM-receptor interaction pathway, while the
   up-regulated genes were enriched in PPAR signaling pathway and fatty
   acid degradation pathway. This study provides clues and basis for
   further understanding on the mechanism of PPAR α/δ agonist on NASH.

Introduction

   Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disease
   usually accompanied by obesity, type 2 diabetes, metabolic syndrome and
   cardiovascular diseases [[42]1, [43]2]. NASH is a progressive disease
   from NAFLD, which characterized by steatosis, hepatocellular
   ballooning, lobular inflammation and fibrosis. Without any effective
   intervention, NASH could develop into cirrhosis and hepatocellular
   carcinoma [[44]3].

   Lipid toxicity results in the liver injury and hepatitis [[45]4].
   Persistent inflammatory injury over activates injury repair process,
   thus leading to hepatic fibrosis [[46]5]. Nuclear receptors PPARs are
   ligand activated transcription factors regulating lipid metabolism and
   inflammation. PPAR α, which is mainly expressed in liver, kidney, heart
   and skeletal muscle, modulates lipid metabolism by regulating fatty
   acid transportation and β-oxidation [[47]6]. Activating of PPAR α could
   decrease the lipid accumulation and oxidative stress in liver [[48]7].
   Similar to PPAR α, PPAR δ is also involved in lipoprotein metabolism
   and plays an important role in the anti-inflammatory effect. Study has
   showed that the PPAR δ agonist could improve hepatic steatosis in NASH
   [[49]8]. GFT505, a dual PPAR α/δ agonist, has been reported to be
   effective in clinical trials of NASH [[50]9]. A short-term (4–8 weeks)
   phase II study showed that GFT505 reduced the plasma triglyceride
   level, increased high-density lipoprotein-cholesterol (HDL-c) and
   reduced low-density lipoprotein- cholesterol (LDL-c) in prediabetic
   patients [[51]10]. Other studies in abdominal obese patient reported
   that GFT505 improved peripheral insulin sensitivity and significantly
   reduced the liver enzyme concentrations of AST and ALT [[52]11,
   [53]12]. These clinical studies demonstrated that GFT505 is a potential
   drug for NASH treatment by regulating lipid and glucose metabolism.

   In this study, in order to further understand the mechanism of the PPAR
   α/δ agonist on NASH, the pharmacodynamics and transcriptome regulation
   of GFT505 were evaluated in CDAHFD fed mouse NASH model. The effects of
   GFT505 on fatty acid metabolism, inflammation and fibrosis were
   determined by histological and immunohistochemical analysis. The
   differential gene expression regulated by GFT505 in NASH was analyzed
   by RNA-sequencing. Bioinformatics analysis was used to investigate the
   effects of GFT505 on biological processes and biological pathways. The
   representative different expressed genes (DEGs) were validated by
   RT-qPCR.

Materials and methods

Animals, drug and cell line

   C57BL/6J mice (male, 4-week-old) were obtained from the Vital-River
   Animal Ltd (Beijing, China). All experimental procedures were approved
   by the institutional animal care and treatment committee of West China
   Hospital in Sichuan University (approval no. 2019273A), according to
   the National Institutes of Health guide for the care and use of
   laboratory animals. Animals were housed in a specific pathogen-free
   environment and maintained on standard diet for 4 weeks at a
   temperature of 24 ± 1°C and 12/12 h light/dark cycles, with free access
   to food and water. Mice at 9 weeks of age were fed with normal diet or
   the CDAHFD diet (60 kcal% fat, Research Diets Inc, New Brunswick, New
   Jersey, USA, A06071302).

   Normal human hepatic cell line LO2 was obtained from the Chinese
   Academy of Sciences (Shanghai, China). The cells were cultured in
   RPMI-1640 medium (Gibco, CAT# C11875500BT) supplemented with 10% fetal
   bovine serum (ZETA, CAT# Z7185FBS-500), 100 U/mL
   penicillin/streptomycin (Gibco, CAT# 15140–122). GFT505 (Med Chem
   Express, Shanghai, China) were dissolved in the solvents (1%
   carboxymethyl cellulose with 0.1% tween 80) for animal experiments and
   in DMSO for cell experiments.

CDAHFD-fed mouse NASH model

   Mice were divided into five groups (10 mice in each group): Control
   group (mice fed on normal diet), vehicle group (mice fed on CDAHFD diet
   treated with vehicle), and GFT505 treatment groups (mice fed on CDAHFD
   diet and treated with 3 mpk, 10 mpk, 30 mpk GFT505). The GFT505
   administration started at the fifth week and ended at the twelfth week.
   The blood glucose was analyzed once a week before the end of the
   experiment, terminal blood sample was collected from the heart in
   non-fasted mice and used for serum biochemical analysis. Animals were
   anesthetized with 3% pentobarbital sodium by intraperitoneal injection.
   The terminal blood samples were obtained from the eyelids for
   biochemical analysis at the twelfth week and the tissues were collected
   for histological and immunohistochemical analysis. Liver tissues for
   RNA-seq were stored in liquid nitrogen before RNA extraction. Serum
   chollesterol levels detected by Cholesterol Gen.2 (CHOL2) kit (Roche,
   CAT# 03039773190). Serum TG levels detected by Triglycerides (TRIGL)
   kit (Roche, CAT# 06380115190). Serum AST levels detected by Aspartate
   Aminotransferase (ASTL) kit(Roche, CAT# 06380115190). Serum ALT levels
   detected by Alanine aminotransferase acc. to IFCC (ALTL) kit (Roche,
   CAT# 04718569190).

Histological and immunohistochemical assays

   Liver tissues were fixed in 10% neutral formalin, embedded in paraffin,
   and cut into 5 μm sections. Hematoxylin and eosin (H&E) staining was
   used to evaluate liver steatosis, inflammation. Hepatocellular
   steatosis, ballooning and inflammation of the specimens were scored
   according to the standards as described before [[54]13]. Hepatic
   fibrosis were evaluated by Sirius red staining and analyzed by Nano
   Zoomer Digital Pathology S210 and Image-Pro Plus 6.0. The Sirius Red
   staining positive areas were presented as the percentage of the total
   area of the specimen.

   For immunohistochemistry, liver sections were incubated with primary
   monoclonal antibodies against α-smooth muscle actin (Abcam, CAT#
   ab3471) and collagen I (Huaan Biotechnology, CAT# ET1607-53). The
   expression of α-SMA and collagen I in the liver tissue were measured
   and quantified by Image-Pro Plus 6.0.

Sample preparation, library construction and RNA-seq

   The total RNA extraction, library construction and sequencing of liver
   tissues (n = 3) were performed at the Beijing Novogene Corporation.
   Tiangen RNA prep Pure Plant Kit (Tiangen, CAT# DP441) was used to
   extract total liver RNA from the three mice of each group. The
   extracted RNA quantity and quality were verified by Qubit^® RNA Assay
   Kit (Life Technologies, CAT# [55]Q32852) in Qubit^® 2.0 Fluorometer
   (Life Technologies, CA, USA) and the RNA Nano 6000 Assay Kit (Agilent
   Technologies, CAT# 5067–1511) of the Bioanalyzer 2100 system (Agilent
   Technologies, CA, USA). The cDNA library construction and RNA
   sequencing were performed as described before [[56]13].

Differential gene expression and bioinformatics analysis

   Clean data were obtained from raw data by RNA-seq after removing reads
   containing adapter, ploy-N and with low quality. DEGs analysis was
   based on clean data. Bowtie v2.0.6 was used to build the indexes of the
   reference genome and TopHat v2.0.9 was used to align the paired-end
   clean reads and reference genome. HTSeq v0.6.1 was used to count the
   read numbers mapped of each gene. DESeq R package (1.10.1) was used to
   analyze the differential expressed genes between two groups. The
   p-values of each result were adjusted by the Benjamini and Hochberg’s
   approach to control the false discovery rate. The gene with an adjusted
   P-value < 0.05 was identified as DEG by DESeq. GO seq R package were
   used for the Gene Ontology (GO) analysis. p< 0.05 was identified as the
   significant enriched GO terms. KOBAS software was applied to the Kyoto
   encyclopedia of genes and genomes (KEGG) pathway enrichment analysis.

Quantitative real-time polymerase chain reaction assay (RT-qPCR)

   Total RNA of liver tissues of mice (n = 3) in each group were extracted
   using TRIzol (Invitrogen, CAT# R1100). RNA degradation and
   contamination were monitored on 1% agarose gels. RNA purity was checked
   using the Nano Photometer^® spectrophotometer (IMPLEN, CA, USA). RNA
   concentration was measured using Qubit^® RNA Assay Kit (Life
   Technologies, CAT# [57]Q32852) in Qubit^® 2.0 Flurometer (Life
   Technologies, CA, USA). RNA integrity was assessed using the RNA Nano
   6000 Assay Kit (Agilent Technologies, CAT# 5067–1511) of the
   Bioanalyzer 2100 system (Agilent Technologies, CA, USA). First-strand
   cDNA was synthesized using HiScriptII Q RT Supermix for qPCR (Vazyme,
   CAT# R223-01). Quantitative RT-PCR was carried out using Aceq QPCR SYBR
   Green Master Mix (Vazyme, CAT# Q111-02/03). Both of them were carried
   out according to the instructions of manufacturer. The corresponding
   primer sequences were listed in [58]S5 Table.

Lipid accumulation cell model

   Human LO2 cell line were seeded into 6-well plates and maintained in
   RPMI 1640 (Gibco, CAT# C11875500BT) medium supplemented with 10% fetal
   bovine serum (ZETA, CAT# Z7185FBS-500) and 100 U/mL
   penicillin/streptomycin (Gibco, CAT# 15140–122) for 24 h. The LO2 cells
   were treated with oleic acid/palmitate mixture (2:1 M ratio, final
   concentration 0.8 mM) for 24 h. And then cells were exposed to 7.5 or
   15 μM GFT505. TG content (Nanjing jiancheng Biological Engineering
   institute, CAT# A110-1) and Oil red O staining (Sigma, CAT# O1391) were
   performed to evaluate the lipid accumulation according to the
   manufacturer´s instruction.

Statistics analysis

   The data of all experiment results were presented as mean ± SEM. The
   difference among groups was analyzed by Student’s t-test or one-way
   ANOVA followed by LSD multiple comparison analysis (GraphPad prism 6.0,
   La Jolla, CA). p< 0.05 was considered to be significant.

Results

GFT505 improved the liver metabolism in the CDAHFD-induced NASH mice model

   As shown in [59]Fig 1A and 1B, although there was no difference in body
   weight between the GFT505 treatment groups and vehicle group, the ratio
   of liver weight to body weight kept increasing in a dose-dependent
   manner. And treatment with GFT505 also increased the concentration of
   serum cholesterol ([60]Fig 1C), but had no effect on serum TG
   expression ([61]Fig 1D). Importantly, the concentrations of AST were
   decreased at the dosages of 10 and 30 mpk of GFT505 ([62]Fig 1E) and
   ALT were significantly reduced after treated with all the dosages of
   GFT505 (3, 10 and 30 mpk) ([63]Fig 1F).

Fig 1. Metabolic changes observed following long-term treatment with GFT505
in C57BL/6 mice.

   [64]Fig 1
   [65]Open in a new tab

   (A) Percentage change in body weight for 12 weeks. (B) Ratio of liver
   weight to body weight. (C) Serum CHO levels detected by Cholesterol
   Gen.2 (CHOL2) kit. (D) Serum TG levels detected by Triglycerides
   (TRIGL) kit. (E) Serum AST levels detected by Aspartate
   Aminotransferase (ASTL) kit. (F) Serum ALT levels detected by Alanine
   aminotransferase acc. to IFCC (ALTL) kit. Control, normal diet; Vehicle
   and GFT505 groups, CDAHFD diet. Statistic analysis were performed using
   one-way ANOVA; Values were means ± SEM; *p < 0.05, **p < 0.001,
   comparing with vehicle group; ^#p < 0.05, ^##p < 0.001, comparing with
   control group; n = 10.

GFT505 attenuated the liver steatosis, inflammation and fibrosis in NASH

   To determine the effects of GFT505 on liver steatosis, inflammation and
   fibrosis in NASH, hematoxylin and eosin (H&E) staining and Sirius red
   staining were applied. The results of H&E staining demonstrated that
   GFT505 inhibited the steatosis and inflammation of NASH in a
   dose-dependent manner ([66]Fig 2A). Sirius red staining showed that
   GFT505 attenuated the fibrosis in NASH mice model induced by CDAHFD
   ([67]Fig 2B). Pathological score of H&E staining results were presented
   in [68]Fig 2C and 2D. GFT505 of 10 and 30 mpk attenuated liver
   steatosis by 46% and 53%, respectively ([69]Fig 2C). And GFT505 at the
   doses of 3, 10 and 30 mpk suppressed the CDAHFD-induced inflammation by
   33%, 44% and 50% respectively ([70]Fig 2D). Furthermore, the
   pathological scores of Sirius red staining showed that GFT505
   suppressed fibrosis by 65% (10 mpk) and 58% (30 mpk) ([71]Fig 2E).
   RT-qPCR results showed that the CD45 (M1-macrophage marker) was higher
   in the vehicle group compared with the control group and the GFT505 (30
   mpk) group ([72]Fig 2F). The CD163 (M2-macrophage marker) was lower in
   the vehicle group compared with the control group and the GFT505 (30
   mpk) group ([73]Fig 2G). The effects of GFT505 on liver steatosis and
   fibrosis were further confirmed by Oil Red O staining and
   immunohistochemistry. Treating with 30 mpk GFT505 reduced lipid
   droplets accumulation ([74]Fig 3A) and the quantification was showed in
   [75]Fig 3C. Decreased protein concentrations of α-SMA ([76]Fig 3B and
   3D) and collagen I ([77]Fig 3B and 3E) were demonstrated after GFT505
   treatment.

Fig 2. GFT505 attenuated hepatic steatosis, inflammation and fibrosis in
CDAHFD-fed C57BL/6 mice.

   [78]Fig 2
   [79]Open in a new tab

   (A) Results of liver Hematoxylin and Eosin (H&E) staining. (B) Results
   of liver Sirius Red staining. (C, D) NASH/NAFLD Clinical Research
   Network scoring system and the scores in steatosis and inflammation.
   (E) Sirius red stained area quantification. (F) Relative mRNA
   expression of the CD45 by RT-qPCR. (G) Relative mRNA expression of the
   CD163 by RT-qPCR; Control, normal diet; Vehicle and GFT505 groups,
   CDAHFD diet; Statistic analysis were performed using one-way ANOVA;
   Values were means ± SEM; *p < 0.05, **p < 0.001, compared with vehicle
   group; ^#p < 0.05, ^##p < 0.001, comparing with control group; n = 10.

Fig 3. GFT505 attenuated hepatic lipid accumulation and the expression of
α-SMA and collagen I in CDAHFD-fed C57BL/6 mice.

   [80]Fig 3
   [81]Open in a new tab

   (A) Results of Oil red O staining. (B) Immunohistochemical analysis of
   α-SMA and collagen I in mice liver sections. (C) Oil red O stained area
   quantification. (D) α-SMA immunohistochemical area quantification. (E)
   The collagen I immunohistochemical area quantification. Control,
   standard diet; Vehicle and GFT505 groups, CDAHFD diet; Statistic
   analyses were performed using one-way ANOVA; Values were means ± SEM;
   *p < 0.05, **p < 0.001, compared with vehicle group; ^#p < 0.05, ^##p <
   0.001, compared with control group; n = 10.

GFT505 regulated the expression of genes involved in lipid metabolism,
inflammation and fibrosis

   Total RNA was extracted from the liver tissues of mice in the GFT505
   groups, vehicle group. There were 3995 up-regulated genes and 3576
   down-regulated genes of 7571 DEGs in GFT505 treatment group compared
   with vehicle group ([82]Fig 4A). To better understand the transcriptome
   regulation of GFT505 treatment in CDAHFD-induced NASH model, we further
   performed Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes
   and Genomes (KEGG) pathway analysis to classify DEGs into relevant
   functions. The results of ten most significant Biological Process by GO
   analysis were listed in [83]S1 Table. The lipid metabolism processes,
   such as fatty acid metabolic process and lipid biosynthetic process,
   were enriched. The results of the Reactom enrichment analysis is
   similar to GO analysis ([84]S2 Table). The top 10 enriched KEGG
   pathways were presented in [85]Fig 4B and listed in [86]S3 Table. Among
   which the enriched PPAR signaling pathway and Fatty acid degradation
   pathway were showed in [87]Fig 4C and [88]S1 Fig. Besides, the
   ECM-receptor interaction pathway ([89]Fig 4D) and Cytokine-cytokine
   receptor interaction pathway ([90]S2 Fig) were enriched as well. As
   shown in [91]S1 Fig, Ehhadh and Acaa2 were up-regulated in fatty acid
   degradation pathway. And the Cytokine-cytokine receptor interaction
   pathway genes involved in inflammation were down-regulated, such as
   Cxcl1, Cxcl2, Cxcl5, Cxcl4, Ccl21, Ccl22, Il6r, Il7r, Tnf and Ccr3. As
   for ECM-receptor interaction pathway, Collagen and Laminin were
   significantly down-regulated. Furthermore, heat map was made to present
   DEGs related to lipid metabolism, inflammation and fibrosis ([92]Fig
   5A), and RT-qPCR was performed to confirmed these related mRNA
   expressions ([93]Fig 5B and 5D). The changes of the related genes
   expression of lipid metabolism, inflammation reaction and fibrosis in
   RNA-SEQ or qRT-PCR were showed in [94]S4 Table. The primers sequences
   for RT-qPCR were showed in [95]S5 Table. In summary, GFT505 increased
   the expression of genes involved in lipid metabolism and decreased
   inflammation and fibrosis related gene expression in CDAHFD-induced
   NASH model.

Fig 4. Effect of GFT505 on the genes expression of PPAR signaling pathway and
ECM-receptor interaction pathway.

   [96]Fig 4
   [97]Open in a new tab

   (A) The differentially expressed genes in GFT505 (30mpk) group,
   compared with vehicle group. Red dots represent up-regulated DEGs, the
   green dots represent down-regulated DEGs, and blue dots represent
   non-DEGs. A total of 7571 unigenes were identified as DEGs (p < 0.005
   and log[2] [fold change] > 1) between treatment and vehicle group. (B)
   KEGG enrichment analysis of DEGs. Top 20 significantly enriched KEGG
   pathway. (C) DEGs enriched in “PPAR Signaling pathway”. (D), DEGs
   enriched in “ECM-Receptor interaction”. Vehicle and GFT505 group:
   CDAHFD diet. DEGs, Differentially Expressed Genes; KEGG, Kyoto
   Encyclopedia of Genes and Genomes; ECM, Extracellular matrix.

Fig 5. The different genes expression about lipid metabolism, inflammation
reaction, and fibrosis after GFT505 30 mpk treatment.

   [98]Fig 5
   [99]Open in a new tab

   (A) The hot map of lipid related-, inflammation related-, and fibrosis
   related-DEGs. (B, C, D) The mRNA expression of lipid metabolism
   related- and inflammation related-DEGs were confirmed by RT-qPCR.
   Vehicle and GFT505 group: CDAHFD diet. DEGs, Differentially Expressed
   Genes. Statistic analyses were performed using one-way ANOVA; Values
   were means ± SEM; **p < 0.0001, comparing with vehicle group; ^##p <
   0.0001, comparing with control group; n = 3.

GFT505 inhibited lipid accumulation in LO2 cell

   To further validate the effect of GFT505 treatment in lipid
   accumulation, the human normal liver cell LO2 was treated with palmitic
   and oleate acid for 24 h. After treating with palmitic and oleate acid,
   LO2 cell showed massive lipid droplets accumulation in vehicle group.
   The lipid accumulation was alleviated by GFT505 in a dose-dependent
   manner ([100]Fig 6A). The quantification of Oil red O staining was
   showed in [101]Fig 6B. The analysis of TG concentration was showed in
   [102]Fig 6C and the result was consistent with Oil red O staining. In
   conclusion, GFT505 treatment reduced lipid accumulation through LO2
   cell Oil red O staining and TG concentration analysis in vitro.

Fig 6. GFT505 reduced lipid accumulation in LO2 cell.

   [103]Fig 6
   [104]Open in a new tab

   (A) GFT505 reduced lipid accumulation in LO2 cell by Oil red O
   staining. (B) The quantification of Oil red O staining. (C) TG content
   was quantified by microplate reader in LO2 cell. Statistical analyses
   were performed using one-way ANOVA; Values were presented as means ±
   SEM; ****p < 0.0001, comparing with vehicle group; ^####p < 0.0001,
   comparing with control group; n = 3.

Discussion

   NASH is a progressive disease usually accompanied by metabolic
   syndrome, insulin resistance and glucose tolerance. The evaluation of
   therapeutic effects about drugs in NASH should include insulin
   resistance, steatosis, inflammation, oxidative stress, mitochondrial
   dysfunction and fibrosis. PPARs, a family of ligand-activated nuclear
   receptors, are highly expressed in the liver and adipose tissue
   regulating the lipid and glucose metabolism. Several specific
   subtype-selective PPARs agonists have been applied to reduce
   experimental fibrotic steatohepatitis [[105]14–[106]16]. The efficacy
   of PPAR α/δ dual agonist GFT505 had been evaluated in several animal
   models previously, including western diet-fed human APOE2 transgenic
   mice, MCD diet-fed db/db mice and CCL4-induced fibrosis rats [[107]9,
   [108]17]. In this study, we firstly evaluated the pharmacodynamics of
   PPAR α/δ agonist GFT505 and profile the regulatory network of GFT505 in
   the CDAHFD induced NASH model.

   Many diets induced NASH animal model were reported. They replicate
   different characteristic of human NASH [[109]18]. High-fat diet (HFD)
   model showed similar features with human NASH such as obesity, IR,
   steatosis, inflammation, but the hepatic pathologic degree is mild and
   takes longer than other diet models [[110]19, [111]20].
   Methionine-choline-deficient diet (MCD) induces steatosis, hepatic
   inflammation and fibrosis in a short feeding time [[112]18, [113]21].
   However, this model is usually not accompanied by obesity and
   peripheral IR. Moreover, the decreased body weight limits the use of
   MCD model [[114]22, [115]23]. The choline-deficient, L-amino
   acid-defined (CDAA) model is similar to MCD model except for more
   serious histological changes [[116]18]. The choline-deficient, L-amino
   acid-defined, high-fat diet (CDAHFD) model was established by the
   combination of CDAA and HFD models. CDAHFD mouse model can not only
   simulate human NASH disease to develop steatosis, inflammation and
   fibrosis in a short period, but also overcome the body weight loss
   [[117]24]. Therefore, the novel CDAHFD mouse model was selected in our
   study to investigate the PD and mechanism of PPAR α/δ dual agonist
   GFT505.

   In CDAHFD animal model, the levels of serum ALT and AST increased with
   hepatomegaly. Besides, serum levels of TG in vehicle group were lower
   than that in control group GFT505. The histology analysis on steatosis
   (H&E staining), fibrosis (SR staining) and α-SMA and collagen I
   staining (immunohistochemical analysis) of liver tissue demonstrated
   that GFT505 can alleviate steatosis, inflammation and fibrosis. In
   brief, PPAR α/δ agonist GFT505 improved liver function, prevented
   intrahepatic lipid accumulation and reduced liver inflammation and
   fibrosis in CDAHFD animal model.

   Furthermore, high-throughput comparative RNA-Seq analysis was used to
   evaluate the gene expression profiles after GFT505 treatment.
   High-throughput sequence and DEGs analysis showed that there were 3995
   up-regulated genes and 3576 down-regulated genes in GFT505 treatment
   group. Lipid metabolism related genes Cpt1b, Acox1, Ehhadh, Fabp3,
   Fabp4, Srebf1 Acaa2 and Pparα were up-regulated compared with the
   vehicle group. The down-regulation of Cpt1b and Acox1 genes could lead
   to lipid accumulation in liver, thus slowing down fatty acid synthesis
   and β-oxidation [[118]25, [119]26]. Down-regulation of Ehhadh gene
   expression decreased very low density lipoprotein (VLDL) transport rate
   [[120]27]. Pparα is involved in free fatty acid transport, oxidation,
   liver lipids and plasma lipoprotein metabolism [[121]28]. Fabp4 is
   highly expressed in adipocytes and also exists in macrophages and
   dendritic cells, which is related to diseases such as obesity and
   insulin resistance. A recent study confirmed that in Fabp4 knockout
   mice, the IKK (IκB kinase) and NF-κB (nuclear factor-κB) activities
   were inhibited and the accumulation of cholesterol, fatty acids and
   lipid droplets was reduced, showing the reduced inflammation [[122]29].
   GFT505 could upregulate the expression of Fabp4 gene, thus regulating
   fat metabolism and inflammation. The effect of changes in Fabp4 gene
   expression on CDAHFD model mice deserves further study. Changes in
   lipid metabolism related genes led to significant enrichment of fatty
   acid metabolic related processes and upregulation of fatty acid
   degradation pathway. Furthermore, the effect of GFT505 on ameliorating
   fat accumulation was verified on LO2 cell model in vitro. A short-term
   phase II clinical study showed that GFT505 reduced plasma triglyceride
   levels, increased high-density lipoprotein cholesterol levels, and
   decereased low-density lipoprotein cholesterol serum levels in
   prediabetic patients [[123]10]. Besides, inflammation-related genes
   such as Ly6e, Tlr9 and Il17d were also down-regulated. Among them, Ly6e
   (lymphocyte antigen 6 complex, locus E) plays an important role in
   regulating immunity, T cell physiological characteristics and tumor
   formation and the increased expression of Ly6e lead to the increased
   risk of human infection with the virus [[124]30]. Il17d plays a key
   role in the recruitment of human immune cells [[125]31]. Studies have
   reported that Tlr9 caused acute injury by activating IL-17A [[126]32].
   In addition, the expression of genes such as Ccl6 (C-C Motif Chemokine
   Receptor 6), Ccl9 (C-C Motif Chemokine Receptor 9), Cxcl14 (C-X-C Motif
   Chemokine Ligand 14) and Il21r (interleukin 21 receptor) were also
   significantly down-regulated after treatment with GFT505. This may
   contribute to the decreased inflammation. Due to the downregulation of
   inflammation-related genes expression, cytokine-cytokine receptor
   interaction pathway was also down-regulated. Furthermore,
   fibrosis-related genes Timp1, Lamc3 (Laminin Subunit Gamma3), Timp2,
   Col3a1, Col1a2, Col1a1, Hapln4 (Hyaluronan And Proteoglycan Link
   Protein 4), Timp3, Pik3r5, Pdgfα, Pdgfβ, Tgfβ1 and Tgfβ2 were
   significantly down-regulated, which caused the downregulation of
   ECM-receptor interaction pathway after GFT505 treatment.

   In summary, the PPAR α/δ dual agonist GFT505 showed efficacy on
   improving serum biochemistry and alleviating steatosis, inflammation
   and fibrosis of liver at both protein and gene expression levels in
   CDAHFD animal model. Lipid metabolism-related genes Cpt1b, Acox1,
   Ehhadh, Fabp3, Srebf1 and Acaa2, inflammation-related genes Ly6e, Tlr9,
   Il17d, Ccl6, Ccl9, Cxcl14 and Il21r, and fibrosis-related genes Timp1,
   Lamc3, Timp2, Col3a1, Col1a2, Col1a1, Hapln4, Timp3, Pik3r5, Pdgfα,
   Pdgfβ, Tgfβ1 and Tgfβ2 regulated by GFT505 are identified in vivo. This
   study further confirmed the effect of PPAR α/δ dual agonist in NASH,
   and also provided clues for further elucidating the mechanism of PPAR
   α/δ agonists in the treatment of NASH. However, not considering the
   effects of GFT505 on normal mice was a major limitation of our study
   design. We will further explore the influence of GFT505 on control
   group.

Supporting information

   S1 Fig. GFT505 Treatment up-regulates “fatty acid degradation” pathway
   related genes.

   (TIF)
   [127]Click here for additional data file.^ (14.4MB, tif)
   S2 Fig. GFT505 Treatment down-regulates “cytokine-cytokine receptor
   interaction” pathway related genes.

   (TIF)
   [128]Click here for additional data file.^ (4.8MB, tif)
   S1 Table. GFT505 treatment most significantly affect 10 biological
   process by Gene Ontology enrichment analysis.

   (DOC)
   [129]Click here for additional data file.^ (37.5KB, doc)
   S2 Table. GFT505 treatment most significantly affect 10 process by
   Reactom enrichment analysis.

   (DOC)
   [130]Click here for additional data file.^ (36.5KB, doc)
   S3 Table. GFT505 treatment most significantly affect 10 pathways by
   Kyoto encyclopedia of genes and genomes enrichment analysis.

   (DOC)
   [131]Click here for additional data file.^ (36.5KB, doc)
   S4 Table. The change of lipid metabolism related genes, inflammation
   reaction related genes and fibrosis related genes expression.

   (DOC)
   [132]Click here for additional data file.^ (43KB, doc)
   S5 Table. Primers sequences used for RT-qPCR.

   (DOC)
   [133]Click here for additional data file.^ (59.5KB, doc)

Acknowledgments