The pharmacodynamic and differential gene expression analysis of PPAR α/δ agonist GFT505 in CDAHFD-induced NASH model

Linfu Liu; Chuang Liu; Manyu Zhao; Qianru Zhang; Ying Lu; Ping Liu; Hua Yang; Jinliang Yang; Xiaoxin Chen; Yuqin Yao

doi:10.1371/journal.pone.0243911

. 2020 Dec 16;15(12):e0243911. doi: 10.1371/journal.pone.0243911

The pharmacodynamic and differential gene expression analysis of PPAR α/δ agonist GFT505 in CDAHFD-induced NASH model

Linfu Liu ^1,^2,^#, Chuang Liu ^1,^2,^#, Manyu Zhao ¹, Qianru Zhang ^2,³, Ying Lu ², Ping Liu ⁴, Hua Yang ¹, Jinliang Yang ^2,⁴, Xiaoxin Chen ^3,^4,^*, Yuqin Yao ^1,^2,^*

Editor: Jonathan M Peterson⁵

¹West China School of Public Health and West China Fourth Hospital, Sichuan University, Chengdu, China

²Collaborative Innovation Center for Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu, China

³Guangdong Zhongsheng Pharmaceutical Co., Ltd., Guangdong, China

⁴Department of Gynecology and Obstetrics, West China Second Hospital, Sichuan University, Chengdu, Sichuan, China

⁵East Tennessee State University, UNITED STATES

Competing Interests: All the authors declare that they have no conflict of interests. And all the institutions and companies declare that they have no conflict of interests. In this study, Sichuan University, including Collaborative Innovation Center for Biotherapy and Cancer Center, West China School of Public Health and West China Fourth Hospital, provided platforms for performing the experiments, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Guangdong Zhongsheng Pharmaceutical Co., Ltd employs [Qianru Zhang and Xiaoxin Chen] but does not fund the study and alter our adherence to PLOS ONE policies on sharing data and materials. The specific roles of the authors are articulated in the ‘author contributions’ section.

^✉

* E-mail: yuqin_yao@scu.edu.cn (YY); chenzhenyu2000@zspcl.com (XC)

Contributed equally.

Roles

Linfu Liu: Data curation, Formal analysis, Investigation, Visualization, Writing – original draft, Writing – review & editing

Chuang Liu: Data curation, Formal analysis, Investigation, Methodology, Software, Visualization, Writing – original draft, Writing – review & editing

Manyu Zhao: Data curation, Formal analysis, Visualization, Writing – review & editing

Qianru Zhang: Data curation, Formal analysis, Investigation, Methodology, Software, Visualization, Writing – original draft, Writing – review & editing

Ying Lu: Data curation, Formal analysis, Visualization, Writing – review & editing

Ping Liu: Project administration, Resources

Hua Yang: Resources, Supervision

Jinliang Yang: Funding acquisition, Project administration, Resources, Supervision

Xiaoxin Chen: Funding acquisition, Resources, Supervision

Yuqin Yao: Funding acquisition, Methodology, Project administration, Resources, Supervision, Visualization, Writing – review & editing

Jonathan M Peterson: Editor

PMCID: PMC7743980 PMID: 33326461

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 [1, 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 [3].

Lipid toxicity results in the liver injury and hepatitis [4]. Persistent inflammatory injury over activates injury repair process, thus leading to hepatic fibrosis [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 [6]. Activating of PPAR α could decrease the lipid accumulation and oxidative stress in liver [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 [8]. GFT505, a dual PPAR α/δ agonist, has been reported to be effective in clinical trials of NASH [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 [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 [11, 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 [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# 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 [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# 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 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 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 (Fig 1C), but had no effect on serum TG expression (Fig 1D). Importantly, the concentrations of AST were decreased at the dosages of 10 and 30 mpk of GFT505 (Fig 1E) and ALT were significantly reduced after treated with all the dosages of GFT505 (3, 10 and 30 mpk) (Fig 1F).

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 (Fig 2A). Sirius red staining showed that GFT505 attenuated the fibrosis in NASH mice model induced by CDAHFD (Fig 2B). Pathological score of H&E staining results were presented in Fig 2C and 2D. GFT505 of 10 and 30 mpk attenuated liver steatosis by 46% and 53%, respectively (Fig 2C). And GFT505 at the doses of 3, 10 and 30 mpk suppressed the CDAHFD-induced inflammation by 33%, 44% and 50% respectively (Fig 2D). Furthermore, the pathological scores of Sirius red staining showed that GFT505 suppressed fibrosis by 65% (10 mpk) and 58% (30 mpk) (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 (Fig 2F). The CD163 (M2-macrophage marker) was lower in the vehicle group compared with the control group and the GFT505 (30 mpk) group (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 (Fig 3A) and the quantification was showed in Fig 3C. Decreased protein concentrations of α-SMA (Fig 3B and 3D) and collagen I (Fig 3B and 3E) were demonstrated after GFT505 treatment.

Fig 2 — (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 — (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 (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 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 (S2 Table). The top 10 enriched KEGG pathways were presented in Fig 4B and listed in S3 Table. Among which the enriched PPAR signaling pathway and Fatty acid degradation pathway were showed in Fig 4C and S1 Fig. Besides, the ECM-receptor interaction pathway (Fig 4D) and Cytokine-cytokine receptor interaction pathway (S2 Fig) were enriched as well. As shown in 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 (Fig 5A), and RT-qPCR was performed to confirmed these related mRNA expressions (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 S4 Table. The primers sequences for RT-qPCR were showed in 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 — (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 log₂ [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 — (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 (Fig 6A). The quantification of Oil red O staining was showed in Fig 6B. The analysis of TG concentration was showed in 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 — (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 [14–16]. 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 [9, 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 [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 [19, 20]. Methionine-choline-deficient diet (MCD) induces steatosis, hepatic inflammation and fibrosis in a short feeding time [18, 21]. However, this model is usually not accompanied by obesity and peripheral IR. Moreover, the decreased body weight limits the use of MCD model [22, 23]. The choline-deficient, L-amino acid-defined (CDAA) model is similar to MCD model except for more serious histological changes [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 [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 [25, 26]. Down-regulation of Ehhadh gene expression decreased very low density lipoprotein (VLDL) transport rate [27]. Pparα is involved in free fatty acid transport, oxidation, liver lipids and plasma lipoprotein metabolism [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 [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 [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 [30]. Il17d plays a key role in the recruitment of human immune cells [31]. Studies have reported that Tlr9 caused acute injury by activating IL-17A [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.

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S2 Fig. GFT505 Treatment down-regulates “cytokine-cytokine receptor interaction” pathway related genes.

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S1 Table. GFT505 treatment most significantly affect 10 biological process by Gene Ontology enrichment analysis.

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S2 Table. GFT505 treatment most significantly affect 10 process by Reactom enrichment analysis.

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S3 Table. GFT505 treatment most significantly affect 10 pathways by Kyoto encyclopedia of genes and genomes enrichment analysis.

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S4 Table. The change of lipid metabolism related genes, inflammation reaction related genes and fibrosis related genes expression.

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S5 Table. Primers sequences used for RT-qPCR.

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Acknowledgments

The authors thank the research platform provided by Public Health and Preventive Medicine Provincial Experiment Teaching Center at Sichuan University and Food Safety Monitoring and Risk Assessment Key Laboratory of Sichuan.

Data Availability

The data has been uploaded to NCBI Sequence Read Archive and the relevant accession number is PRJNA657593.

Funding Statement

This work was supported by the National Major Scientific and Technological Special Project (Nos. 2018ZX09201002, 2018ZX09711001-011 and 2019ZX09201001) and the National Science Foundation of China (No. 81773375). The authors thank the research platform provided by Public Health and Preventive Medicine Provincial Experiment Teaching Center at Sichuan University.

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PLoS One. doi: 10.1371/journal.pone.0243911.r001

Decision Letter 0

Jonathan M Peterson

8 Jun 2020

PONE-D-20-10913

The pharmacodynamic and differential gene expression analysis of PPAR α/δ agonist GFT505 in CDAHFD-induced NASH model

PLOS ONE

Dear Dr. Yao,

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Both of the reviewers believed the study was interesting and a valuable addition to the literature. However there were a number of issues that need to be address as listed by the reviewers. Additionally, the The RNAseq datasets should be put in a database to be readily accessed upon publication and their were a number of grammatical issues that need to be addressed.

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Reviewer #1: Yes

Reviewer #2: Partly

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: No

Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #1: The authors assessed the effects of the PPARa/d dual agonist GFT505 (elafibranor) on liver histology and gene expression in a mouse model of NASH and fibrosis induced by a choline-deficient diet.

Remarks:

-GFT505 is now termed elafibranor. The authors may want to use its generic name.

-Since choline deficiency impacts on hepatic VLDL output, the impact of GFT505 on plasma lipid and lipoprotein parameters is difficult to evaluate. Moreover, it is unclear how the authors assessed plasma HDL and LDL. This reviewer assumes it is cholesterol in these lipoproteins that was measured? If this was done using kits for lipoprotein cholesterol and triglyceride measurement in humans, the results may incorrectly be termed LDL-C and HDL-C. The authors should either eliminate these data from the paper or perform more detailed cholesterol and triglyceride distribution profile analysis on plasma of the animals in order to have a clearer view on the plasma lipid changes.

-The effects of PPARa agonism on liver mass are due to peroxisome proliferation (erroneously mentioned as liver swelling). This only occurs in rodents and therefore should not be classified as side effect.

Reviewer #2: The manuscript entitled "The pharmacodynamic and differential gene expression analysis of PPAR α/δ agonist GFT505 in CDAHFD-induced NASH model" by Liu et al. demonstrates that GFT505 reduces lipid accumulation, inflammation and fibrosis in a NASH mouse model. The histological data is clear. However, the manuscript presents several issues:

Major comments:

1. This study lacks a mechanism by which GFT505 reduces fat accumulation in hepatocytes, inflammation and fibrosis.

2. Relevance to human disease is missing.

3. The manuscript has many English typos.

Specific comments:

1. Abstract: please revise the sentence starting with “The significant regulation genes…”, it is quite confusing.

2. The authors should revise the English. Example: Introduction, line 9, please replace “PPAR α, which mainly expresses…” by “PPAR α, which is mainly expressed”.

3. Why only male mice are used in this study? The authors should also use female mice.

4. When authors state that mice were divided in 5 groups, does the n=10 mean 10 mice/group or 10 mice in total? Please specify.

5. Figure 1A: why vehicle decreases the body weight compared to control?

6. The authors should specify in the figure panels which groups received normal diet and which received CDAHFD.

7. Figure legends are not very explanatory. They should describe briefly the experiment for each panel.

8. Figure 3: it would be nice to confirm these results by performing WB, which gives a better idea on the levels of Col1a1 and aSMA.

9. Figure 4 is hard to read, the letters are too small and the resolution of the figure is not ideal.

10. Does the target genes show the same trend in human NASH livers compared to healthy individuals as in the NASH model?

11. What types of inflammatory cells are reduced after GFT505 treatment?

12. In this study, is GFT505 used as a preventive or a curative treatment?

13. What are the effects of GFT505 in control diet-fed mice?

**********

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2020 Dec 16;15(12):e0243911. doi: 10.1371/journal.pone.0243911.r002

Author response to Decision Letter 0

27 Aug 2020

Dear Editor and Reviewers,

Thanks for your professional and valuable comments on the manuscript (PONE-D-20-10913) entitled "The pharmacodynamic and differential gene expression analysis of PPAR α/δ agonist GFT505 in CDAHFD-induced NASH model". We have carefully addressed the editors’ and reviewers’ comments and revised the manuscript. The point-to-point corrections we did are following.

Criticisms: The point-to-point corrections we did are following.

Editor’s advice:

1. Both of the reviewers believed the study was interesting and a valuable addition to the literature. However, there were a number of issues that need to be address as listed by the reviewers. Additionally, the RNAseq datasets should be put in a database to be readily accessed upon publication and there were a number of grammatical issues that need to be addressed.

Reply: thanks for your advice. We have uploaded our RNAseq data to NCBI Sequence Read Archive and have revised the grammar errors throughout the manuscript.

Reply: thanks for your suggestions. We have uploaded the data to NCBI Sequence Read Archive and the relevant accession number is PRJNA657593.

3. Thank you for including your competing interests statement; "The authors have declared that no competing interests exist."

We note that one or more of the authors are employed by a commercial company: Guangdong Zhongsheng Pharmaceutical Co., Ltd

Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form. Please also include the following statement within your amended Funding Statement“The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.” Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc. Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests). If this adherence statement is not accurate and there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

Reply: thanks for your suggestion. We have updated the competing interest statement, author contributions and funding statement. Moreover, we have added competing interest statement and funding statement in the manuscript.

Although there were two authors employed by Guangdong Zhongsheng Pharmaceutical Co., Ltd involved in our research, the company didn’t fund the study. The specific roles of these authors are articulated in the ‘author contributions’ section.” We fell so sorry for not explaining the conflict of interests about this.

Funding Statement

Competing interest statement

All the authors declare that they have no conflict of interests. And all the institutions and companies declare that they have no conflict of interests.

In this study, Sichuan University, including Collaborative Innovation Center for Biotherapy and Cancer Center, West China School of Public Health and West China Fourth Hospital, provided platforms for performing the experiments, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. And Guangdong Zhongsheng Pharmaceutical Co., Ltd does not fund the study and alter our adherence to PLOS ONE policies on sharing data and materials. The specific roles of the authors were articulated in the ‘author contributions’ section.

Author contributions

Qianru Zhang, Manyu Zhao, Linfu Liu and Chuang Liu performed most of the experiments. Ying Lu and Hua Yang collected and interpreted data. Linfu Liu and Chuang Liu wrote this paper. Qianru Zhang, Yuqin Yao and Jinliang Yang revised the paper. Yuqin Yao, Jinliang Yang, and Xiaoxin Chen were responsible for fund collection and revised this paper. Qianru Zhang and Xiaoxin Chen are employees of Guangdong Zhongsheng Pharmaceutical Co., Ltd.. Qianru Zhang is an on-the-job ph.d.student of Sichuan University

4. Please include both an updated Funding Statement and Competing Interests Statement in your cover letter. We will change the online submission form on your behalf.

Reply: thanks for your advice. We have updated Funding Statement and Competing Interests Statement in our cover letter.

5. To comply with PLOS ONE submission requirements, in your Methods section, please provide additional information regarding the experiments involving animals and ensure you have included details on (1) methods of sacrifice, (2) methods of anesthesia and/or analgesia, and (3) efforts to alleviate suffering.

Reply: thanks for your suggestions. Animals were anesthetized with 3% pentobarbital sodium by intraperitoneal injection. And then the terminal blood samples were obtained from the eyelids for biochemical analysis (mice were sacrificed in this way). We have added these details in the section of “CDAHFD-fed mouse NASH model”.

Reviewers' comments:

Remarks:

-GFT505 is now termed elafibranor. The authors may want to use its generic name.

Reply: thanks for your review. We have descibed the generic name of GFT505 in abstract.

Reply: thanks for your kind comments. The serum HDL levels was detected by HDL-Cholesterol Gen.4 (Roche, Basel, Switzerland) and serum LDL levels was detected by LDL-Cholesterol Gen.3 (Roche, Basel, Switzerland). Considering your suggestion, we have eliminated these data.

Reply: sincerely thanks for your useful suggestion. We have deleted this desciption.

Major comments:

1. This study lacks a mechanism by which GFT505 reduces fat accumulation in hepatocytes, inflammation and fibrosis.

Reply: thanks for your kind comments. The objective of our study was to profile the regulatory network of PPAR α/δ agonist in NASH and test the pharmacodynamics of elafibranor. And we found that GFT505 could regulate the 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), which were confirmed by qRT-PCR. The down-regulated genes were significantly 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. These results suggest that GFT505 may play a role in inhibiting fat accumulation, inflammation and fibrosis in NASH by regulating these genes and signaling pathways. And we will futher investigate the exact mechanism of GFT505 on NASH regualting the specific gene or signaling pathway. Thanks for your useful comments again.

2. Relevance to human disease is missing.

Reply: thanks for your review. NASH is a progressive disease from NAFLD, which characterized by steatosis, hepatocellular ballooning, lobular inflammation and fibrosis. Because of the slow progress of NASH, although the incidence rate is increasing, the overall incidence rate is still low, and clinical samples are difficult to obtain. And our study is only a preclinical study of NASH and the aims of our study is to investigate the pharmacodynamics and transcriptome regulation of GFT505 in vivo and in vitro.

3. The manuscript has many English typos.

Reply: thanks for your advice. We have correted our grammatical errors throughout the manuscript.

Specific comments:

1. Abstract: please revise the sentence starting with “The significant regulation genes…”, it is quite confusing.

Reply: thanks for your advice. We have revised this sentence.

2. The authors should revise the English. Example: Introduction, line 9, please replace “PPAR α, which mainly expresses…” by “PPAR α, which is mainly expressed”.

Reply: thanks for your carefully review. We have revised the grammatical errors in our manuscript, e.g., “PPAR α, which mainly expresses…” have been corrected into “PPAR α, which is mainly expressed”.

3. Why only male mice are used in this study? The authors should also use female mice.

Reply: thanks for your advice. We chose male mice from the effectiveness of the NASH model. Studies have shown that estrogens can protect female mice from liver steatosis and fibrosis in female mice(1). Moreover, female specific white adipose tissue browning helps protect female mice from fatty liver disease induced by MCD diet(2).

Reference:

1. Ballestri S, Nascimbeni F, Baldelli E, Marrazzo A, Romagnoli D, Lonardo A. NAFLD as a Sexual Dimorphic Disease: Role of Gender and Reproductive Status in the Development and Progression of Nonalcoholic Fatty Liver Disease and Inherent Cardiovascular Risk. Advances in therapy. 2017;34(6):1291-326.

2. Lee YH, Kim SH, Kim SN, Kwon HJ, Kim JD, Oh JY, et al. Sex-specific metabolic interactions between liver and adipose tissue in MCD diet-induced non-alcoholic fatty liver disease. Oncotarget. 2016;7(30):46959-71.

4. When authors state that mice were divided in 5 groups, does the n=10 mean 10 mice/group or 10 mice in total? Please specify.

Reply: thanks for your suggestion. We are so sorry for not describing animal amounts clearly. “n=10” means 10 mice in each group. We have specified this in the section of “CDAHFD-fed mouse NASH model”.

5. Figure 1A: why vehicle decreases the body weight compared to control?

Reply: in our study, Model group was given CDAHFD, GFT505 groups were given GFT505 and CDAHFD, control group was given normal diet. GFT505 was dissolved by vehicle. The 1% carboxymethyl cellulose sodium and 0.1% tween 80 are the components of vehicle, which have been widely used as solvent of drugs(1,2) and studies have shown that they have no obvious effect on mice weight(2). Compared with the control group, the body weight of vehicle and GFT505 groups decreased, and there was no difference between GFT505 groups and model group, which mainly indicated that bleomycin caused the weight loss of mice.

Reference:

1. Camilleri C, Beiter RM, Puentes L, Aracena-Sherck P, Sammut S. Biological, Behavioral and Physiological Consequences of Drug-Induced Pregnancy Termination at First-Trimester Human Equivalent in an Animal Model. Frontiers in neuroscience. 2019;13:544.

2. Chassaing B, Koren O, Goodrich JK, Poole AC, Srinivasan S, Ley RE, et al. Dietary emulsifiers impact the mouse gut microbiota promoting colitis and metabolic syndrome. Nature. 2015;519(7541):92-6.

6. The authors should specify in the figure panels which groups received normal diet and which received CDAHFD.

Reply: thanks for your suggestions. We have specified which groups received normal diet and which received CDAHFD both in methods section and figure legends.

7. Figure legends are not very explanatory. They should describe briefly the experiment for each panel.

Reply: thanks for your kind comments. We have revised the figure legends and described the experiment for each panel.

8. Figure 3: it would be nice to confirm these results by performing WB, which gives a better idea on the levels of Col1a1 and aSMA.

Reply: thanks for your professional comments. The WB results of Col1a1 and a-SMA were shown in R-figure1, which is writen in the rebuttal letter. The protein level of Collagen 1 in GFT505 group was lower than that in the Vehicle group, but there was no statistical significance. And the level of α-SMA was decreased by GFT505 treatment compared with Vehicle group. The WB results of Collagen 1 was not consistent with the results of immunohistochemistry. The reason may be that there is steatosis in NASH animal model, and fat has a great influence on the extraction of tissue protein, which will lead to the loss of two proteins. In addition, Collagen 1 is a kind of secreted protein, which is not suitable for Western blot detection and is easy to degrade (our tissue samples were extracted in 2017). In addition, immunohistochemistry and Sirius Red staining are the standard methods for fibrosis evaluation.

R-figure 1. The protein expression levels of Collagen 1 and α-SMA. A. The results of the protein expression levels of Collagen 1 and α-SMA detected by western blotting. B. Quantification of A (Collagen 1). C. Quantification of A (α-SMA).

9. Figure 4 is hard to read, the letters are too small and the resolution of the figure is not ideal.

Reply: thanks for you review. We have uploaded the high resolution of figure4.

10. Does the target genes show the same trend in human NASH livers compared to healthy individuals as in the NASH model?

Reply: thanks for your review. Our study is a preclinical study of NASH. The samples used for RNA-sequecing were obtained from mouse tissue samples. We will futher investigate whether these target genes showing the same trend in human NASH.

11. What types of inflammatory cells are reduced after GFT505 treatment?

Reply: thanks for your professional comments. This is a shortcoming of our research. In our study, we used H&E staining to evaluate the effect of GFT505 on NASH inflammation, and did not detect which inflammatory cells decreased after GFT505 treatment. We will detect inflammatory cells in future experiments. Truly thanks for your useful comments.

12. In this study, is GFT505 used as a preventive or a curative treatment?

Reply: in our study, GFT505 was used as curative treatment.

13. What are the effects of GFT505 in control diet-fed mice?

Reply: thanks for your review. We haven’t test the effects of GFT505 in control diet-fed mice in our study. This is also one of our research shortcomings. We did not consider the side effects or good effects of GFT505 on normal mice. In the future, we will design related experimental groups.

Attachment

Submitted filename: rebuttal letter.docx

Click here for additional data file.^{(480KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0243911.r003

Decision Letter 1

Jonathan M Peterson

22 Sep 2020

PONE-D-20-10913R1

The pharmacodynamic and differential gene expression analysis of PPAR α/δ agonist GFT505 in CDAHFD-induced NASH model

PLOS ONE

Dear Dr. Yao,

What was the normal control diet?

Please add the catalog numbers for the commercially purchased kits (cholesterol, triglyceride, etc...) and antibodies used. Many companies have multiple version of similar products.

I agree with the reviewer that the lack of a control group treated with the GFT505 is a major limitation of the study designed and needs to be addressed in the discussion as a limitation to the interpretation of the study findings.

I agree with your response to the reviewer comments that histological staining is the standard for fibrosis evaluation, I suggest probing via western blot for an additional marker of fibrosis (or try another collagen antibody), further these data should be included in the manuscript along with the description of the methodology for the tissue processing for western blots. Especially as the alpha-SMA western data showed a reduction.

It would be a nice addition to the study if the RNA-seq data showed changes to the type of inflammatory cells present in the liver, or as the reviewer suggests run RT-PCR for markers such as CD45+ and CD163+ (or histological staining for markers for inflammatory cells), while these experiments are not absolutely necessary I agree with the reviewer that this addition would greatly strengthen the manuscript

Please submit your revised manuscript by Nov 06 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Jonathan M Peterson, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

What was the normal control diet?

Please add the catalog numbers for the commercially purchased kits (cholesterol, triglyceride, etc...) and antibodies used. Many companies have multiple version of similar products.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #2: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #2: I thank the authors for their responses. However, I think they did not address some of, what I think are important, suggestions. For this paper to be scientifically rigorous and sound, these suggestions need to be addressed. Please, find these suggestions below:

1. What are the effects of GFT505 in control diet-fed mice?

2. What types of inflammatory cells are reduced after GFT505 treatment? You can check these by simple qPCR for markers of inflammatory cells.

3. Figure 3 and R-figure 1: it is understandable that for NASH model, collagen WB is difficult to get. However, collagen protein levels are a standard way to check liver fibrosis. Therefore, either the authors could try to get a good collagen antibody or perform hydroxy proline assay. Please include these panels in the main Figure 3.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

PLoS One. 2020 Dec 16;15(12):e0243911. doi: 10.1371/journal.pone.0243911.r004

Author response to Decision Letter 1

20 Nov 2020

Editor’s advice:

1. Please add the catalog numbers for the commercially purchased kits (cholesterol, triglyceride, etc...) and antibodies used. Many companies have multiple version of similar products.

Reply: thanks for your advice. We have add the catalog numbers for the commercially purchased kits and antibodies used in our manuscript.

2.I agree with the reviewer that the lack of a control group treated with the GFT505 is a major limitation of the study designed and needs to be addressed in the discussion as a limitation to the interpretation of the study findings.

Reply: Thanks for your suggestion. We haven’t consider the effects of GFT505 in control diet-fed mice in our study, which is also neglected by many studies about GFT505 (1,2,3). This is also one of our research shortcomings and we have addressed this in the discussion as a limitation of the study. In the future, we will design related experimental groups.

3.I agree with your response to the reviewer comments that histological staining is the standard for fibrosis evaluation, I suggest probing via western blot for an additional marker of fibrosis (or try another collagen antibody), further these data should be included in the manuscript along with the description of the methodology for the tissue processing for western blots. Especially as the alpha-SMA western data showed a reduction.

Reply:Thanks for your advice. We bought a new antibody of collagen 1 (HuaBio, CAT# RT1152). The results of western blot was showed in R-figure 1 and there wasn’t clear bind of collagen 1 in our results. It is difficult for to evaluate the protein level of collagen 1 by western blot in NASH animal model. One of the characteristics of NASH is steatosis and fat has a great influence on the extraction of tissue protein. Actually, collagen 1 evaluation in many studies about NASH is mainly dependent on Immunohistochemistry and Sirus red staining (1,3). Moreover, while the evaluation of collagen 1 or collagen 3 is most dependent on the tertiary/quaternary structures, the Immunohlistochemistry (IHC) is the standard method for collagen evaluation (2). Considering the importance of Collagen1 levels and the concerns of editor and reviewer, we run the RT-qPCR of collagen 1 (R-figure 2) in three groups based on our collagen subtype mRNA expression including col1a1, col1a2, col3a1 as a supplement to the IHC of collagen 1.

4.It would be a nice addition to the study if the RNA-seq data showed changes to the type of inflammatory cells present in the liver, or as the reviewer suggests run RT-PCR for markers such as CD45+ and CD163+ (or histological staining for markers for inflammatory cells), while these experiments are not absolutely necessary I agree with the reviewer that this addition would greatly strengthen the manuscript.

Reply: Thanks for your professional comments. The results of RT-qPCR experiment (R-figure 3) showed that the mRNA expression of CD45 in the Vehicle group was higher than that in the Control group, which was decreased after the treatment of GFT505. The mRNA expression of CD163 in the Vehicle group was lower compared with control group, which was increased after the treatment of GFT505.

Macrophages are heterogeneous and their phenotype and functions are regulated by the surrounding micro-environment (1). Macrophages commonly exist in two distinct subsets: classically (M1) activated and alternatively (M2) activated (2,3). M1-macrophages are pro-inflammatory and M2-macrophages are anti-inflammatory (1). CD45 is one of the makers of M1-macrophage and CD163 is one of the makers of M2-macrophage. Our results shows that the vehicle group has the highest CD45 mRNA expression and the lowest CD163 mRNA expression, which are corresponding to previous studies(2,3,4).

Reviewers' comments:

1.What are the effects of GFT505 in control diet-fed mice?

2.What types of inflammatory cells are reduced after GFT505 treatment? You can check these by simple qPCR for markers of inflammatory cells.

Reply:Thanks for your advice. We evaluated the CD45-represented M1-macrophages and CD163-represented M2-macrophages via RT-qPCR. The results of RT-qPCR experiments (R-figure 3) showed that the mRNA expression of CD45 in the Vehicle group was higher than that in the Control group, which was decreased after the treatment of GFT505. The mRNA expression of CD163 in the Vehicle group was lower compared with control group, which was increased after the treatment of GFT505. Macrophages are heterogeneous and their phenotype and functions are regulated by the surrounding micro-environment (1). Macrophages commonly exist in two distinct subsets: classically (M1) activated and alternatively (M2) activated (2,3). M1-macrophages are pro-inflammatory and M2-macrophages are anti-inflammatory (1). CD45 is one of the makers of M1-macrophage and CD163 is one of the makers of M2-macrophage. Our results shows that the vehicle group has the highest CD45 mRNA expression and the lowest CD163 mRNA expression, which are corresponding to previous studies(2,3,4).

3.Figure 3 and R-figure 1: it is understandable that for NASH model, collagen WB is difficult to get. However, collagen protein levels are a standard way to check liver fibrosis. Therefore, either the authors could try to get a good collagen antibody or perform hydroxyproline assay. Please include these panels in the main Figure 3.

Reply: Thanks for your suggestion. According to your suggestion, we bought a new Hydroxyproline assay kit (Nanjing Jiancheng Bioengineering Institute, A030-2-1) and performed a hydroxyproline assay (R-figure 4). The results showed that there was no difference between vehicle group and GFT505 group. Hydroxyproline is essential to collagen synthesis, thus the level of hydroxyproline may be an indirect index for fibrosis (1). In fact, many studies of NASH didn’t choose hydroxyproline as a fibrosis evaluation index. Moreover, fibrosis evaluation is comprehensive and dependent on multiple indexes. According to the results in Figure 2B, 3D and 5D, GFT505 has a favorable effect in inhibiting fibrosis. We bought a new antibody of collagen 1 (HuaBio, CAT# RT1152). The result of western blot was showed in R-figure 1 and there wasn’t clear bind of collagen 1 in our results. It is difficult for to evaluate the protein level of collagen 1 by western blot in NASH animal model. One of the characteristics of NASH is steatosis and fat has a great influence on the extraction of tissue protein. Actually, collagen 1 evaluation in many studies about NASH is mainly dependent on Immunohistochemistry and Sirus red staining (1,3). Moreover, while the evaluation of collagen 1 or collagen 3 is most dependent on the tertiary/quaternary structures, the Immunohlistochemistry (IHC) is the standard method for collagen evaluation(2). Considering the importance of Collagen1 levels and the concerns of editor and reviewer, we run the RT-qPCR of collagen 1 (R-figure 2) in three groups based on our collagen subtype mRNA expression including col1a1, col1a2, col3a1 as a supplement to the IHC of collagen 1.

PLoS One. doi: 10.1371/journal.pone.0243911.r005

Decision Letter 2

Jonathan M Peterson

1 Dec 2020

The pharmacodynamic and differential gene expression analysis of PPAR α/δ agonist GFT505 in CDAHFD-induced NASH model

PONE-D-20-10913R2

Dear Dr. Yao,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Jonathan M Peterson, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #2: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #2: Thank you for the responses. The authors have responded to all my comments and I have no further suggestion.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

PLoS One. doi: 10.1371/journal.pone.0243911.r006

Acceptance letter

Jonathan M Peterson

7 Dec 2020

PONE-D-20-10913R2

The pharmacodynamic and differential gene expression analysis of PPAR α/δ agonist GFT505 in CDAHFD-induced NASH model

Dear Dr. Yao:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr Jonathan M Peterson

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

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

(TIF)

Click here for additional data file.^{(14.4MB, tif)}

S2 Fig. GFT505 Treatment down-regulates “cytokine-cytokine receptor interaction” pathway related genes.

(TIF)

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)

Click here for additional data file.^{(37.5KB, doc)}

S2 Table. GFT505 treatment most significantly affect 10 process by Reactom enrichment analysis.

(DOC)

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)

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)

Click here for additional data file.^{(43KB, doc)}

S5 Table. Primers sequences used for RT-qPCR.

(DOC)

Click here for additional data file.^{(59.5KB, doc)}

Attachment

Submitted filename: rebuttal letter.docx

Click here for additional data file.^{(480KB, docx)}

Data Availability Statement

The data has been uploaded to NCBI Sequence Read Archive and the relevant accession number is PRJNA657593.

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PERMALINK

The pharmacodynamic and differential gene expression analysis of PPAR α/δ agonist GFT505 in CDAHFD-induced NASH model

Linfu Liu

Chuang Liu

Manyu Zhao

Qianru Zhang

Ying Lu

Ping Liu

Hua Yang

Jinliang Yang

Xiaoxin Chen

Yuqin Yao

Roles

Abstract

Introduction

Materials and methods

Animals, drug and cell line

CDAHFD-fed mouse NASH model

Histological and immunohistochemical assays

Sample preparation, library construction and RNA-seq

Differential gene expression and bioinformatics analysis

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

Lipid accumulation cell model

Statistics analysis

Results

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

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

GFT505 attenuated the liver steatosis, inflammation and fibrosis in NASH

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

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

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

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

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

GFT505 inhibited lipid accumulation in LO2 cell

Fig 6. GFT505 reduced lipid accumulation in LO2 cell.

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Jonathan M Peterson

Roles

Author response to Decision Letter 0

Decision Letter 1

Jonathan M Peterson

Roles

Author response to Decision Letter 1

Decision Letter 2

Jonathan M Peterson

Roles

Acceptance letter

Jonathan M Peterson

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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