Pirfenidone Is an Agonistic Ligand for PPARα and Improves NASH by Activation of SIRT1/LKB1/pAMPK

Abstract

Nonalcoholic steatohepatitis (NASH) is recognized by hepatic lipid accumulation, inflammation, and fibrosis. No studies have evaluated the prolonged-release pirfenidone (PR-PFD) properties on NASH features. The aim of this study is to evaluate how PR-PFD performs on metabolic functions, and provide insight on a mouse model of human NASH. Male C57BL/6J mice were fed with either normo diet or high-fat/carbohydrate diet for 16 weeks and a subgroup also fed with PR-PFD (300 mg/kg/day). An insulin tolerance test was performed at the end of treatment. Histological analysis, determination of serum hormones, adipocytokines measurement, and evaluation of proteins by western blot was performed. Molecular docking, in silico site-directed mutagenesis, and in vitro experiments using HepG2 cultured cells were performed to validate PR-PFD binding to peroxisome proliferator-activated receptor alpha (PPAR-α), activation of PPAR-α promoter, and sirtuin 1 (SIRT1) protein expression. Compared with the high-fat group, the PR-PFD-treated mice displayed less weight gain, cholesterol, very low density lipoprotein and triglycerides, and showed a significant reduction of hepatic macrosteatosis, inflammation, hepatocyte ballooning, fibrosis, epididymal fat, and total adiposity. PR-PFD restored levels of insulin, glucagon, adiponectin, and resistin along with improved insulin resistance. Noteworthy, SIRT1-liver kinase B1-phospho-5' adenosine monophosphate-activated protein kinase signaling and the PPAR-α/carnitine O-palmitoyltransferase 1/acyl-CoA oxidase 1 pathway were clearly induced in high fat + PR-PFD mice. In HepG2 cells incubated with palmitate, PR-PFD induced activation and nuclear translocation of both PPARα and SIRT1, which correlated with increased SIRT1 phosphorylated in serine 47, suggesting a positive feedback loop between the two proteins. These results were confirmed with both synthetic PPAR-α and SIRT1 activators and inhibitors. Finally, we found that PR-PFD is a true agonist/ligand for PPAR-α. Conclusions: PR-PFD provided an anti-steatogenic effect and protection for inflammation and fibrosis.

Figure 1

Animal weight and glucose determination. (A) Weekly weight gain. Switch diet and SDP groups changed diet at week 8, with dramatically diminishing weight. (B) Weight at sacrifice. (C) Epididymal adipose fat pad weight. The HFP group showed a statistical diminution in animal and fat weight compared with the HF animals (P < 0.01). (D) Fasting glucose at week 16. HFP animals presented a slight decrease in serum glucose compared with the HF group. (E) ITT. The HFP group showed insulin sensitivity comparable to the ND group. (F) The AUC for ITT is improved in HFP animals (P < 0.01). Data are expressed as the media of the group ± SEM. Abbreviations: ns, not significant; SD, switch diet.

Figure 2

Histological analysis of liver in NASH obesity–induced animals treated with PR‐PFD. (A) HF animals had a 1‐fold increase in inflammation nodules (P < 0.001). Arrows indicate inflammation nodules. Oil Red O staining in fresh samples showed a decrease in fat staining in HFP animals (P < 0.01). Percentage of macrosteatosis was dramatically diminished in liver samples of animals treated with PR‐PFD (P < 0.0001). Representative pictures are displayed at ×10. Total bridges were counted by an independent pathologist. Representative pictures are shown at ×20. Periportal (B) and centrilobulillar (C) areas were evaluated for fibrosis (P < 0.01 and P < 0.05, respectively); ×40. Data are expressed as the median of the group ± SEM.

Figure 3

Biochemical markers and adipokines serum levels. (A) Resistin levels demonstrated a 5‐fold decrease in the HFP animals (P < 0.01). (B) Adiponectin (P < 0.001) increases in the HFP group (C) Leptin showed no statistical difference between high‐fat groups. (D) insulin was increased in HF and HFD animals compared with the ND group (P < 0.01). (E) Glucagon showed no statistical difference between groups. (F) IL‐17 levels decreased (P < 0.0001) in the HFP animals. Data are expressed as the median of the group ± SEM. Abbreviation: ns, not significant.

Figure 4

PR‐PFD modifies the adiposity and gene expression, and induces PPAR‐α signaling and SIRT1. (A) Fat pads in mice were diminished in the HPF groups. (B) Representative pictures of adipose tissue stained with H&E are displayed at ×20. Fewer adipocytes in the ND, switch diet, SDP, and HFP animals compared with the HF mice. Morphometric analysis showed a statistically significant diminution in adipocyte area in HFP (P < 0.05). (C) IL6‐mRNA levels showed a tendency to decrease. Tumor necrosis factor α, SREBP1, RIPK3, and MLKL gene expression are diminished in mice treated with PR‐PFD (P < 0.05, P < 0.01, P < 0.01 and P < 0.001, respectively). (D) Representative western blots (three of six liver tissues of animals) and analysis of SIRT1, LKB1, AMPK, pAMPK, PPAR‐α, CPT1A, ACOX1, and beta‐actin in the mouse model. Abbreviations: MLKL, mixed lineage kinase domain‐like; ns, not significant; RIPK3, receptor‐interacting serine/threonine‐protein kinase 3; SD, switch diet.

Figure 5

PR‐PFD induces PPAR‐α signaling and SIRT1 both in vivo and in vitro. (A) Representative microphotographs and analysis of Oil Red O staining of HepG2 cells incubated with 1‐mM palmitate with or without PFD. Scale bars = 20 μm. (B) In HepG2 cells, SIRT1 and LKB1 are overexpressed after 4 and 8 hours of PFD incubation, whereas NAM kept the expression down‐regulated. (C) PFD increases SIRT1 expression to similar levels than those obtained with SIRT1720 activator, whereas EX527 as a specific SIRT1 inhibitor decreased SIRT1; diminished SIRT1 phosphorylation in Ser47 is also shown. (D) Positive co‐localization in nuclei of phosphorylated SIRT1(Ser47) and PPAR‐α is observed in cells incubated with PFD. (E) Effect of PPAR‐α agonist (GW7647) and antagonist (GW9662) on HepG2 cell protein expression. (F) Representative confocal images. Scale bars = 10 µm. Data are expressed as the median of the group ± SEM. Abbreviations: CTL, control; PA, palmitate.

Figure 6

Molecular docking analysis of PFD interaction with PPAR‐α. (A) Chemical structure of PFD and schematic representation PPAR‐α, and 3D structure of the PLBD. (B) SwissDock 3D reconstruction of all possible molecular interactions of PFD with PPAR‐α PLBD. Covalent bonds established between the carbonyl group of FFB (C) and PFD (D) with amino acids MET220, ALA333, and TYR334 of PPAR‐α. Sequence and 3D visualizations of PFD interactions in MET220, ALA333, and TYR334 of PPAR‐α PLBD (E) and annulled interactions when these amino acids are mutated to glycine (F).

Figure 7

PFD induces activation of PPAR‐α promoter and expression in vitro of PPAR‐α, CPT1, and ACOX1. (A) Schematic representation of human PPAR‐α promoter constructs transfected in HepG2 cells and subsequently treated with PFD or 0.1% DMSO (−PDF). Luciferase expression was quantified using a luminometer. (B) Silver‐stained EMSA. (C) Western blots for quantification of luciferase expression. FFB was used as positive control. (D) PPAR‐α, CPT1, and ACOX1 protein expression in HepG2 cells. Values represent the mean ± SD. ****P < 0.0001 and ***P < 0.001. Abbreviations: CTL, control; NT, Non Treated cells.