Animals: All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals, and were approved by the VA Nebraska-Western Iowa Health Care System’s Institutional Animal Care and Use Committee. Male C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME, USA) aged 8 weeks were maintained with free access to food and water for the duration of the study. The mice were randomly assigned to treatment groups (
n = 6 per group). To induce hepatic fibrosis, carbon tetrachloride (CCl
4) diluted 1:4 in sunflower oil was injected intraperitoneally at a dose of 1 mL/kg body weight twice a week for 6 weeks, as described previously [19 (
link)]. The control mice were injected with oil alone. For the final 2 weeks, the mice received daily oral administration of SR1664 (Cayman Chemical, Ann Arbor, MI, USA) mixed with peanut butter to deliver a dose of 20 mg/kg body weight, which was previously shown to be an optimal dose to reduce insulin resistance in mice [18 (
link)]. The control mice received peanut butter alone. After 6 weeks, 72 h after the last CCl
4 injection, the mice were humanely euthanized for blood and tissue collection.
Serum and tissue determinations: Serum alanine transaminase (ALT) and aspartate transaminase (AST) were measured by the VA Nebraska-Western Iowa Health Care System’s Clinical Chemistry Service using standard assays. The serum total adiponectin level was determined using an ELISA kit (Alpco, Salem, NH, USA) according to the manufacturer’s instructions. Liver hydroxyproline content was determined as described previously [19 (
link)].
Histology and Immunohistochemistry: Formalin-fixed, paraffin-embedded sections were prepared from liver tissue, then stained with hematoxylin and eosin or picrosirius red using standard techniques. For immunohistochemistry, antigen retrieval was performed by heating in 10 mM sodium citrate buffer, with a pH of 6.0, then specimens were incubated overnight with primary antibodies. The antibodies used were specific for smooth muscle α-actin (clone 1A4, #A5228, SMA, Sigma Chemical, St. Louis, MO, USA)) at 1:2000, or collagen type 1 (Abcam, Cambridge, MA, USA) #ab21286) at 1:250. Secondary horseradish peroxidase polymer-conjugated antibodies and detection were carried out using the EnVision kit (Agilent Dako, Santa Clara, CA, USA). Images were collected using a Nikon Eclipse 80i microscope and DS-Fi2 camera. For quantification of staining intensity, low-power images (2× objective) were subjected to histomorphometry using ImageJ software to apply color deconvolution and threshold functions, and data are expressed as the percent staining per total tissue area.
Cell culture: The LX-2 cell line, derived from human hepatic stellate cells, was generously provided by Dr. Scott Friedman (Icahn School of Medicine at Mount Sinai, New York, USA) [20 (
link)]. The cells were maintained in a humidified 5% CO
2 environment in Dulbecco’s modified Eagle’s medium supplemented with 2% fetal bovine serum. For experiments, cells were activated with 0.1 nM recombinant human TGF-β (R&D Systems, Minneapolis, MN, USA) overnight, then treated with 1 µM SR1664 or vehicle (0.05% dimethylformamide) for 16 h.
Gene expression assays: RNA was extracted from cell lysates using the PureLink Mini Kit (ThermoFisher, Waltham, MA, USA). The RNA was treated with DNase to remove the contaminating genomic DNA. The integrity and purity were determined by visualization on agarose gels. The RNA was quantified using the RiboGreen assay (ThermoFisher, Waltham, MA, USA), and 2 µg RNA was converted to cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA) in a total volume of 20 µL. For gene expression assays, 40 ng cDNA was used with TaqMan Universal Master Mix in a 20 µL reaction volume using a CFX Connect PCR system (Bio-Rad, Hercules, CA, USA). All primers and/or probe sets were designed to span introns. The human gene expression assays used (all from ThermoFisher, Waltham, MA, USA) included collagen type 1 (
COL1A2), SMA (
ACTA2), tissue inhibitor of matrix metalloproteinases-1 (
TIMP1), tissue inhibitor of matrix metalloproteinases-2 (
TIMP2), plasminogen activator inhibitor-1 (
SERPINE1), transforming growth factor-β (
TGFB), matrix metalloproteinase-1 (
MMP1), matrix metalloproteinase-3 (
MMP3), and glucuronidase-β (
GUSB). The gene assay numbers and probe context sequences are provided in
Table 1. The gene expression was normalized to that of
GUSB, and the data expressed as expression relative to that of the control sample using the ΔΔCt method.
Western blotting: Whole-cell lysates were prepared using RIPA lysis buffer (20 mM Tris (pH 7.5) containing 150 mM NaCl, 1% Nonidet P40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and Halt Phosphatase and Protease Inhibitor Cocktail (ThermoFisher Waltham, MA, USA). The total protein was quantified using the bicinchoninic acid (BCA) assay (ThermoFisher Waltham, MA, USA). A total of 25 µg protein was resolved on 10% polyacrylamide gels, then transferred to Immobilon-FL membranes (Millipore). Immunoblotting was performed using mouse anti-SMA antibody (#A5228, 1:2000, Sigma Chemical, St. Louis, MO, USA)) and rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (#G9545, 1:10,000, Sigma Chemical, St. Louis, MO, USA), as well as secondary goat anti-mouse and goat anti-rabbit secondary antibodies labeled with IRDye 800 or IRDye 680, respectively (Li-Cor Biosciences, Lincoln, NE, USA). The blots were imaged using an Odyssey near-infrared scanner (Li-Cor Biosciences, Lincoln, NE, USA).
Cell proliferation assay: LX-2 cells were serum-deprived overnight in DMEM + 0.2% BSA (bovine serum albumin) in the presence of 1 µM SR1664 or vehicle. The cells were then stimulated with recombinant human platelet-derived growth factor BB (PDGF-BB, R&D Systems, Minneapolis, MN, USA) at 10 ng/mL, or vehicle (DMEM + 0.2% BSA) overnight. For the final 3 h of incubation,
3H-thymidine was added to the culture, then the DNA was precipitated using 10% trichloroacetic acid, solubilized in 0.1 M NaOH/0.1% SDS, mixed with Ultima Gold scintillation fluid, and counted in a liquid scintillation counter (PerkinElmer, Hopkinton, MA, USA).
Statistical analysis: All data are expressed as mean ± standard error. All graphs and statistical analyses were performed using Prism 8.0 software (GraphPad). Comparisons across multiple groups were conducted using one-way analysis of variance (ANOVA) with the Tukey post-test. Comparisons between two groups were made using the two-tailed Student
t-test. In all cases, a value of
p < 0.05 was considered significant.
McVicker B.L., Hamel F.G., Simpson R.L, & Bennett R.G. (2020). A Selective PPARγ Modulator Reduces Hepatic Fibrosis. Biology, 9(7), 151.
