Journal: Antioxidants

Article Title: Pirfenidone Sensitizes Hepatic Stellate Cells to Ferroptosis by Reprogramming Glutamine and Serine Metabolism for GSH Depletion

doi: 10.3390/antiox15050552

Figure Lengend Snippet: Pirfenidone suppresses activation markers and inhibits the proliferation of aHSCs. Primary rat HSCs were treated with day 1 (qHSC), day 3 (activating), and day 7 (aHSC) post-isolation. The cells were exposed to a dose range of pirfenidone (PFD, 10–1000 μM) or the glutaminase 1 (GLS1) inhibitor CB-839 (0.1–10 μM) for 72 h. ( A – C ) Western blot and quantitative analysis of the activation markers collagen type I and alpha-smooth muscle actin (α-SMA) in qHSCs (Day 1; n = 5), activating HSCs (Day 3; n = 4) and aHSCs (Day 7; n = 4). ( D ) Quantification of aHSC proliferation by bromodeoxyuridine (BrdU) incorporation assay following treatment with PFD or CB-839 (5 μM) ( n = 3 per group). ( E ) Real-time monitoring of aHSC proliferation using the xCELLigence Real-Time Cell Analysis (RTCA) system, presented as a Normalized Cell Index. Data are presented as mean ± SD. The statistical significance of differences was analyzed by one-way ANOVA followed by Tukey’s post hoc test. * p < 0.05, ** p < 0.01.

Article Snippet: Pirfenidone (PFD), erastin (ERA), and ferrostatin-1 (Fer-1) were purchased from MedChemExpress (Sollentuna, Sweden).

Techniques: Activation Assay, Isolation, Western Blot, BrdU Incorporation Assay, Cell Analysis

Journal: Antioxidants

Article Title: Pirfenidone Sensitizes Hepatic Stellate Cells to Ferroptosis by Reprogramming Glutamine and Serine Metabolism for GSH Depletion

doi: 10.3390/antiox15050552

Figure Lengend Snippet: Pirfenidone minimally affects the transcription of key activation markers. Primary rat HSCs at quiescent (Day 1), intermediate (Day 3), and fully activated (Day 7) states were treated with the indicated doses of pirfenidone (PFD) or CB-839 for 72 h ( n = 4 per group). ( A – C ) Relative mRNA levels of collagen type I ( Col1a1 ), alpha-smooth muscle actin ( Acta2 ), and matrix metallopeptidase 9 ( Mmp9 ) in qHSCs. ( D – F ) Relative mRNA levels of the same markers in activating HSCs. ( G – I ) Relative mRNA levels in aHSCs. Data are presented as mean ± SD. The statistical significance of differences was analyzed by one-way ANOVA followed by Tukey’s post hoc test. * p < 0.05, ** p < 0.01.

Article Snippet: Pirfenidone (PFD), erastin (ERA), and ferrostatin-1 (Fer-1) were purchased from MedChemExpress (Sollentuna, Sweden).

Techniques: Activation Assay

Journal: Antioxidants

Article Title: Pirfenidone Sensitizes Hepatic Stellate Cells to Ferroptosis by Reprogramming Glutamine and Serine Metabolism for GSH Depletion

doi: 10.3390/antiox15050552

Figure Lengend Snippet: Pirfenidone treatment reduces protein levels of key metabolic enzymes in HSCs. Primary rat HSCs at quiescent (qHSCs, Day 1), intermediate (Day 3), and fully activated (aHSCs, Day 7) states were treated with pirfenidone (PFD, 10–1000 μM) or CB-839 for 72 h. ( A – C ) Western blot and corresponding quantitative analysis of glutaminase 1 (GLS1), serine hydroxymethyltransferase 2 (SHMT2), and pyrroline-5-carboxylate synthase (P5CS) protein levels in qHSCs ( n = 5), intermediate activating HSCs ( n = 4), and aHSCs ( n = 4). Data are presented as mean ± SD. The statistical significance of differences was analyzed by one-way ANOVA followed by Tukey’s post hoc test. * p < 0.05, ** p < 0.01.

Article Snippet: Pirfenidone (PFD), erastin (ERA), and ferrostatin-1 (Fer-1) were purchased from MedChemExpress (Sollentuna, Sweden).

Techniques: Western Blot

Journal: Antioxidants

Article Title: Pirfenidone Sensitizes Hepatic Stellate Cells to Ferroptosis by Reprogramming Glutamine and Serine Metabolism for GSH Depletion

doi: 10.3390/antiox15050552

Figure Lengend Snippet: Pirfenidone prevents ATF4 nuclear accumulation and suppresses mitochondrial respiration in aHSCs. aHSCs were treated with pirfenidone (PFD, 10–1000 μM) or the glutaminase 1 (GLS1) inhibitor CB-839 (5 μM) for 72 h. For activating transcription factor 4 (ATF4) analysis, tunicamycin and 4-phenylbutyrate (4-PBA) were administered as positive and negative controls, respectively, during the final 12 h of treatment. ( A ) Representative immunofluorescence images of ATF4 localization (scale bar: 25 μm). ( B – F ) Quantification of mitochondrial respiration parameters in treated aHSCs, measured using a Seahorse XF96 Analyzer. The analysis shows ( C ) basal respiration, ( D ) ATP-linked respiration, ( E ) maximal respiration, and ( F ) proton leak, derived from the oxygen consumption rate (OCR) profile after sequential injections of oligomycin (Oligo), dinitrophenol (DNP), and a mixture of rotenone and antimycin A (R + A). Data are presented as mean ± SD. The statistical significance of differences was analyzed by one-way ANOVA followed by Tukey’s post hoc test. * p < 0.05, ** p < 0.01.

Article Snippet: Pirfenidone (PFD), erastin (ERA), and ferrostatin-1 (Fer-1) were purchased from MedChemExpress (Sollentuna, Sweden).

Techniques: Immunofluorescence, Derivative Assay

Journal: Antioxidants

Article Title: Pirfenidone Sensitizes Hepatic Stellate Cells to Ferroptosis by Reprogramming Glutamine and Serine Metabolism for GSH Depletion

doi: 10.3390/antiox15050552

Figure Lengend Snippet: Pirfenidone sensitizes aHSCs to ferroptosis by disrupting glutathione homeostasis and promoting lipid peroxidation. aHSCs were treated with pirfenidone (PFD) for 72 h. During the final 24 h of incubation, cells were co-treated with either the ferroptosis inducer erastin (ERA, 10 μM), the ferroptosis inhibitor ferrostatin-1 (Fer-1, 1 μM), or the antioxidant N-acetylcysteine (NAC, 2 mM). ( A ) Western blot and quantitative analysis of collagen type I ( n = 6) and α-SMA ( n = 5) protein levels. ( B – D ) Quantification of total glutathione (GSH), oxidized glutathione (GSSG), and the free GSH pool. ( E ) Protein expression levels of the lipid peroxidation marker 4-hydroxynonenal (4-HNE), presented as fold change relative to untreated controls ( n = 3 per group). Data are presented as mean ± SD. The statistical significance of differences was analyzed by one-way ANOVA followed by Tukey’s post hoc test. * p < 0.05, ** p < 0.01.

Article Snippet: Pirfenidone (PFD), erastin (ERA), and ferrostatin-1 (Fer-1) were purchased from MedChemExpress (Sollentuna, Sweden).

Techniques: Incubation, Western Blot, Expressing, Marker

Journal: Antioxidants

Article Title: Pirfenidone Sensitizes Hepatic Stellate Cells to Ferroptosis by Reprogramming Glutamine and Serine Metabolism for GSH Depletion

doi: 10.3390/antiox15050552

Figure Lengend Snippet: Pirfenidone potentiates erastin-induced ROS production, labile iron accumulation, and cell death in aHSCs. aHSCs were treated as described in . ( A ) Representative fluorescence images and corresponding intensity quantification of intracellular reactive oxygen species (ROS) using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) staining (scale bar: 100 μm). ( A’ ) Data were quantified from 8 random fields per group. ( B ) Representative fluorescence images and corresponding intensity quantification of the intracellular labile iron pool using FerroOrange staining (scale bar: 100 μm). ( B’ ) Data were quantified from 8 random fields per group. ( C ) Cell viability was assessed by Cell Counting Kit-8 (CCK-8) assay ( n = 3 per group). Data are presented as mean ± SD. The statistical significance of differences was analyzed by one-way ANOVA followed by Tukey’s post hoc test. * p < 0.05, ** p < 0.01.

Article Snippet: Pirfenidone (PFD), erastin (ERA), and ferrostatin-1 (Fer-1) were purchased from MedChemExpress (Sollentuna, Sweden).

Techniques: Fluorescence, Staining, Cell Counting, CCK-8 Assay

Journal: Antioxidants

Article Title: Pirfenidone Sensitizes Hepatic Stellate Cells to Ferroptosis by Reprogramming Glutamine and Serine Metabolism for GSH Depletion

doi: 10.3390/antiox15050552

Figure Lengend Snippet: Schematic of the mechanism by which pirfenidone remodels metabolism and sensitizes hepatic stellate cells to ferroptosis. Pirfenidone inhibits the nuclear accumulation of the metabolic transcription factor ATF4. This suppression downregulates the expression of key metabolic enzymes including glutaminase 1 (GLS1), serine hydroxymethyltransferase 2 (SHMT2), and pyrroline-5-carboxylate synthase (P5CS). This coordinated downregulation has two consequences for HSC fate. First, it impairs glutaminolysis, de novo glycine synthesis, and proline synthesis, thereby limiting the availability of precursors for collagen production and inhibiting HSC activation. Second, the reduced supply of glutamate and glycine, which are essential substrates for glutathione (GSH) synthesis, leads to GSH depletion. This depletion sensitizes the cells to lipid peroxidation and subsequent ferroptosis. Red arrows indicate points of downregulation (↓) or upregulation (↑) within the metabolic network following pirfenidone treatment. This figure was created in BioRender. Li, J. (2026), https://BioRender.com/35yqyyj (accessed on 20 March 2026).

Article Snippet: Pirfenidone (PFD), erastin (ERA), and ferrostatin-1 (Fer-1) were purchased from MedChemExpress (Sollentuna, Sweden).

Techniques: Expressing, Activation Assay

Journal: Chest

Article Title: Clinical Molecular Imaging of Pulmonary CXCR4 Expression to Predict Outcome of Pirfenidone Treatment in Idiopathic Pulmonary Fibrosis.

doi: 10.1016/j.chest.2020.08.2043

Figure Lengend Snippet: Figure 3 – A-H, Targeted PET/CT scan detects early changes in pulmonary CXCR4 expression in IPF patients after commencing treatment with pirfenidone. Early change in pulmonary CXCR4 expression was quantified using CXCR4-targeted 68Ga-pentixafor PET scanning. A, B, Exemplary images of a patient with decreasing pulmonary CXCR4 signal after commencing pirfenidone treatment and stable FVC at 12 months: before treatment at baseline (A) and after 6 weeks of pirfenidone treatment (B). C-H, After initiation of pirfenidone therapy, the decrease in CXCR4 signal between the initial and the follow-up PET scan is less in patients with worse outcome (C, D). Patients with worse outcome demonstrate an increase in CXCR4 expression at the follow-up PET scan (E, F). Early change in CXCR4 signal between the initials and the follow-up PET scan is statistically significantly correlated with outcome, as determined by FVC decline after 12 months of pirfenidone treatment (G, H). See Figure 1 and 2 legends for expansion of other abbreviations.

Article Snippet: 1x106 BAL cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 Medium (ThermoFisher Scientific #11879020) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich #TMS-013-B) and 1% penicillin/streptomycin (Biochrom #A2212) for 24h in an incubator (5% CO2, 37°C) w/wo 2mM pirfenidone (Santa Cruz Biotechnology, #sc-203663) or 10μM SB-431542 (a selective and potent inhibitor of the TGF-β/Activin/NODAL pathway, STEMCELLTM technologies #72234) treatment.

Techniques: Positron Emission Tomography-Computed Tomography, Expressing

Journal: Arthritis Research & Therapy

Article Title: The senescence-like activity of BMS-470539 is associated with anti-fibrotic actions in models of dermal fibrosis

doi: 10.1186/s13075-025-03635-3

Figure Lengend Snippet: In vitro anti-fibrotic activity of BMS-470539 on cultured human dermal fibroblasts. ( A ) Systemic sclerosis (SSc) fibroblasts were treated with 10 µM BMS-470539, every other day for 6 days and αSMA expression analysed by immunofluorescence. ( B ) To address migratory activity, a scratch was created on cells previously treated with 10 µM BMS-470539 for 6 days and incubated for further 24 h in fresh media. Images were acquired on a light microscope and gap size quantified using Fiji imaging processing tool. ( C ) SSc fibroblasts were treated with 10 µM BMS-470539, 0.5 mM pirfenidone, 0.5 µM nintedanib or appropriate vehicle control (PBS or DMSO), every other day for 6 days. Cytokines were measured in supernatants by ELISA. Data ( A , B ) represent mean ± SEM of n = 8–9 and was analysed by paired t-test * p < 0.05, *** p < 0.001. Fluorescent images were captured at 10X and scale bars represent 200 μm. Data ( C ) represent mean ± SEM of n = 3 and was analysed by paired one-way ANOVA vs. control followed by Dunnett’s multiple comparisons test - * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The compounds used in this study were: BMS-470539 - CAS No. 2341796-82-3 (Tocris [for in vitro experiments] and Cayman Chemical [for in vivo experiments ] ), bleomycin (Cayman Chemical), alpha-melanocyte stimulating hormone ⍺MSH (Tocris), dersimelagon - CAS No. 1835256-48-8 (Merk), dimethyl sulfoxide (Merck), nintedanib (Tocris) and pirfenidone (Tocris).

Techniques: In Vitro, Activity Assay, Cell Culture, Expressing, Immunofluorescence, Incubation, Light Microscopy, Imaging, Control, Enzyme-linked Immunosorbent Assay

Journal: Journal of ethnopharmacology

Article Title: Gracillin relieves pulmonary fibrosis by suppressing the STAT3 axis.

doi: 10.1016/j.jep.2023.116704

Figure Lengend Snippet: Fig. 2. Safety profile of gracillin in the treatment of pulmonary edema and inflammatory exudation. Mice were treated with BLM (3 mg/kg) or saline to induce PF. Following treatment with gracillin (1 or 2 mg/kg every other day) or pirfenidone (50 mg/kg/day) from day 2 to day 28, mice were sacrificed, and parameters indicating safety and efficacy were determined. (A) Wet/dry weight ratio of lung. n = 5. (B) Live/total cell ratio in BLAF. n = 5. (C) BLAF total protein concentration. n = 5. (D–H) The secretion of (D) TGF-β1 and the inflammatory factors (E) IL-1β, (F) IL-6 and (G) TNF-α in BALF and the secretion of (H) TGF-β1 in serum were determined by ELISA. n = 5. (I) HE staining of heart (upper), liver (middle) and kidney (lower) sections, 200 × magnification. Scale bar: 50 μm. (J) Body

Article Snippet: Pirfenidone (PFD; #HY-B0673) was purchased from MedChemExpress (Princeton, New Jersey, USA), and was re-suspended in 0.5% sodium carboxymethyl cellulose (CMC-Na) for treatment.

Techniques: Saline, Protein Concentration, Enzyme-linked Immunosorbent Assay, Staining

Journal: Human Reproduction (Oxford, England)

Article Title: Endometriotic tissue fragments are viable after cryopreservation in an ex vivo tissue model recapitulating the fibrotic microenvironment

doi: 10.1093/humrep/deae164

Figure Lengend Snippet: RT-PCR analysis of ex vivo treated tissue fragments . Cryopreserved and thawed tissue fragments from two patients were cultured for 72 h: without treatment (condition 1); with 5 mM metformin (condition 2); with 250 μM pirfenidone (condition 3). ( A ) RT-PCR signal of Cell Migration Inducing Protein (CEMIP) is less intense compared to reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in both pirfenidone treated (condition 3) patient samples compared to untreated tissue (condition 1). ( B ) RT-qPCR analysis of the same samples. Fold changes of RT-qPCR are calculated using the ΔΔCt-method. The fold change for COL1A1 was 0.45 and for CEMIP 0.16 compared to untreated tissue. Lines represent means and circles and squares represent individual values.

Article Snippet: Next, the fragments were cultured for 72 h in DMEM, 10% FBS, and 40 μg/ml gentamicin, with or without the addition of 250 μM pirfenidone (Pirfenidone CRS PHRS, Merck, Germany) or 5 mM metformin (Metformin 317240-5GM, Merck, Germany).

Techniques: Reverse Transcription Polymerase Chain Reaction, Ex Vivo, Cell Culture, Migration, Quantitative RT-PCR

Journal: Human Reproduction (Oxford, England)

Article Title: Endometriotic tissue fragments are viable after cryopreservation in an ex vivo tissue model recapitulating the fibrotic microenvironment

doi: 10.1093/humrep/deae164

Figure Lengend Snippet: RT-PCR analysis of myofibroblasts isolated from cryopreserved and thawed, ex vivo -treated tissue fragments . Sections were cut from one tissue fragments after 72-h culture: without treatment (condition 1); with 5 mM metformin (condition 2); with 250 μM pirfenidone (condition 3). ( A ) Areas rich in myofibroblasts were selected (left panel, yellow dotted line) and isolated by laser capture microdissection (right panel, after laser cutting). Arrowheads point out endometriotic glands. Note that individual cells are difficult to distinguish due to the thickness of the sections. Scale bar = 100 μm. ( B ) RT-PCR signal of Cell Migration Inducing Protein (CEMIP) is less intense compared to reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in metformin-treated (condition 2) tissue compared to untreated tissue (condition 1).

Article Snippet: Next, the fragments were cultured for 72 h in DMEM, 10% FBS, and 40 μg/ml gentamicin, with or without the addition of 250 μM pirfenidone (Pirfenidone CRS PHRS, Merck, Germany) or 5 mM metformin (Metformin 317240-5GM, Merck, Germany).

Techniques: Reverse Transcription Polymerase Chain Reaction, Isolation, Ex Vivo, Laser Capture Microdissection, Migration