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Figure Lengend Snippet: Standard-dose bleomycin increases pulmonary vascular permeability in EC- S1pr1 −/− mice. ( A ) Evans blue dye assay was performed on Day 0 and Day 7 after standard-dose intratracheal (IT) bleomycin (1.0 U/kg). ( B ) Quantification of vascular permeability was calculated as an Evans blue (EB) index defined as the ratio of lung Evans blue to plasma Evans blue. n = 3–7 mice per group. ( C ) Hematoxylin and eosin and Masson’s trichrome staining of lungs was performed on Day 7 after standard dose bleomycin. Representative images are shown from n = 3 mice per group. Scale bars, 50 μm. ( D ) Survival of mice receiving standard-dose (1.0 U/kg) bleomycin. n = 5 mice per group. * P < 0.05 comparing survival of EC- S1pr1 −/− mice to littermate controls S1pr1 f/f . *** P < 0.001 comparing EB index in EC- S1pr1 −/− mice to littermate controls S1pr1 f/f . H&E = hematoxin and eosin; Trich = trichrome.

Article Snippet: Fibrosis was induced by single-dose intratracheal (IT) injection of bleomycin (Fresenius Kabi) at a standard dose of 1.0 units/kg and also at a reduced dose of 0.5 units/kg, and quantified by measuring hydroxyproline content, per our usual methods ( 20 ).

Techniques: Permeability, Clinical Proteomics, Staining

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Figure Lengend Snippet: Low-dose bleomycin induces fibrosis in EC- S1pr1 −/− mice. ( A ) Survival of mice receiving low-dose (0.5 U/kg) bleomycin. n = 5 mice per group. ( B ) Total protein content in the BAL was quantified at Days 0, 7, 14, and 21 after low-dose bleomycin. BAL from EC- S1pr1 −/− mice was compared with intact littermate controls S1pr1 f/f . n = 3–5 mice per group. ( C ) Hematoxylin and eosin and Masson’s trichrome staining of lungs were performed on Day 21 after low-dose bleomycin. Representative images are shown from n = 3 mice per group. Scale bars, 50 μm. ( D ) Fibrosis was quantified at Days 0, 7, 14, 21, and 42 after low-dose IT bleomycin by measuring hydroxyproline levels in the mouse lungs. n = 3–15 mice per group. ( E ) Ashcroft scoring performed on trichrome stained lung sections from EC- S1pr1 −/− mice at Days 0 and 14 or 21 was compared with intact littermate controls S1pr1 f/f . n = 3–4 mice per group. * P < 0.05, ** P < 0.01, and *** P < 0.001 for comparisons of total protein or hydroxyproline in EC- S1pr1 −/− mice compared with intact littermate control mice S1pr1 f/f . OHP = hydroxyproline.

Article Snippet: Fibrosis was induced by single-dose intratracheal (IT) injection of bleomycin (Fresenius Kabi) at a standard dose of 1.0 units/kg and also at a reduced dose of 0.5 units/kg, and quantified by measuring hydroxyproline content, per our usual methods ( 20 ).

Techniques: Staining, Control

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Article Title:

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Figure Lengend Snippet: Endothelial-specific S1pr1 deletion leads to reduced intercellular junction proteins and increased extravascular coagulation. ( A ) Mice were injected with FITC-labeled albumin to identify albumin extravasated into the lung tissue after bleomycin. Sequential sectioning and staining of the lung tissue were performed with Masson’s trichrome stain. Representative images are shown from n = 3 mice per group. Scale bars, 100 μm. ( B ) D-dimer ELISA was performed on BAL fluid collected from mice on days 0, 7, 14, and 21 after low-dose IT bleomycin (0.5 U/kg). n = 4 mice per group. ( C ) D-dimer ELISA was performed on homogenized lung tissue collected from mice on days 0, 7, 14, and 21 after low-dose IT bleomycin (0.5 U/kg). n = 4 mice per group. ( D ) Junctional proteins were assessed through immunoblotting for VE-Cadherin in lung tissue homogenates harvested at days 0 and 14 after low-dose bleomycin challenge. Densitometry was performed to quantify differences in protein expression at each time point. n = 2–3 mice per group. * P < 0.05 and *** P < 0.001 for comparisons of D-dimer in the BAL and lung tissue of EC- S1pr1 −/− mice as compared with intact littermate controls S1pr1 f/f .

Article Snippet: Fibrosis was induced by single-dose intratracheal (IT) injection of bleomycin (Fresenius Kabi) at a standard dose of 1.0 units/kg and also at a reduced dose of 0.5 units/kg, and quantified by measuring hydroxyproline content, per our usual methods ( 20 ).

Techniques: Coagulation, Injection, Labeling, Staining, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing

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Figure Lengend Snippet: Immune cell populations in the BAL of mice lacking endothelial S1pr1 after bleomycin. ( A ) Total cell counts were quantified from BAL obtained on Day 0 and Day 7 after low-dose IT bleomycin. n = 3–4 mice per group. Flow cytometry was performed on BAL collected 7 days after bleomycin challenge in EC- S1pr1 −/− and control mice to assess the number of ( B ) alveolar macrophages, ( C ) dendritic cells, ( D ) NK cells, ( E ) monocyte-derived dendritic cells (DCs), ( F ) inflammatory monocytes, ( G ) CD4 + T cells, ( H ) CD8 + T cells, and ( I ) T regulatory cells. In contrast, no differences were detected in the quantities of BAL ( J ) B cells, ( K ) eosinophils, ( L ) γδ T cells, or ( M ) neutrophils. n = 3–4 mice per group. * P < 0.05, ** P < 0.01, and *** P < 0.001 for comparisons of BAL cells in EC- S1pr1 −/− mice as compared with intact control mice S1pr1 f/f . γδ T cells = GD T cells.

Article Snippet: Fibrosis was induced by single-dose intratracheal (IT) injection of bleomycin (Fresenius Kabi) at a standard dose of 1.0 units/kg and also at a reduced dose of 0.5 units/kg, and quantified by measuring hydroxyproline content, per our usual methods ( 20 ).

Techniques: Flow Cytometry, Control, Derivative Assay

Journal:

Article Title:

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Figure Lengend Snippet: Bleomycin-induced lung injury reduces endothelial cell populations in the lung. ( A ) Immunoblotting was performed using protein from lung homogenates of C57BL/6 wild-type mice (WT) at Days 0, 1, 3, 7, and 14 after IT bleomycin (0.5 U/kg). Blots were probed for S1PR1, and protein expression levels were compared using GAPDH as a loading control. n = 2–3 mice per group. Densitometry was performed using Image J software (NIH). ( B ) Flow cytometry to identify CD31 + cells was performed on lung tissue from WT mice on Days 0, 7, and 14 after standard-dose IT bleomycin. n = 4 mice per group. ( C ) Flow cytometry of CD31 + cells expressing S1PR1. n = 4 mice per group. ( D ) The percentage of endothelial cells expressing S1PR1 at each time point after bleomycin was quantified. ( E ) Mean fluorescence intensity of S1PR1 in CD31 + cells after bleomycin was quantified. ( F ) Plasma S1P levels in WT mice were quantified at Days 0, 3, 7, 10, and 14 after IT bleomycin. n = 3–4 mice per group. ( G ) Immunoflourescence co-staining on lungs from naive and bleomycin treated mice at Day 14 was performed for S1PR1 (red), endothelial CDH5/VE-cadherin (green), and nuclear DAPI (blue). White box indicates a fibrotic region. Scale bars, 50 μm. * P < 0.05, ** P < 0.01, and *** P < 0.001 for comparisons of cells in WT mice at various time points after bleomycin injury.

Article Snippet: Fibrosis was induced by single-dose intratracheal (IT) injection of bleomycin (Fresenius Kabi) at a standard dose of 1.0 units/kg and also at a reduced dose of 0.5 units/kg, and quantified by measuring hydroxyproline content, per our usual methods ( 20 ).

Techniques: Western Blot, Expressing, Control, Software, Flow Cytometry, Fluorescence, Clinical Proteomics, Staining

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Figure Lengend Snippet: Increased circulating S1P is insufficient to protect mice from bleomycin-induced pulmonary fibrosis. ( A ) Transgenic mice overexpressing human apolipoprotein M (ApoM Tg + ) were bred, and Evans blue dye assay was performed at Day 7 after standard-dose IT bleomycin. ( B ) Evans blue indices were calculated using Evans blue dye indices in the lungs and plasma of ApoM Tg + mice and littermate controls (WT). n = 4 mice per group. ( C ) BAL total protein was quantified at Day 14 after standard dose bleomycin. ( D ) Histology at Days 0 and 14 after standard-dose IT bleomycin were stained with hematoxylin and eosin and Masson’s trichrome staining. Representative images are shown from n = 3 mice per group. Scale bars, 100 μm. ( E ) Quantification of fibrosis was performed with hydroxyproline assay. ( F ) S1P levels were quantified by mass spectrometry from circulating plasma at baseline (Day 0), Day 7, and Day 14 after standard-dose bleomycin. n = 3–6 mice per group. n = 3–8 mice per group. *** P < 0.001, and **** P < 0.0001 for comparisons of S1P between ApoM-Tg + and control mice.

Article Snippet: Fibrosis was induced by single-dose intratracheal (IT) injection of bleomycin (Fresenius Kabi) at a standard dose of 1.0 units/kg and also at a reduced dose of 0.5 units/kg, and quantified by measuring hydroxyproline content, per our usual methods ( 20 ).

Techniques: Transgenic Assay, Clinical Proteomics, Staining, Hydroxyproline Assay, Mass Spectrometry, Control

Journal: Toxicology Reports

Article Title: Phloridzin mitigates bleomycin-elicited lung fibrosis in Wistar rats: The interplay between antioxidant defenses, inflammatory processes, transforming growth factor beta 1, and autophagy

doi: 10.1016/j.toxrep.2026.102267

Figure Lengend Snippet: Phloridzin dose-dependently mitigated the effect of bleomycin on BALF LDH activity. Values are expressed as mean ± standard deviation. Number of animals = 15 rats in each group. One-way analysis of variance (ANOVA) was used to assess the differences between the different groups, and then Tukey's multiple comparisons post-hoc test was employed. A p-value less than 0.05 was considered statistically significant. BLM: bleomycin; LDH: lactate dehydrogenase; NS: non-significant; PZ: phloridzin.

Article Snippet: Bleomycin, a white powder with a purity of ≥ 98%, a product of Celon Laboratories Ltd., Hyderabad, Telangana, India (CAS # 9041–93–4).

Techniques: Activity Assay, Standard Deviation

Journal: Toxicology Reports

Article Title: Phloridzin mitigates bleomycin-elicited lung fibrosis in Wistar rats: The interplay between antioxidant defenses, inflammatory processes, transforming growth factor beta 1, and autophagy

doi: 10.1016/j.toxrep.2026.102267

Figure Lengend Snippet: Phloridzin dose-dependently exhibited antioxidant effects in bleomycin-treated animals. Values are expressed as mean ± standard deviation. Number of animals = 15 rats in each group. One-way analysis of variance (ANOVA) was used to assess the differences between the different groups, and then Tukey's multiple comparisons post-hoc test was employed. A p-value less than 0.05 was considered statistically significant. BLM: bleomycin; GSH: reduced glutathione; MDA: malondialdehyde; NS: non-significant; PZ: phloridzin.

Article Snippet: Bleomycin, a white powder with a purity of ≥ 98%, a product of Celon Laboratories Ltd., Hyderabad, Telangana, India (CAS # 9041–93–4).

Techniques: Standard Deviation

Journal: Toxicology Reports

Article Title: Phloridzin mitigates bleomycin-elicited lung fibrosis in Wistar rats: The interplay between antioxidant defenses, inflammatory processes, transforming growth factor beta 1, and autophagy

doi: 10.1016/j.toxrep.2026.102267

Figure Lengend Snippet: Phloridzin dose-dependently abrogated the effect of bleomycin on the inflammatory markers in the lung tissues. Values are expressed as mean ± standard deviation. Number of animals = 15 rats in each group. One-way analysis of variance (ANOVA) was used to assess the differences between the different groups, and then Tukey's multiple comparisons post-hoc test was employed. A p-value less than 0.05 was considered statistically significant. BLM: bleomycin; IL-1β: interleukin-1 beta; NF-κB: nuclear factor kappa B; NS: non-significant; PZ: phloridzin.

Article Snippet: Bleomycin, a white powder with a purity of ≥ 98%, a product of Celon Laboratories Ltd., Hyderabad, Telangana, India (CAS # 9041–93–4).

Techniques: Standard Deviation

Journal: Toxicology Reports

Article Title: Phloridzin mitigates bleomycin-elicited lung fibrosis in Wistar rats: The interplay between antioxidant defenses, inflammatory processes, transforming growth factor beta 1, and autophagy

doi: 10.1016/j.toxrep.2026.102267

Figure Lengend Snippet: Phloridzin dose-dependently ameliorated the effect of bleomycin on TGF-β1 levels in the lung tissues. Values are expressed as mean ± standard deviation. Number of animals = 15 rats in each group. One-way analysis of variance (ANOVA) was used to assess the differences between the different groups, and then Tukey's multiple comparisons post-hoc test was employed. A p-value less than 0.05 was considered statistically significant. BLM: bleomycin; NS: non-significant; PZ: phloridzin; TGF-β1: transforming growth factor beta 1.

Article Snippet: Bleomycin, a white powder with a purity of ≥ 98%, a product of Celon Laboratories Ltd., Hyderabad, Telangana, India (CAS # 9041–93–4).

Techniques: Standard Deviation

Journal: Toxicology Reports

Article Title: Phloridzin mitigates bleomycin-elicited lung fibrosis in Wistar rats: The interplay between antioxidant defenses, inflammatory processes, transforming growth factor beta 1, and autophagy

doi: 10.1016/j.toxrep.2026.102267

Figure Lengend Snippet: A photomicrograph demonstrating the immunohistochemical staining of the lung tissue specimens for cleaved caspase-3 (x200, scale bar=40 µm) from A) and B) the control group and PZ-alone treated group respectively showing minimal positive immune expression of cleaved caspase-3 (Arrow); C) the group treated with bleomycin alone showing strong positive immune expression of cleaved caspase-3 (Arrows); D) the bleomycin group treated with phloridzin 60 mg/kg/day showing moderate positive immune expression of cleaved caspase-3 (Arrows); E) the bleomycin group treated with phloridzin 120 mg/kg/day showing mild positive immune expression of cleaved caspase-3 (Arrows); and F) The percentage effect of phloridzin (60 and 120 mg/kg/day, p.o) on the immune expression of cleaved caspase-3 in the lung tissues of bleomycin-treated rats. Values are expressed as mean ± standard deviation. Number of animals = 15 rats in each group. One-way analysis of variance (ANOVA) was used to assess the differences between the different groups, and then Tukey's multiple comparisons post-hoc test was employed. A p-value less than 0.05 was considered statistically significant. BLM: bleomycin; NS: non-significant; PZ: phloridzin.

Article Snippet: Bleomycin, a white powder with a purity of ≥ 98%, a product of Celon Laboratories Ltd., Hyderabad, Telangana, India (CAS # 9041–93–4).

Techniques: Immunohistochemical staining, Staining, Control, Expressing, Standard Deviation

Journal: Toxicology Reports

Article Title: Phloridzin mitigates bleomycin-elicited lung fibrosis in Wistar rats: The interplay between antioxidant defenses, inflammatory processes, transforming growth factor beta 1, and autophagy

doi: 10.1016/j.toxrep.2026.102267

Figure Lengend Snippet: A photomicrograph demonstrating the immunohistochemical staining of the lung tissue specimens for beclin-1 (×200, scale bar = 40 µm) from A) and B) the control group and PZ-alone treated group respectively showing strong positive immune expression of beclin-1 (Arrows); C) the group treated with bleomycin alone showing minimal positive immune expression of beclin-1 (Arrows); D) the bleomycin group treated with phloridzin 60 mg/kg/day showing moderate positive immune expression of beclin-1 (Arrows); E) the bleomycin group treated with phloridzin 120 mg/kg/day showing strong positive immune expression of beclin-1 (Arrows); and F) The percentage effect of phloridzin (60 and 120 mg/kg/day, p.o) on the immune expression of beclin-1 in the lung tissues of bleomycin-treated rats. Values are expressed as mean ± standard deviation. Number of animals = 15 rats in each group. One-way analysis of variance (ANOVA) was used to assess the differences between the different groups, and then Tukey's multiple comparisons post-hoc test was employed. A p-value less than 0.05 was considered statistically significant. BLM: bleomycin; NS: non-significant; PZ: phloridzin.

Article Snippet: Bleomycin, a white powder with a purity of ≥ 98%, a product of Celon Laboratories Ltd., Hyderabad, Telangana, India (CAS # 9041–93–4).

Techniques: Immunohistochemical staining, Staining, Control, Expressing, Standard Deviation

Journal: Toxicology Reports

Article Title: Phloridzin mitigates bleomycin-elicited lung fibrosis in Wistar rats: The interplay between antioxidant defenses, inflammatory processes, transforming growth factor beta 1, and autophagy

doi: 10.1016/j.toxrep.2026.102267

Figure Lengend Snippet: A photomicrograph of hematoxylin and eosin-stained sections (×100, scale bar = 100 µm) from the lung tissues of A) and B) the control group and PZ-alone treated group respectively showing normal lung morphological structure with intact alveoli (Thin arrows) and bronchioles (Thick arrows) with normal blood vessels (BV); C) and D) the group treated with bleomycin alone showing significant perturbation of the lung architecture as evidenced by destruction of the interalveolar septa (Thin arrows), distortion of the walls of the bronchi (Thick arrow), perinuclear vacuolation with pyknotic nuclei in the epithelial lining of the bronchioles (wavy green arrows), massive inflammatory cellular infiltration (Arrow heads), severe congestion of the vascular bed (V), with massive interstitial hemorrhage (IH); E) and F) bleomycin group treated with phloridzin (60 mg/kg/day) showing significant decline in the alveolar walls' destruction (Thin arrows), moderate inflammatory cellular infiltration (Arrow head), moderate vascular congestion (V), and significant decrease in the interstitial haemorrhage (IH); G) and H) bleomycin group treated with phloridzin (120 mg/kg/day) showing apparently normal alveoli (Thin arrows) with significant diminution of the inflammatory cellular infiltration (Arrow head) and minimal vascular congestion (V) with restoration of the bronchiolar wall (Thick arrow).

Article Snippet: Bleomycin, a white powder with a purity of ≥ 98%, a product of Celon Laboratories Ltd., Hyderabad, Telangana, India (CAS # 9041–93–4).

Techniques: Staining, Control

Journal: Toxicology Reports

Article Title: Phloridzin mitigates bleomycin-elicited lung fibrosis in Wistar rats: The interplay between antioxidant defenses, inflammatory processes, transforming growth factor beta 1, and autophagy

doi: 10.1016/j.toxrep.2026.102267

Figure Lengend Snippet: A photomicrograph of Masson’s trichrome stained sections (×200, scale bar = 100 µm) from the lung tissues of A) and B) the control group and PZ-alone treated group respectively showing minimal collagen fibers deposits around the bronchioles and the pulmonary blood vessels (Arrows); C) the group treated with bleomycin alone showing massive deposition of the collagen fibers around the alveoli, bronchioles, and pulmonary blood vessels in addition to the interalveolar septa (Arrows); D) bleomycin group treated with phloridzin (60 mg/kg/day) exhibiting a significant decline in the deposition of the collagen fibers around the pulmonary blood vessels and the alveoli, and in the interalveolar septa (Arrows); E) bleomycin group treated with phloridzin (120 mg/kg/day) exhibiting minimal deposition of the collagen fibers around the pulmonary blood vessels and the alveoli, in addition to the interalveolar septa (Arrows), and F) The effect of phloridzin (60 and 120 mg/kg/day, p.o) on the fibrosis score in the lung tissues harvested from bleomycin-treated rats. Values were expressed as median (Interquartile range, IQR) and compared using Kruskal-Wallis followed by Dunn's test. Number of animals = 15 rats in each group. A p-value less than 0.05 was considered statistically significant. BLM: bleomycin; NS: non-significant; PZ: phloridzin.

Article Snippet: Bleomycin, a white powder with a purity of ≥ 98%, a product of Celon Laboratories Ltd., Hyderabad, Telangana, India (CAS # 9041–93–4).

Techniques: Staining, Control