Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: S-Adenosylmethionine Inhibits Plasminogen-Activating Inhibitor-1 and Protects Male Mice from FOLFOX-Induced Liver Injury

doi: 10.1016/j.jcmgh.2025.101513

Figure Lengend Snippet: SAMe treatment prevents FOLFOX, IL-1β-, and TNF-α-induced PAI-1 expression and p65 nuclear translocation. Primary mouse hepatocytes (0.5 x 10 6 /6-well plate) were treated with 2 mM SAMe, 10 ng/mL IL-1β, 10 ng/mL TNF-α alone or in combination for 24 hours. ( A ) The mouse promoter was aligned to the human -785 bp promoter using BLAST tool. The identified NF-κB p65-binding site in both promoters is boxed ( red ). Total mRNA level of Serpine1 was analyzed by real-time polymerase chain reaction ( B ), whereas ( C ) PAI-1 and p65 protein levels were analyzed by Western blotting. The densitometric ratios are represented as % of control and summarized below the blots. Data represent mean ± standard error of the mean from n = 3. ∗ P < .01 versus control; † P < .01 versus IL-1β; ‡ P < .02 versus TNF-α. ( D ) Protein lysates were extracted from livers of control and FOLFOX ± SAMe-treated mice to measure PAI-1, p65, p50, and IκBα protein levels by Western blotting. Densitometric ratios normalized to actin are indicated under each blot. Results are expressed as % of control (mean ± standard error of the mean) from 3 mice/group. ∗ P < .02 versus control; † P < .01 versus FOLFOX. ( E ) Nuclear and cytoplasmic proteins were extracted from livers of FOLFOX ± SAMe-treated mice and analyzed by Western blotting, with densitometric values summarized below the blots, and ( F ) immunofluorescence to measure p65 protein level and subcellular localization ( green ), respectively. Cell nuclei were stained with DAPI ( blue ) (x400). Data represent mean ± standard error of the mean from n = 3. ∗ P < .02 versus control and † P < .01 versus FOLFOX for nuclear fraction; ∗ P < .01 versus control and † P < .05 versus FOLFOX for cytoplasmic fraction.

Article Snippet: Finally, stained cells and sections were mounted using Vectashield mounting media with DAPI (cat.# H-1300-10; Vector laboratories, Inc, Burlingame, CA) and imaged with the Keyence BZX-700 microscope.

Techniques: Expressing, Translocation Assay, Binding Assay, Real-time Polymerase Chain Reaction, Western Blot, Control, Immunofluorescence, Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: S-Adenosylmethionine Inhibits Plasminogen-Activating Inhibitor-1 and Protects Male Mice from FOLFOX-Induced Liver Injury

doi: 10.1016/j.jcmgh.2025.101513

Figure Lengend Snippet: Loss of PAI-1 protects from FOLFOX-induced liver damage in mice. PAI-1 knockout mice were treated with FOLFOX as indicated in . ( A ) Histology and immunohistochemistry of mouse liver tissues under magnification x200. Hematoxylin-eosin (H&E), F4/80 immunofluorescence, PAI-1, and TUNEL staining of liver sections. Boxed areas are further magnified. Arrowheads point to TUNEL+ staining. Fluorescence and IHC staining intensity were quantified by ImageJ and summarized in the box below. ( B ) Plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. Data represent mean ± standard error of the mean from n = 7. ( C ) RNA was extracted from livers and expression of SOS gene markers ( Mmp9 , Cx c 1 , Vwf , Tnf-α ) was analyzed by real-time polymerase chain reaction. Data represent mean ± standard error of the mean from n = 8. ∗ P < .01 versus control. ( D ) Nuclear and cytoplasmic proteins were extracted from livers of FOLFOX-treated mice and analyzed by Western blotting with densitometric values summarized below the blots, and ( E ) immunofluorescence to measure p65 protein level and subcellular localization ( green ), respectively. Cell nuclei were stained with DAPI ( blue ) (x400). Data represent mean ± standard error of the mean from n = 3. ( F ) Body, ( G ) liver weight loss, and ( H ) liver/body weight ratios in PAI-1 KO mice control or treated with FOLFOX for 5 weeks. Values are expressed as mean ± standard error of the mean from n = 8 per group.

Article Snippet: Finally, stained cells and sections were mounted using Vectashield mounting media with DAPI (cat.# H-1300-10; Vector laboratories, Inc, Burlingame, CA) and imaged with the Keyence BZX-700 microscope.

Techniques: Knock-Out, Immunohistochemistry, Immunofluorescence, TUNEL Assay, Staining, Fluorescence, Clinical Proteomics, Expressing, Real-time Polymerase Chain Reaction, Control, Western Blot

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: S-Adenosylmethionine Inhibits Plasminogen-Activating Inhibitor-1 and Protects Male Mice from FOLFOX-Induced Liver Injury

doi: 10.1016/j.jcmgh.2025.101513

Figure Lengend Snippet: PAI-1 activates NF-κB in mouse hepatocytes and causes hepatocytes to release factors that induce CD31 expression in LSECs and inflammatory cytokines in KCs. Primary mouse hepatocytes were treated with 100 nM rPAI-1 for 24 hours. ( A ) mRNA levels of Serpine 1 , Rela , and Tnf-α were measured by real-time polymerase chain reaction in primary mouse hepatocytes. Data represent mean ± standard error of the mean from n = 3. ∗ P < .01 versus control. ( B ) Protein levels of PAI-1, p-p65 (S536), and p65 were analyzed by Western blotting. Actin was used as housekeeping. Densitometric values are summarized below the blots. Data represent mean ± standard error of the mean from n = 3. ∗ P < .02 versus control. ( C ) Immunofluorescence of p65 ( red ). Cell nuclei were stained with DAPI ( blue ) (x400). ( D ) LSECs, KCs, and HSCs were cultured for 24 hours with conditioned media from rPAI-1-treated primary mouse hepatocytes. RNA was extracted to measure the gene expression of Cd31 and Cd32 in LSECs, Tnf-α , IL-1β, and IL-6 in KCs, and Col I and Acta2 in HSCs by real-time polymerase chain reaction. Data represent mean ± standard error of the mean. ∗ P < .01 versus control in LSECs (n = 5); ∗ P < .04 and ∗∗ P < .01 versus control in KCs (n = 3); HSCs (n = 4).

Article Snippet: Finally, stained cells and sections were mounted using Vectashield mounting media with DAPI (cat.# H-1300-10; Vector laboratories, Inc, Burlingame, CA) and imaged with the Keyence BZX-700 microscope.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Western Blot, Immunofluorescence, Staining, Cell Culture, Gene Expression