serpine1 inhibitor tm5441  (MedChemExpress)

Journal: JHEP Reports

Article Title: Serpine1 as a potential therapeutic target in pyrrolidine alkaloids-induced hepatic sinusoidal obstruction syndrome

doi: 10.1016/j.jhepr.2026.101736

Figure Lengend Snippet: Transcriptomic signatures in PA-HSOS mouse livers. (A) Volcano plot of differential gene expression reveals dysregulation of genes critical to PA-HSOS pathogenesis, with log 2 fold-change and statistical significance plotted to identify transcriptional alterations between PA-HSOS and control livers (n = 6/group). (B) KEGG pathway enrichment analysis identifies dysregulated biological pathways in PA-HSOS livers (n = 6/group). (C) Heatmap of key differentially expressed genes demonstrates distinct transcriptomic signatures distinguishing PA-HSOS from control samples (n = 6/group). (D) Single-cell RNA-seq UMAP clustering delineates distinct hepatic cell populations and their transcriptional heterogeneity in PA-HSOS vs. control livers (n = 3/group). (E) Violin plots of Serpine1 expression in LSECs (n = 3/group). (F) Ridge plot visualization further characterizes the dynamic range of Serpine1 expression in LSECs (n = 3/group). All data represent means ± SEM. LSEC, liver sinusoidal endothelial cell; PA-HSOS, pyrrolizidine alkaloid–induced hepatic sinusoidal obstruction syndrome; RNA-seq, RNA sequencing; UMAP, uniform manifold approximation and projection.

Article Snippet: Following the 28-day establishment of the PA-HSOS model, C57BL/6 mice underwent treatment with the Serpine1 inhibitor TM5441 (Cat# HY-101761, MCE).

Techniques: Gene Expression, Control, Single Cell, RNA Sequencing, Expressing

Journal: JHEP Reports

Article Title: Serpine1 as a potential therapeutic target in pyrrolidine alkaloids-induced hepatic sinusoidal obstruction syndrome

doi: 10.1016/j.jhepr.2026.101736

Figure Lengend Snippet: The Serpine1/p53 pathway activation and associated pathologies in vivo . (A) qPCR analysis demonstrates upregulation of Serpine1 and p5 3 mRNA in mouse LSECs from PA-HSOS mice (n = 6/group). (B) Western blot analysis of mouse LSECs confirms elevated Serpine1 protein alongside increased p53, p21, and p16 expression. (C) Flow cytometry-based cell cycle analysis reveals G1/S phase arrest in PA-HSOS LSECs (n = 6/group). (D) qPCR quantification of senescence-associated molecules in mouse LSECs shows increased expression of key senescence markers (n = 6/group). (E) Immunofluorescence intensity of p16 in ERG-positive cells in mouse livers localizes senescent endothelial cells within hepatic sinusoids (n = 6/group). Scale bars, 50 μm. (F) TUNEL staining reveals enhanced endothelial cell apoptosis in PA-HSOS livers compared to controls (n = 6/group). Scale bars, 200 μm (or 50 μm for enlarged regions). (G) Flow cytometry of mouse LSECs quantifies apoptotic populations (n = 6/group). (H) Western blot analysis of Bax and cleaved caspase-3 in mouse LSECs confirms activation of the intrinsic apoptotic pathway. (I) In vivo endothelial permeability assay using tail-vein injection of 40 kDa FITC-dextran demonstrates compromised sinusoidal barrier function in PA-HSOS mice, with quantified fluorescence intensity reflecting enhanced vascular leakage into hepatic tissue (n = 6/group). Scale bars, 100 μm (or 50 μm for enlarged regions). (J) Western blot of eNOS and VE-cadherin in mouse LSECs reveals reduced expression of these vascular integrity markers. (K) Serum Serpine1 levels are significantly elevated in PA-HSOS mice and humans compared to respective controls (mice: n = 6/group; humans: n = 16/group). All data represent means ± SEM. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 by Mann-Whitney U test.

Article Snippet: Following the 28-day establishment of the PA-HSOS model, C57BL/6 mice underwent treatment with the Serpine1 inhibitor TM5441 (Cat# HY-101761, MCE).

Techniques: Activation Assay, In Vivo, Western Blot, Expressing, Flow Cytometry, Cell Cycle Assay, Immunofluorescence, TUNEL Assay, Staining, Permeability, Injection, Fluorescence, MANN-WHITNEY

Journal: JHEP Reports

Article Title: Serpine1 as a potential therapeutic target in pyrrolidine alkaloids-induced hepatic sinusoidal obstruction syndrome

doi: 10.1016/j.jhepr.2026.101736

Figure Lengend Snippet: PA-induced endothelial dysfunction and senescence in a hepatocyte-LSEC co-culture system. (A) Schematic representation of the hepatocyte-LSEC co-culture system, with hepatocytes cultured in the upper Transwell compartment and LSECs in the lower chamber, enabling paracrine signaling and physiologically relevant PA metabolite exposure. (B) PA treatment impairs LSEC morphology and viability in co-culture, as demonstrated by phase-contrast microscopy and quantified by CCK-8 assay (n = 3 independent experiments). Scale bars, 100 μm. (C) qPCR analysis reveals transcriptional upregulation of Serpine1 and p53 alongside their downstream effectors p21 and p16 in PA-exposed LSECs (n = 3 independent experiments). (D) Western blot analysis demonstrates corresponding increases in Serpine1, p53, p21, and p16 protein levels in LSECs following PA treatment. (E) SA-β-Gal staining quantifies senescent LSECs following PA exposure, providing morphological evidence of endothelial senescence induction and confirming functional engagement of the senescence program (n = 3 independent experiments). (F) qPCR profiling of inflammatory cytokines and SASP genes demonstrates coordinated upregulation of pro-inflammatory mediators and senescence markers in PA-treated LSECs (n = 3 independent experiments). (G) Western blot analysis of Serpine1, p53, p21, and p16 in PA-exposed LSECs treated with si-Serpine1 demonstrates that Serpine1 knockdown attenuates p53-pathway activation and senescence markers. All data represent means ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 by Mann-Whitney U test. n = 3/group (B-G). LSEC, liver sinusoidal endothelial cell; PA, pyrrolizidine alkaloid; qPCR, quantitative PCR; SA-β-Gal, senescence-associated β-galactosidase; SASP, senescence-associated secretory phenotype; si, small-interfering.

Article Snippet: Following the 28-day establishment of the PA-HSOS model, C57BL/6 mice underwent treatment with the Serpine1 inhibitor TM5441 (Cat# HY-101761, MCE).

Techniques: Co-Culture Assay, Cell Culture, Microscopy, CCK-8 Assay, Western Blot, Staining, Functional Assay, Knockdown, Activation Assay, MANN-WHITNEY, Real-time Polymerase Chain Reaction

Journal: JHEP Reports

Article Title: Serpine1 as a potential therapeutic target in pyrrolidine alkaloids-induced hepatic sinusoidal obstruction syndrome

doi: 10.1016/j.jhepr.2026.101736

Figure Lengend Snippet: Serpine1 mediates LSEC dysfunction through cell cycle arrest, fenestration loss, and apoptosis in PA-HSOS. (A) Representative flow cytometry histograms and quantification of propidium iodide-stained LSECs reveal G1/S arrest in PA-exposed LSECs, which are reversed by Serpine1 knockdown. (B) Scanning electron microscopy visualizes LSEC fenestrations in control and PA-treated conditions, with yellow arrows highlighting characteristic pores. Fenestration density (number per unit area) is significantly reduced in PA-HSOS LSECs but restored upon Serpine1 inhibition. Scale bar, 5 μm. (C) Immunofluorescence shows disrupted VE-cadherin organization in PA-treated LSECs, with reduced VE-cadherin expression partially recovered by Serpine1 knockdown. Scale bar, 100 μm. (D) In vitro permeability assay using 40 kDa FITC-dextran leakage quantifies barrier dysfunction in PA-exposed LSECs, demonstrating enhanced paracellular flux that reflects compromised endothelial integrity and is attenuated by Serpine1 knockdown. (E) Western blot analysis of endothelial function-related proteins (including eNOS and VE-cadherin) and apoptotic markers, including pro-apoptotic factors (cleaved caspase-3 and Bax) reveals downregulated endothelial function and activation of the apoptotic cascade in PA-treated LSECs, which is suppressed by Serpine1 inhibition. (F) Representative flow cytometry analysis delineates that PA-induced LSEC apoptosis is significantly reduced by Serpine1 knockdown. Data represent means ± SEM (n = 3/group). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 by Mann-Whitney U test. n = 3/group independent experiments. LSEC, liver sinusoidal endothelial cell; PA, pyrrolizidine alkaloid; PA-HSOS, pyrrolizidine alkaloid–induced hepatic sinusoidal obstruction syndrome.

Article Snippet: Following the 28-day establishment of the PA-HSOS model, C57BL/6 mice underwent treatment with the Serpine1 inhibitor TM5441 (Cat# HY-101761, MCE).

Techniques: Flow Cytometry, Staining, Knockdown, Electron Microscopy, Control, Inhibition, Immunofluorescence, Expressing, In Vitro, Permeability, Western Blot, Activation Assay, MANN-WHITNEY

Journal: JHEP Reports

Article Title: Serpine1 as a potential therapeutic target in pyrrolidine alkaloids-induced hepatic sinusoidal obstruction syndrome

doi: 10.1016/j.jhepr.2026.101736

Figure Lengend Snippet: Molecular interaction between Serpine1 and p53 in PA-HSOS pathogenesis. (A) Western blot analysis of p53 and Serpine1 in LSECs treated with si-Serpine1 or si-p53, showing reciprocal regulation of p53 and Serpine1 protein levels under PA exposure. (B) qPCR analysis showing that Serpine1 or p53 knockdown does not significantly alter p53 or Serpine1 mRNA levels in PA-treated LSECs. (C) Cycloheximide (CHX) chase assay combined with autophagy inhibitor (3-MA) or proteasome inhibitor (MG-132) demonstrating that Serpine1 stabilizes p53 protein by inhibiting proteasomal degradation. (D) Co-immunoprecipitation analysis of human liver samples showing enhanced interaction between Serpine1 and p53 in PA-HSOS compared with control tissue. (E) Immunofluorescence staining demonstrating subcellular colocalization of Serpine1 and p53 in LSECs under PA-HSOS conditions. Scale bar, 5 µm. (F) Co-immunoprecipitation analysis showing that Serpine1 interferes with the interaction between p53 and MDM2. (G) Ubiquitination assay demonstrating increased p53 polyubiquitination following Serpine1 knockdown, indicating enhanced p53 degradation. (H) Western blot analysis showing that Serpine1 knockdown promotes MDM2-dependent p53 degradation, whereas intact Serpine1 expression stabilizes p53 protein levels. Data represent means ± SEM (n = 3/group). LSEC, liver sinusoidal endothelial cell; PA-HSOS, pyrrolizidine alkaloid–induced hepatic sinusoidal obstruction syndrome; qPCR, quantitative PCR; si, small-interfering.

Article Snippet: Following the 28-day establishment of the PA-HSOS model, C57BL/6 mice underwent treatment with the Serpine1 inhibitor TM5441 (Cat# HY-101761, MCE).

Techniques: Western Blot, Knockdown, Immunoprecipitation, Control, Immunofluorescence, Staining, Ubiquitin Proteomics, Expressing, Real-time Polymerase Chain Reaction

Journal: JHEP Reports

Article Title: Serpine1 as a potential therapeutic target in pyrrolidine alkaloids-induced hepatic sinusoidal obstruction syndrome

doi: 10.1016/j.jhepr.2026.101736

Figure Lengend Snippet: Therapeutic efficacy of TM5441 in ameliorating PA-HSOS. (A) Schematic overview of the experimental design for PA-HSOS mouse model treatment with TM5441, a selective p53 activator. (B) Body weight trajectories demonstrate that TM5441 treatment significantly attenuates PA-induced weight loss. (C) Hemodynamic analysis reveals that TM5441 restores abnormal hemodynamic parameters, including PP, MAP, and PBF. (D) Comprehensive biochemical assessment demonstrates that TM5441 treatment ameliorates liver dysfunction (ALT, AST, ALP), coagulation abnormalities (PT, aPTT, D-dimer), systemic inflammation (IL-1β, TNF-α), and hematological dysregulation (platelets, RBC, WBC). (E) Histopathological analysis including H&E, Masson trichrome, IL-1β immunohistochemistry, and DAPI/CD31/VE-cadherin immunofluorescence demonstrates that TM5441 reduces sinusoidal RBC accumulation, hepatic fibrosis, inflammatory infiltration, and endothelial dysfunction. Black arrows indicate RBC accumulation within hepatic sinusoids. Scale bars, 200 μm (or 50 μm for enlarged regions). (F) FITC-dextran permeability assay with immunofluorescence visualization demonstrates that TM5441 restores sinusoidal barrier integrity and reduces vascular leakage. Scale bars, 100 μm (or 50 μm for enlarged regions). (G) qPCR profiling reveals that TM5441 suppresses inflammatory cytokine expression and reduces senescence-associated gene upregulation in liver tissue. (H) Western blot analysis of LSEC function and apoptotic markers shows that TM5441 reduces pro-apoptotic protein levels and restores anti-apoptotic factors. (I) Flow cytometry demonstrates that TM5441 significantly reduces apoptosis rates in hepatic endothelial cells. (J) Western blot of the Serpine1-p53 pathway reveals that TM5441 downregulates Serpine1 and p53 protein levels alongside reduced expression of downstream senescence effectors, including p21 and p16. (K) Immunofluorescence of ERG and p16 in hepatic tissue reveals reduced coexpression in TM5441-treated mice. Scale bar, 50 μm. (L) Flow cytometry-based cell cycle analysis demonstrates that TM5441 reverses PA-induced G1/S phase arrest in hepatic endothelial cells. Data represent means ± SEM (n = 6/group). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 by Mann-Whitney U test. ALP, alkaline phosphatase; ALT, alanine aminotransferase; aPTT, activated partial thromboplastin time; AST, aspartate aminotransferase; LSEC, liver sinusoidal endothelial cell; MAP, mean arterial pressure; PA-HSOS, pyrrolizidine alkaloid–induced hepatic sinusoidal obstruction syndrome; PBF, portal blood flow; PP, portal pressure; PT, prothrombin time; qPCR, quantitative PCR; RBC, red blood cell; WBC, white blood cell.

Article Snippet: Following the 28-day establishment of the PA-HSOS model, C57BL/6 mice underwent treatment with the Serpine1 inhibitor TM5441 (Cat# HY-101761, MCE).

Techniques: Drug discovery, Coagulation, Immunohistochemistry, Immunofluorescence, FITC-Dextran Permeability Assay, Expressing, Western Blot, Flow Cytometry, Cell Cycle Assay, MANN-WHITNEY, Real-time Polymerase Chain Reaction

Journal: Molecular Therapy. Nucleic Acids

Article Title: Inducible miR-1224 silences cerebrovascular Serpine1 and restores blood flow to the stroke-affected site of the brain

doi: 10.1016/j.omtn.2022.12.019

Figure Lengend Snippet: Serpine1 is a target of miR-1224 (A) Angiogenic protein array showing the difference in control mimic- and miR-1224 mimic-transfected bEnd.3 cells. The Proteome Profiler Mouse Angiogenesis Array Kit (cat. no. ARY015) was used to simultaneously assess the relative levels of 53 mouse angiogenesis-related proteins. (B) SERPINE1 protein (n = 9) and (C) Serpine1 transcript abundance in miR-1224 mimic-delivered cells (n = 6). (D) Binding site to mouse transcript ENSMUST00000041388-3′ UTR position 652–674 to miR-1224-5p (MIMAT0005460); binding energy −30.5 kcal/mol. Proposed binding sites for miR-1224 were identified in the SERPINE1 3′ UTR that was inserted into the Gaussia luciferase plasmid vector (wild type). The pEZX-MT05 vector also contains the secreted Gaussia luciferase (GLuc) open reading frame, driven by the SV40 promoter, as a reporter of the 3′ UTR expression, and a secreted alkaline phosphatase (SEAP) reporter, driven by a CMV promoter, as an internal control. (E) Quantification of luciferase reporter assay (n = 12) of wild-type and mutated versions of the miR-1224 binding site on the SERPINE1 3′ UTR. GLuc activity and alkaline phosphatase activity were assayed after 48 h of transfection using GeneCopoeia’s Secrete-Pair dual luminescence assay kit. Percentage change in GLuc activity was calculated after normalizing to alkaline phosphatase activity. Data are shown as mean ± SEM.

Article Snippet: Mice were treated with the small-molecule SERPINE1 inhibitor TM5441 (R&D Systems, Minneapolis, MN).

Techniques: Protein Array, Control, Transfection, Binding Assay, Luciferase, Plasmid Preparation, Expressing, Reporter Assay, Activity Assay, Luminescence Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: Inducible miR-1224 silences cerebrovascular Serpine1 and restores blood flow to the stroke-affected site of the brain

doi: 10.1016/j.omtn.2022.12.019

Figure Lengend Snippet: Downregulation of Serpine1 induced angiogenic response in mouse primary brain microvascular endothelial cells Matrigel tube formation was visualized by phase-contrast microscopy at 18–24 h in control or SERPINE1 siRNA-transfected cells. pMBMECs transfected with nontargeting siRNA (control siRNA) or SERPINE1 siRNA were plated on Matrigel 72 h following transfection. (A) Tube formation in control and SERPINE1 siRNA-transfected pMBMECs. Scale bars, 100 μm. (B) SERPINE1 protein (ELISA) after transfection of SERPINE1 siRNA (n = 12). Standard angiogenic parameters were quantified (n = 7) using ImageJ and the Angiogenesis Analyzer plug-in tool. (C) Total tube length, (D) number of nodes, (E) number of junctions, and (F) number of meshes. Data shown as mean ± SEM. (G–K) Alternative studies were conducted with the SERPINE1 inhibitor TM5441. (G) Tube formation in control and TM5441 (10 μM, 24 h)-treated pMBMECs. Scale bars, 100 μm. Angiogenic parameters were quantified (n = 8) using ImageJ and the Angiogenesis Analyzer plug-in tool. (H) Total tube length, (I) number of nodes, (J) number of junctions, and (K) number of meshes. Data are shown as mean ± SEM.

Article Snippet: Mice were treated with the small-molecule SERPINE1 inhibitor TM5441 (R&D Systems, Minneapolis, MN).

Techniques: Microscopy, Control, Transfection, Enzyme-linked Immunosorbent Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: Inducible miR-1224 silences cerebrovascular Serpine1 and restores blood flow to the stroke-affected site of the brain

doi: 10.1016/j.omtn.2022.12.019

Figure Lengend Snippet: Small-molecule SERPINE1 inhibition increased cerebrovascular blood flow at the stroke-affected site and protected against stroke (A) Timeline of TM5441 treatment, MCAO, LSI, MRI, and brain harvest. (B) Real-time, noninvasive, cerebrovascular perfusion images of the dorsal surface of the brain were acquired in mice during occlusion and at 48 h post-stroke. (C) Perfusion quantification in TM5441-treated mice showed significantly higher perfusion in stroke-affected brain (n = 7 and 8). The SERPINE1 inhibitor TM5441 improved FITC-lectin perfusion to the S1 cortex at the stroke-affected site at 48 h post-stroke. (D) Fluorescence micrographs of stroke-affected S1 cortex of control and TM5541-treated mice. The inset images (indicated by white dashed lines) show a zoomed-in view of the selected region on the right. Scale bars, 50 μm. (E) Quantification of lectin-perfused vessel length (μm 2 area, n = 7). (F) Representative 9.4 T MRI (48 h post-stroke) images and (G) percentage hemisphere lesion volume calculated based on T2-weighted MRI at 48 h post-stroke (n = 7). (H) Representative mobility track plots from baseline and 48 h post-stroke of control or TM5441-treated mice. Tracks start at blue dots and end at red dots. (I) TM5441 treatment significantly improved 48 h post-stroke distance traveled, mean speed, and time mobile (n = 6 and 8) compared with control. Data are shown as mean ± SEM.

Article Snippet: Mice were treated with the small-molecule SERPINE1 inhibitor TM5441 (R&D Systems, Minneapolis, MN).

Techniques: Inhibition, Fluorescence, Control