Journal: Inflammation

Article Title: Hepatocyte-Derived IL-25 Promotes Macrophage Extracellular Trap Formation and Drives Liver Fibrosis Progression

doi: 10.1007/s10753-026-02506-6

Figure Lengend Snippet: Upregulated hepatocyte-derived IL-25 promotes the formation of METs in liver fibrosis. A qPCR analysis of 31 kinds of Interleukins expression in 4 W LFib mice liver tissues, The heatmap and bar graph visually represent the fold changes of IL-25 and other interleukins. ( n = 6 per group). B qPCR analysis of 31 kinds of Interleukins expression in AML-12, stimulated with culture medium containing 0.2% CCl₄ for 12 h. The heatmap and bar graph visually represent the fold changes of IL-25 and other interleukins. ( n = 6 per group). C Double immunofluorescence staining of mouse liver tissue sections using HNF4α and Il-25 antibodies, with DAPI for nuclear counterstaining with the semi-quantitative results of the processed images from each group, yellow arrows indicate hepatocyte-derived IL-25 (HNF4α⁺Il-25⁺). ( n = 6 per group). Mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Student’s t-test

Article Snippet: RAW 264.7 and BMDMs were stimulated with 100 nM phorbol 12-myristate 13-acetate (PMA; Cat. No. G2272-100UL, Servicebio) for 8 h and IL-25 (Cat. No. HY- P73198 ; MedChemExpress; NJ, USA) 50 ng/ml for 24 h to induce METs formation.

Techniques: Derivative Assay, Expressing, Double Immunofluorescence Staining

Journal: Inflammation

Article Title: Hepatocyte-Derived IL-25 Promotes Macrophage Extracellular Trap Formation and Drives Liver Fibrosis Progression

doi: 10.1007/s10753-026-02506-6

Figure Lengend Snippet: IL-25 promotes the formation of METs in vitro. A CCK-8 assay was used to detect the proliferation of RAW cells stimulated with IL-25, as indicated by relative cell viability ( n = 6 per group). B qPCR analysis of Tnf-α expression in RAW cells stimulated with Il-25 for 24 h ( n = 6 per group). C Observation of METs formation in RAW cells under scanning electron microscopy, red arrows indicate METs formation. ( n = 4 per group). D Observation of METs formation in RAW cells by SYTOX Green staining, with Hoechst for nuclear counterstaining with the semi-quantitative results of the processed images from each group, red arrows indicate METs formation. ( n = 6 per group). E Observation of METs formation in RAW cells by CitH3 IF staining, with Hoechst for nuclear counterstaining with the semi-quantitative results of the processed images from each group, CitH3-positive structures were defined as those with perinuclear staining co-localized with Hoechst, red arrows indicate extracellular fibrous CitH3⁺ structures with characteristic chromatin decondensation ( n = 6 per group). F WB analysis of CitH3 expression in RAW cells with the semi-quantitative results from the grayscale analysis of protein bands for each group, representative western blots from three independent experiments are shown, each lane represents a biological replicate, and a subset of samples is displayed for clarity ( n = 6 per group). For IL-17RB blockade, cells were pre-incubated with IL-17RBb 50 ng/ml for 1 h prior to stimulation with IL-25. The inhibitor was present throughout the subsequent stimulation period. Mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Student’s t-test

Article Snippet: RAW 264.7 and BMDMs were stimulated with 100 nM phorbol 12-myristate 13-acetate (PMA; Cat. No. G2272-100UL, Servicebio) for 8 h and IL-25 (Cat. No. HY- P73198 ; MedChemExpress; NJ, USA) 50 ng/ml for 24 h to induce METs formation.

Techniques: In Vitro, CCK-8 Assay, Expressing, Electron Microscopy, Staining, Western Blot, Incubation

Journal: Inflammation

Article Title: Hepatocyte-Derived IL-25 Promotes Macrophage Extracellular Trap Formation and Drives Liver Fibrosis Progression

doi: 10.1007/s10753-026-02506-6

Figure Lengend Snippet: Hepatocyte-derived IL-25 promote the progression of liver fibrosis. A IL-25CKO mice create strategy. B Flowchart of liver fibrosis modeling in IL-25 fl/fl ALB-icre ki/wt mice ( n = 10 per group). C Flowchart of liver fibrosis modeling induced by IL-25 treatment in C57 mice ( n = 10 per group). D Measurement of ALT and AST levels in mouse peripheral blood serum ( n = 6 per group). E qPCR analysis of Col1a1 and α-SMA expression in mice liver tissues ( n = 6 per group). F Representative histological staining of liver tissues in a mouse model of liver fibrosis, including H&E, Sirius Red, and α-SMA IHC staining with the semi-quantitative results of the processed images from each group ( n = 6 per group). G Mice liver entity photo ( n = 6 per group). H Double immunofluorescence staining of mouse liver tissue sections using F4/80 and Cith3 antibodies, with DAPI for nuclear counterstaining with the semi-quantitative results of the processed images from each group ( n = 6 per group). Mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Student’s t-test

Article Snippet: RAW 264.7 and BMDMs were stimulated with 100 nM phorbol 12-myristate 13-acetate (PMA; Cat. No. G2272-100UL, Servicebio) for 8 h and IL-25 (Cat. No. HY- P73198 ; MedChemExpress; NJ, USA) 50 ng/ml for 24 h to induce METs formation.

Techniques: Derivative Assay, Expressing, Staining, Immunohistochemistry, Double Immunofluorescence Staining

Journal: Inflammation

Article Title: Hepatocyte-Derived IL-25 Promotes Macrophage Extracellular Trap Formation and Drives Liver Fibrosis Progression

doi: 10.1007/s10753-026-02506-6

Figure Lengend Snippet: IL-25 to the IL-25 receptor on macrophages triggers ROS burst via NOX activation and mitophagy, consequently inducing lysosomal activation and MET formation. A Cell ROS assay of RAW264.7 by DCFH-DA with the semi-quantitative results of the processed images from each group ( n = 6 per group). B Observation of METs formation in RAW cells by SYTOX Green staining, with Hochest for nuclear counterstaining with the semi-quantitative results of the processed images from each group ( n = 6 per group). C Observation of lysosome and mitochondrial morphology in RAW cells under transmission electron microscopy, red arrows indicate activated lysosomes and autophagosome formation, and blue arrows indicate METs formation. ( n = 4 per group). D Mitochondrial & Lysosomes staining in RAW cell with the semi-quantitative results of the processed images from each group ( n = 6 per group). E WB analysis of CitH3 expression in RAW cells with the semi-quantitative results from the grayscale analysis of protein bands for each group, representative western blots from three independent experiments are shown, each lane represents a biological replicate, and a subset of samples is displayed for clarity ( n = 6 per group). F-G WB analysis of phosphorylated p47phox protein expression in RAW cells and liver tissues with the semi-quantitative results from the grayscale analysis of protein bands for each group, representative western blots from three independent experiments are shown, each lane represents a biological replicate, and a subset of samples is displayed for clarity ( n = 6 per group). Cells were pretreated with VAS2870 25 μM for 30 min prior to stimulation, NAC 10 μM for 1 h before stimulation, CQ 10 μM for 2 h before stimulation. Mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Student’s t-test

Article Snippet: RAW 264.7 and BMDMs were stimulated with 100 nM phorbol 12-myristate 13-acetate (PMA; Cat. No. G2272-100UL, Servicebio) for 8 h and IL-25 (Cat. No. HY- P73198 ; MedChemExpress; NJ, USA) 50 ng/ml for 24 h to induce METs formation.

Techniques: Activation Assay, ROS Assay, Staining, Transmission Assay, Electron Microscopy, Expressing, Western Blot

Journal: Inflammation

Article Title: Hepatocyte-Derived IL-25 Promotes Macrophage Extracellular Trap Formation and Drives Liver Fibrosis Progression

doi: 10.1007/s10753-026-02506-6

Figure Lengend Snippet: IL-25 can bind to the interleukin-25 receptor on macrophages, subsequently activating the NOX, leading to increased ROS production. Additionally, it impairs mitochondrial function, resulting in a ROS burst, triggers lysosomal dysfunction, causing calcium ion leakage from lysosomes. This, induces chromatin unraveling in macrophages, ultimately leading to macrophage rupture and the release of METs. Finally, METs activate the HSCs and causing accumulation of ECM and liver fibrosis

Article Snippet: RAW 264.7 and BMDMs were stimulated with 100 nM phorbol 12-myristate 13-acetate (PMA; Cat. No. G2272-100UL, Servicebio) for 8 h and IL-25 (Cat. No. HY- P73198 ; MedChemExpress; NJ, USA) 50 ng/ml for 24 h to induce METs formation.

Techniques: