hepatocyte growth factor  (PeproTech)

Journal: Military Medical Research

Article Title: Glucocorticoids trigger muscle-liver crosstalk to attenuate acute liver injury and promote liver regeneration via the FGF6-FGFBP1 axis

doi: 10.1186/s40779-025-00618-y

Figure Lengend Snippet: Skeletal muscle glucocorticoid receptor ( GR ) knockdown via siRNA delivery exacerbates acute liver injury (ALI) in mice. a Schematic of the animal experiments. Briefly, 8-week-old C57BL/6 mice were injected with si Nr3c1 ( Nr3c1 , encoding GR) or siNC into the gastrocnemius (Gas) and tibialis anterior (Ta) muscles and subjected to partial (2/3) hepatectomy (PHx) or acetaminophen (APAP). b mRNA levels of Nr3c1 and Fgf6 in the Gas of mice 24 h after PHx ( n = 6). c Protein levels of GR and FGF6 in the Gas of mice 24 h after PHx ( n = 2). d Western blotting analysis depicting the changes in protein synthesis rates [tracked using the surface sensing of translation (SUnSET) assay] in the liver and Gas of mice 24 h after PHx ( n = 3 independent biological replicates). The levels of puromycin relative to the α-tubulin were shown through quantification, with the level of puromycin in the siNC setting as 1.00. This applied to all puromycin quantification in this figure. e Tissue weight as a percentage of body weight in the liver, Gas, Ta, and quadriceps (Qu) of mice 24 h after PHx ( n = 6). f Representative Ki67 (for indicating the proliferating cells) and β-catenin (for indicating cell size) immunohistochemistry of mouse liver Sections 24 h after PHx, and quantification of hepatocyte area and Ki67 + nuclei ( n = 6). Scale bar = 50 μm. g Western blotting analysis of the protein synthesis rates in the liver and Gas of mice 24 h after APAP dosing ( n = 3 independent biological replicates). h Serum concentrations of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) ( n = 6). i Representative hematoxylin and eosin (H&E) (necrotic areas circled with black lines) and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining (for identifying apoptotic cells) images of mouse liver Sections 24 h after APAP dosing. Scale bar = 50 μm. j Western blotting analysis of hepatic apoptosis related proteins BCL2 associated X (BAX) and cleaved caspase-3 (Cl-CASP3) expression with the corresponding quantification ( n = 4). * P < 0.05, ** P < 0.01, *** P < 0.001. FGF6 fibroblast growth factor 6, Nr3c1 nuclear receptor subfamily 3 group c member 1, DAPI 4’,6-diamidino-2-phenylindole

Article Snippet: A total of 100,000 cells were embedded in 50 μl Matrigel drop on the dishes in advanced DMEM/F12 with 1× B27, 1× N2, 1× GlutaMaX, 5 ng/ml FGF2 (100-18B, Peprotech, NJ, USA), 10 ng/ml VEGF (100–20–10, Peprotech, NJ, USA), 20 ng/ml EGF (AF-100–15, Peprotech, NJ, USA), 3 μmol/L CHIR99021, 0.5 μmol/L A83–01 (2939, Tocris, MO, USA), 50 μg/ml ascorbic acid (A5960, Sigma, MO, USA) and 1% P/S for 4 d. Then the media was switched to advanced DMEM/F12 with 1× B27, 1× N2, 1× GlutaMaX, 2 μmol/L retinoic acid (R2625, Sigma, MO, USA), and 1% P/S and cultured for another 4 d. Finally, organoids were harvested and re-embedded in Matrigel on the ultra-low attachment multiwall plate (3471, Corning, MI, USA) in liver maturation media [hepatocyte culture medium (CC-3198, Lonza, MD, USA) prepared as manufacturer’s instructions, except no EGF, and supplemented with 10 ng/ml hepatocyte growth factor (100–39, PeproTech, NJ, USA), 0.1 μmol/L Dex (D4902, Sigma, MO, USA), 20 ng/ml oncostatin M (300–10, Peprotech, NJ, USA), and 1% P/S] with PBS or rFGFBP1 (25 ng/ml) or rFGFBP1 (25 ng/ml) + rFGF5 (200 ng/ml) for 10 d. Cells were maintained at 37 °C in humidified air with 5% CO 2 , and the medium was added every 2 d. For further details regarding the materials and methods used, please refer to Additional file : Methods.

Techniques: Knockdown, Injection, Muscles, Western Blot, Immunohistochemistry, TUNEL Assay, Staining, Expressing

Journal: Military Medical Research

Article Title: Glucocorticoids trigger muscle-liver crosstalk to attenuate acute liver injury and promote liver regeneration via the FGF6-FGFBP1 axis

doi: 10.1186/s40779-025-00618-y

Figure Lengend Snippet: Fgf6 deficiency protects mice from PHx- and APAP-induced acute liver injury (ALI). a Liver/Body weight ratio determined at the indicated time points ( n = 5–14 per time point and group), and Gas/Body weight ratio as determined at the indicated time points ( n = 5–7 per time point and group). b Western blotting analysis of the protein synthesis rates in the liver and Gas of WT and Fgf6 - KO mice 24 h after partial (2/3) hepatectomy (PHx) ( n = 3 independent biological replicates). The levels of puromycin relative to the α-tubulin were shown through quantification, with the level of puromycin in the WT setting as 1.00. c Representative liver β-catenin (for indicating cell size) immunohistochemistry results and quantification of the hepatocyte area ( n = 5). Scale bar = 50 μm. d Western blotting analysis of the hepatic proliferation-related proteins PCNA and cyclin D1 (G1/S-specific) at the indicated time points after PHx ( n = 3 independent biological replicates). e Representative liver Ki67 (for indicating the proliferating cells) immunohistochemistry results and quantification of Ki67 + nuclei ( n = 6). Scale bar = 50 μm. f Serum concentrations of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in WT and Fgf6 -KO mice 24 and 48 h after APAP dosing (300 mg/kg body weight; n = 6). g Representative liver H&E (necrotic areas circled with black lines), TUNEL (for identifying apoptotic cells), and Ki67 staining results 24 and 48 h after APAP dosing. Scale bars = 100 μm (H&E) or 50 μm (TUNEL and Ki67). h Western blotting analysis of hepatic apoptosis and proliferation-related proteins BAX, Cl-CASP3, PCNA, and cyclin D1 expression ( n = 4). The data ( f ) were log-transformed and then analyzed using an unpaired Student’s t -test. * P < 0.05, ** P < 0.01, *** P < 0.001, ns non-significant. Fgf6 fibroblast growth factor 6, APAP acetaminophen, PCNA proliferating cell nuclear antigen, H&E hematoxylin and eosin, TUNEL terminal deoxynucleotidyl transferase dUTP Nick-End labeling, BAX BCL2 associated X, Cl-CASP3 cleaved caspase-3, FGF6 fibroblast growth factor 6, Gas gastrocnemius, WT wild-type, KO knockout, DAPI 4’,6-diamidino-2-phenylindole

Article Snippet: A total of 100,000 cells were embedded in 50 μl Matrigel drop on the dishes in advanced DMEM/F12 with 1× B27, 1× N2, 1× GlutaMaX, 5 ng/ml FGF2 (100-18B, Peprotech, NJ, USA), 10 ng/ml VEGF (100–20–10, Peprotech, NJ, USA), 20 ng/ml EGF (AF-100–15, Peprotech, NJ, USA), 3 μmol/L CHIR99021, 0.5 μmol/L A83–01 (2939, Tocris, MO, USA), 50 μg/ml ascorbic acid (A5960, Sigma, MO, USA) and 1% P/S for 4 d. Then the media was switched to advanced DMEM/F12 with 1× B27, 1× N2, 1× GlutaMaX, 2 μmol/L retinoic acid (R2625, Sigma, MO, USA), and 1% P/S and cultured for another 4 d. Finally, organoids were harvested and re-embedded in Matrigel on the ultra-low attachment multiwall plate (3471, Corning, MI, USA) in liver maturation media [hepatocyte culture medium (CC-3198, Lonza, MD, USA) prepared as manufacturer’s instructions, except no EGF, and supplemented with 10 ng/ml hepatocyte growth factor (100–39, PeproTech, NJ, USA), 0.1 μmol/L Dex (D4902, Sigma, MO, USA), 20 ng/ml oncostatin M (300–10, Peprotech, NJ, USA), and 1% P/S] with PBS or rFGFBP1 (25 ng/ml) or rFGFBP1 (25 ng/ml) + rFGF5 (200 ng/ml) for 10 d. Cells were maintained at 37 °C in humidified air with 5% CO 2 , and the medium was added every 2 d. For further details regarding the materials and methods used, please refer to Additional file : Methods.

Techniques: Western Blot, Immunohistochemistry, TUNEL Assay, Staining, Expressing, Transformation Assay, Knock-Out

Journal: Military Medical Research

Article Title: Glucocorticoids trigger muscle-liver crosstalk to attenuate acute liver injury and promote liver regeneration via the FGF6-FGFBP1 axis

doi: 10.1186/s40779-025-00618-y

Figure Lengend Snippet: Illustration summarizing the muscle-liver crosstalk in protecting against ALI and promoting liver regeneration. PHx or APAP-induced ALI elevates CORT, which activates muscle GR to suppress Fgf6 transcription. This suppression triggers two downstream effects: 1) inhibits protein synthesis and enhances catabolism, potentially providing substrates for pre-regenerative hepatocyte hypertrophy; and 2) FGF6 regulates Fgfbp1 transcription via the ERK-ATF3 axis. FGFBP1 forms membrane-associated condensates through LLPS. Free HS antagonizes LLPS to promote FGFBP1 release. FGF6 sequesters HS to stabilize LLPS, while FGF6 depletion liberates HS, disrupting LLPS and enhancing FGFBP1 secretion. Circulating FGFBP1 interacts with hepatic FGF5/FGF5s via LLPS to coordinate liver regeneration. CORT corticosterone, PHx partial (2/3) hepatectomy, APAP acetaminophen, GR glucocorticoid receptor, nGRE negative GC response elements, FGF6 fibroblast growth factor 6, FGFR fibroblast growth factor receptor, ERK extracellular signal-regulated kinases, ATF3 activating transcription factor 3, TF transcriptional factor, RNAP II RNA polymerase II, FGFBP1 fibroblast growth factor binding protein 1, LLPS liquid–liquid phase separation, HSPG heparan sulfate proteoglycan, HS heparan sulfate, FGF5 fibroblast growth factor 5, FGF5s short form of FGF5, ROS reactive oxygen species

Article Snippet: A total of 100,000 cells were embedded in 50 μl Matrigel drop on the dishes in advanced DMEM/F12 with 1× B27, 1× N2, 1× GlutaMaX, 5 ng/ml FGF2 (100-18B, Peprotech, NJ, USA), 10 ng/ml VEGF (100–20–10, Peprotech, NJ, USA), 20 ng/ml EGF (AF-100–15, Peprotech, NJ, USA), 3 μmol/L CHIR99021, 0.5 μmol/L A83–01 (2939, Tocris, MO, USA), 50 μg/ml ascorbic acid (A5960, Sigma, MO, USA) and 1% P/S for 4 d. Then the media was switched to advanced DMEM/F12 with 1× B27, 1× N2, 1× GlutaMaX, 2 μmol/L retinoic acid (R2625, Sigma, MO, USA), and 1% P/S and cultured for another 4 d. Finally, organoids were harvested and re-embedded in Matrigel on the ultra-low attachment multiwall plate (3471, Corning, MI, USA) in liver maturation media [hepatocyte culture medium (CC-3198, Lonza, MD, USA) prepared as manufacturer’s instructions, except no EGF, and supplemented with 10 ng/ml hepatocyte growth factor (100–39, PeproTech, NJ, USA), 0.1 μmol/L Dex (D4902, Sigma, MO, USA), 20 ng/ml oncostatin M (300–10, Peprotech, NJ, USA), and 1% P/S] with PBS or rFGFBP1 (25 ng/ml) or rFGFBP1 (25 ng/ml) + rFGF5 (200 ng/ml) for 10 d. Cells were maintained at 37 °C in humidified air with 5% CO 2 , and the medium was added every 2 d. For further details regarding the materials and methods used, please refer to Additional file : Methods.

Techniques: Membrane, Binding Assay