Journal: Hepatology Communications

Article Title: Targeting fatty acid metabolism in hepatic macrophages mitigates acetaminophen-induced liver injury and promotes regeneration in mice

doi: 10.1097/HC9.0000000000000865

Figure Lengend Snippet: HMGB1 released from necrotic hepatocytes promotes inflammation and migration in macrophages by activating FASN-mediated DNL. (A) Serum levels of HMGB1 in the AILI model mouse were detected using ELISA. (B, C) AML12 cells were stimulated with APAP (5 mM) for 24 hours, and levels of HMGB1 protein were measured with a western blot. RAW264.7 cells were stimulated with recombinant HMGB1 (100 ng/mL) for 24 hours. (D, E) The expression of FASN was detected with western blot. (F) Representative images of BODIPY493/503 fluorescent dye staining of intracellular neutral lipids. Bar=50 μm. (G) Cell migration was assessed using a transwell assay. bar=50μm. (H) mRNA levels of Tnf-α and Il-1β . AML12 cells were transfected with siRNA to silence Hmgb1 expression, and the cell culture supernatant of AML12 cells with APAP treatment was collected as the CM. RAW246.7 cells were stimulated with CM. Levels of FASN protein (I, J), migration (K), and mRNA levels of Tnf-α and Il-1β. (L) were measured, respectively, bar=50 μm. Data are means±SEMs. Two-tailed unpaired Student t tests were performed for statistical analysis. * p <0.05, ** p <0.01, *** p <0.001. Abbreviations: APAP, acetaminophen; AILI, APAP-induced liver injury; CM, conditioned media; NC, negative control; SiRNA, small interfering RNA.

Article Snippet: The lower chamber was filled with CM or DMEM containing recombinant mouse HMGB1 (MCE, HY-P700182AF).

Techniques: Migration, Enzyme-linked Immunosorbent Assay, Western Blot, Recombinant, Expressing, Staining, Transwell Assay, Transfection, Cell Culture, Two Tailed Test, Negative Control, Small Interfering RNA

Journal: Hepatology Communications

Article Title: Targeting fatty acid metabolism in hepatic macrophages mitigates acetaminophen-induced liver injury and promotes regeneration in mice

doi: 10.1097/HC9.0000000000000865

Figure Lengend Snippet: HMGB1 enhances the inflammatory response and facilitates the migration of macrophages through the PI3K/AKT-SREBP1/FASN signaling pathway. (A, B) HMGB1 was used to stimulate RAW246.7 cells for the indicated durations. The protein levels of PI3K/p-PI3K and AKT/p-AKT were detected with western blot. RAW246.7 cells were pretreated with the PI3K inhibitor, LY294002 (50 μM), then stimulated with HMGB1. (C, D) The levels of FASN protein were detected with western blot. (E) mRNA levels of Tnf-α and Il-1β . (F, G) The protein levels of SREBP1 in HMGB1-treated RAW264.7 cells were detected with western blot. (H, I) After pretreatment with LY294002, SREBP1 protein levels in RAW246.7 cells exposed to HMGB1 were analyzed using western blot. RAW246.7 cells were pretreated with Fatostatin (10 mM), an inhibitor of SREBP1, and stimulated with HMGB1. (J, K) Protein levels of FASN were analyzed using western blot. (L) mRNA levels of Tnf-α and Il-1β . The data are the means±SEMs. Two-tailed unpaired Student t tests were performed for statistical analysis. * p <0.05, ** p <0.01, *** p <0.001. Abbreviations: FASN, fatty acid synthase; HMGB1, high-mobility group protein B1.

Article Snippet: The lower chamber was filled with CM or DMEM containing recombinant mouse HMGB1 (MCE, HY-P700182AF).

Techniques: Migration, Western Blot, Two Tailed Test

Journal: Biochemistry and Biophysics Reports

Article Title: HMGB1 impairs nasal mucosa epithelial barrier function in allergic rhinitis by promoting BECN1-mediating autophagy

doi: 10.1016/j.bbrep.2025.102351

Figure Lengend Snippet: Inhibition of HMGB1 preserves nasal mucosal epithelial barrier junction integrity in an IL-4-induced inflammatory model . The hNEpiC cells were transfected with sh-NC (negative control) or sh-HMGB1 plasmids. After 4–6 h of incubation, the transfection mixture was replaced with complete culture medium containing 10 ng/ml IL-4 for 48 h to simulate an inflammatory environment. (A) Protein level of HMGB1 in IL-4-induced hNEpiC cells was analyzed by Western blot. ∗∗∗p < 0.001. (B) mRNA level of HMGB1 in hNEpiC cells transfected with sh-NC or sh-HMGB1 was detected by qRT-PCR, confirming efficient knockdown. ∗∗∗p < 0.001. (C) Barrier function was evaluated by trans -epithelial electrical resistance (TEER) in IL-4-induced hNEpiC cells transfected with sh-NC or sh-HMGB1. ∗∗∗∗p < 0.0001 vs. Control; ####p < 0.0001 vs. IL-4 + sh-NC. (D) Paracellular permeability was assessed by FITC-dextran flux in IL-4-treated hNEpiC cells transfected with sh-NC or sh-HMGB1. ∗∗∗∗p < 0.0001 vs. Control; ####p < 0.0001 vs. IL-4 + sh-NC. (E) Representative Western blots and quantitative analysis of HMGB1 and tight junction proteins (ZO-1, Occludin, Claudin-1) in IL-4-induced hNEpiC cells transfected with sh-NC or sh-HMGB1. ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: After incubated for 4–6 h, the transfection solution was removed and replaced with complete culture medium containing 10 ng/ml of IL-4 (HY– P70445 , MCE, USA) with or without 2 μg/ml of recombinant HMGB1 protein (HY– P72797 , MCE) for 48 h.

Techniques: Inhibition, Transfection, Negative Control, Incubation, Western Blot, Quantitative RT-PCR, Knockdown, Control, Permeability

Journal: Biochemistry and Biophysics Reports

Article Title: HMGB1 impairs nasal mucosa epithelial barrier function in allergic rhinitis by promoting BECN1-mediating autophagy

doi: 10.1016/j.bbrep.2025.102351

Figure Lengend Snippet: HMGB1 knockdown attenuates autophagy in IL-4-stimulated nasal mucosal epithelial cells. hNEpiC cells were transfected with sh-NC or sh-HMGB1 plasmids and then treated with 10 ng/ml IL-4 for 48 h to establish an inflammatory microenvironment. (A) Representative Western blots and quantitative analysis of key autophagy-related proteins (BECN1, LC3-I/II, ATG5, and p62) in IL-4-treated hNEpiC cells transfected with sh-NC or sh-HMGB1. Decreased LC3-II/LC3-I ratio, BECN1 and ATG5 levels and increased p62 level indicate impaired autophagy flux upon HMGB1 knockdown. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (B) Immunofluorescence staining of LC3B (green) in IL-4-treated hNEpiC cells. Nuclei were stained with DAPI (blue). HMGB1 knockdown reduced LC3B puncta formation, suggesting suppression of autophagosome accumulation. Scale bar: 20 μm (C) Autophagic flux was monitored using the mCherry-GFP-LC3B tandem reporter. Yellow puncta (mCherry + GFP+) represent autophagosomes; red-only puncta (mCherry + GFP−) indicate autolysosomes. HMGB1 knockdown inhibit autolysosomes formation. Scale bar: 20 μm.

Article Snippet: After incubated for 4–6 h, the transfection solution was removed and replaced with complete culture medium containing 10 ng/ml of IL-4 (HY– P70445 , MCE, USA) with or without 2 μg/ml of recombinant HMGB1 protein (HY– P72797 , MCE) for 48 h.

Techniques: Knockdown, Transfection, Western Blot, Immunofluorescence, Staining

Journal: Biochemistry and Biophysics Reports

Article Title: HMGB1 impairs nasal mucosa epithelial barrier function in allergic rhinitis by promoting BECN1-mediating autophagy

doi: 10.1016/j.bbrep.2025.102351

Figure Lengend Snippet: BECN1 knockdown restores epithelial barrier integrity impaired by HMGB1 overexpression . The hNEpiC cells were transfected with sh-NC or sh-BECN1 plasmids and treated with 10 ng/ml IL-4 in the presence or absence of 2 μg/ml recombinant HMGB1 protein for 48 h to evaluate functional rescue. (A) Protein-protein interaction network generated using the STRING database, showing HMGB1 as a central node interacting with multiple partners including BECN1, suggesting a potential regulatory relationship in autophagy and inflammation pathways. (B) Luciferase reporter assay demonstrating dose-dependent activation of the BECN1 promoter by HMGB1 overexpression in hNEpiC cells, indicating HMGB1 transcriptionally regulates BECN1 expression. ∗p < 0.05, ∗∗∗∗p < 0.0001. (C) Epithelial barrier function assessed by trans -epithelial electrical resistance (TEER) under indicated conditions. BECN1 knockdown significantly ameliorated the barrier disruption induced by HMGB1 overexpression. ∗∗p < 0.01, ∗∗∗∗p < 0.0001. (D) Paracellular permeability evaluated by FITC-dextran flux across the epithelial monolayer. HMGB1 overexpression increased permeability, which was rescued by BECN1 knockdown. ∗p < 0.05, ∗∗∗∗p < 0.0001. (E) Expression levels of tight junction proteins (ZO-1, Occludin, Claudin-1) analyzed by Western blot. BECN1 knockdown reversed the HMGB1-induced downregulation of these critical barrier proteins. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (F) Protein expression of inflammatory mediators (Caspase-1, Caspase-4, TNF-α) measured by Western blot. HMGB1-enhanced inflammation was attenuated by BECN1 knockdown, linking autophagy regulation to inflammatory response. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: After incubated for 4–6 h, the transfection solution was removed and replaced with complete culture medium containing 10 ng/ml of IL-4 (HY– P70445 , MCE, USA) with or without 2 μg/ml of recombinant HMGB1 protein (HY– P72797 , MCE) for 48 h.

Techniques: Knockdown, Over Expression, Transfection, Recombinant, Functional Assay, Generated, Luciferase, Reporter Assay, Activation Assay, Expressing, Disruption, Permeability, Western Blot

Journal: Biochemistry and Biophysics Reports

Article Title: HMGB1 impairs nasal mucosa epithelial barrier function in allergic rhinitis by promoting BECN1-mediating autophagy

doi: 10.1016/j.bbrep.2025.102351

Figure Lengend Snippet: BECN1 knockdown suppresses HMGB1-induced autophagy in hNEpiC cells . The hNEpiC cells were transfected with sh-NC or sh-BECN1 plasmids and treated with 10 ng/ml IL-4 with or without 2 μg/ml recombinant HMGB1 protein for 48 h to evaluate autophagic activity. (A) Representative Western blots and quantitative analysis of autophagy markers. HMGB1 overexpression promoted autophagy as evidenced by increased LC3-II/LC3-I ratio, BECN1 and ATG5 levels and decreased p62 level, while BECN1 knockdown reversed these effects, confirming its essential role in HMGB1-mediated autophagy. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (B) Immunofluorescence analysis of LC3B puncta formation (green). HMGB1 overexpression enhanced LC3B puncta formation, indicating increased autophagosome accumulation, which was suppressed by BECN1 knockdown. Nuclei were counterstained with DAPI (blue). Scale bar: 20 μm (C) Autophagic flux assessment using mCherry-GFP-LC3B reporter. Yellow puncta (mCherry + GFP+) represent autophagosomes while red puncta (mCherry + GFP-) indicate autolysosomes. HMGB1 overexpression increased autolysosomes, whereas BECN1 knockdown predominantly reduced autolysosome formation. Scale bar: 20 μm.

Article Snippet: After incubated for 4–6 h, the transfection solution was removed and replaced with complete culture medium containing 10 ng/ml of IL-4 (HY– P70445 , MCE, USA) with or without 2 μg/ml of recombinant HMGB1 protein (HY– P72797 , MCE) for 48 h.

Techniques: Knockdown, Transfection, Recombinant, Activity Assay, Western Blot, Over Expression, Immunofluorescence