Journal: Acta Pharmaceutica Sinica. B

Article Title: Heme oxygenase 1-mediated ferroptosis in Kupffer cells initiates liver injury during heat stroke

doi: 10.1016/j.apsb.2024.05.007

Figure Lengend Snippet: HMOX-1-specific targeting in KCs induces ferroptosis. (A) Schematic diagram illustrating the influence of HMOX-1 through ZnPP, CoPP, and CoPP + DFO. (B) Survival curves of mice pretreated with 10 mg/kg ZnPP, 5 mg/kg CoPP, or 5 mg/kg CoPP + 100 mg/kg DFO followed by HS ( n = 15 mice per group). Evaluation of hepatocellular function by AST (C), ALT (D), and TBIL (E) ( n = 3–5 mice per group). (F) Representative H&E staining of liver paraffin sections from HS mice (scale bar: 200 μm). Detection of MDA (G), tissue non-heme iron (H) ( n = 3–5 mice per group), and BODIPY 581/591 C11 (I) in liver tissue of HS mice (scale bar: 200 μm). (J) Schematic diagram illustrating the influence of HMOX-1 through specific Hmox1 knockout in KCs. (K) Survival curves of Clec4f-crexHmox1 flox/flox mice or Hmox1 flox/flox mice, followed by HS ( n = 15 mice per group). Measurement of ALT (L), AST (M), and TBIL (N) ( n = 3–5 mice per group). (O) Representative H&E staining of liver paraffin sections of Clec4f-cre x Hmox1 flox/flox mice or Hmox1 flox/flox mice (scale bar: 200 μm). Measurement of MDA (P), tissue non-heme iron (Q) ( n = 3–5 mice per group), and BODIPY 581/591 C11 (R) in liver tissue (scale bar: 200 μm). Summary data are presented as the mean ± SEM. Significance was calculated using a one-way ANOVA with Tukey's post hoc test. Significance in (B) and (K) is determined by the log-rank (Mantel–Cox) test.

Article Snippet: Genetic Modifications: Mice with specific knockout of Hmox1 in KCs were generated by crossing C57BL/6J- Clec4f em1(cre)Glass /J (033296, Jackson lab) with C57BL/6J- Hmox1 em1Cflox /Cya (S–CKO-02911, Cyagen Biosciences) mice, resulting in Clec4f-cre x Hmox1 flox/flox mice.

Techniques: Staining, Knock-Out

Journal: Acta Pharmaceutica Sinica. B

Article Title: Heme oxygenase 1-mediated ferroptosis in Kupffer cells initiates liver injury during heat stroke

doi: 10.1016/j.apsb.2024.05.007

Figure Lengend Snippet: HMOX-1 affects NLRP3 inflammasome activation. (A) Representative immunofluorescence staining images displaying the expression of Clec4F (purple), HMOX-1 (green), NLRP3 (red), and in the liver of mice after HS, with magnified insets (scale bar: 10 μm). (B) Western Blotting analysis of Caspase-1 in liver tissues of HS mice and statistical results ( n = 3) (C). (D) Representative images of immunofluorescence staining for Clec4F (purple), HMOX-1 (green), and NLRP3 (red) in liver tissues of HS mice pretreated with PBS, 10 mg/kg ZnPP, 5 mg/kg CoPP, or 5 mg/kg CoPP + 100 mg/kg DFO, with magnified insets (scale bar: 10 μm). (E) estern blotting analysis of caspase-1 in liver tissues of HS mice and statistical results ( n = 3) (F). (G) Measurement of plasma IL-1 β content by ELISA ( n = 3–5 mice per group). (H) Representative images of immunofluorescence staining for Clec4F (purple), HMOX-1 (green), and NLRP3 (red) in liver tissues of Clec4f-cre x Hmox1 flox/flox mice or Hmox1 flox/flox mice, with magnified insets (scale bar: 10 μm). (I) Western blotting analysis of caspase-1 in liver tissues and statistical results ( n = 3) (J). (K) Measurement of plasma IL-1 β content by ELISA ( n = 3–5 mice per group). Significance was calculated using a one-way ANOVA with Tukey's post hoc test.

Article Snippet: Genetic Modifications: Mice with specific knockout of Hmox1 in KCs were generated by crossing C57BL/6J- Clec4f em1(cre)Glass /J (033296, Jackson lab) with C57BL/6J- Hmox1 em1Cflox /Cya (S–CKO-02911, Cyagen Biosciences) mice, resulting in Clec4f-cre x Hmox1 flox/flox mice.

Techniques: Activation Assay, Immunofluorescence, Staining, Expressing, Western Blot, Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Heme oxygenase 1-mediated ferroptosis in Kupffer cells initiates liver injury during heat stroke

doi: 10.1016/j.apsb.2024.05.007

Figure Lengend Snippet: HMOX-1 affects PI4K β activation in vitro . (A) Representative images of immunofluorescence staining for OSBP-PH-GFP (green), TGN38 (red), and DAPI (blue) in KC2 treated at 43 °C for 3 h and recovered at 37 °C for 0, 6, or 24 h (scale bar: 5 μm), and statistical analysis (B) of co-localization of OSBP-PH-GFP and TGN38 ( n = 20). (C) Representative images of immunofluorescence staining for OSBP-PH-GFP (green), TGN38 (red), and DAPI (blue) in KC2 pretreated with 4 μmol/L ZnPP for 12 h or 4 μmol/L CoPP for 12 h (scale bar: 5 μm), and statistical analysis (D) of co-localization of OSBP-PH-GFP and TGN38 ( n = 20). (E) Representative images of immunofluorescence staining for OSBP-PH-GFP (green), PI4K β (red), and DAPI (blue) in KC2 (scale bar: 5 μm), and statistical analysis (F) of co-localization of OSBP-PH-GFP and PI4K β ( n = 20). (G) Representative images of immunofluorescence staining for OSBP-PH-GFP (green), TGN38 (red), and DAPI (blue) in KC2 pretreated with 5 μmol/L PI4K β inhibitor for 1 h (scale bar: 5 μm), and statistical analysis (H) of co-localization of OSBP-PH-GFP and TGN38 ( n = 20). (I) Representative images of immunofluorescence staining for NLRP3 (red), TGN38 (green), and DAPI (blue) in KC2 (scale bar: 5 μm), and statistical analysis (J) of co-localization of OSBP-PH-GFP and TGN38 ( n = 20). (K) Representative images of immunofluorescence staining for PI4K β (red), TGN38 (green), and DAPI (blue) in KC2 pretreated with 4 μmol/L ZnPP for 12 h, 4 μmol/L CoPP for 12 h or 4 μmol/L CoPP for 12 h + 1 μmol/L Fer-1 for 1 h (scale bar: 5 μm), and statistical analysis (L) of co-localization of PI4K β and TGN38 ( n = 20). (M) Western blotting analysis of caspase-1 in KC2 pretreated with DMSO or PI4K β inhibitor and (N) statistical results ( n = 3). (O) Representative images of immunofluorescence staining for PI4K β (red), TGN38 (green), and DAPI (blue) in vector , sh-Hmox1 or OE-Hmox1 treated with 43 °C for 3 h and recovered at 37 °C for 6 h (scale bar: 5 μm) and (P) statistical analysis of co-localization of PI4K β and TGN38 ( n = 20). Summary data are presented as the mean ± SEM. Significance was calculated using a one-way ANOVA with Tukey's post hoc test.

Article Snippet: Genetic Modifications: Mice with specific knockout of Hmox1 in KCs were generated by crossing C57BL/6J- Clec4f em1(cre)Glass /J (033296, Jackson lab) with C57BL/6J- Hmox1 em1Cflox /Cya (S–CKO-02911, Cyagen Biosciences) mice, resulting in Clec4f-cre x Hmox1 flox/flox mice.

Techniques: Activation Assay, In Vitro, Immunofluorescence, Staining, Western Blot, Plasmid Preparation

Journal: Acta Pharmaceutica Sinica. B

Article Title: Heme oxygenase 1-mediated ferroptosis in Kupffer cells initiates liver injury during heat stroke

doi: 10.1016/j.apsb.2024.05.007

Figure Lengend Snippet: EGR1 regulates the transcription of Hmox1 . (A) Representative images of immunofluorescence staining for HMOX-1 (red), EGR1 (green), and DAPI (blue) in KC2 treated at 43 °C for 3 h and recovered at 37 °C for 0, 6, or 24 h (scale bar: 5 μm). (B) Western blotting analysis of the expression of HMOX-1 after knocking down Egr1 ( sh-Egr1 ) in ImKCs and statistical analysis ( n = 3) (C). (D) Representative images of immunofluorescence staining for PI4K β (red), TGN38 (green), and DAPI (blue) in vector or sh-Egr1 (scale bar: 5 μm). (E) Representative images of immunofluorescence staining for NLRP3 (red), TGN38 (green) and DAPI (blue) (scale bar: 5 μm). (F) Western blotting analysis of caspase-1. (G) Cellular supernatant IL-1 β content detected by ELISA ( n = 3 mice per group). (H) Dual-luciferase experiments in HEK-293T cells transfected with different truncated regions of the Hmox1 promoter ( n = 3). (I) Dual-luciferase experiments in HEK-293T cells transfected with different mutational regions of the Hmox1 promoter ( n = 3). ChIP-qRT-PCR analysis of EGR1 binding to −103 to −93, −1300 to −1290, −1849 to −1839 sites in the Hmox1 promoter region ( n = 3) (J) and fold change of signals in ImKC treated with 43 °C for 3 h and recovered at 37 °C for 6 h ( n = 3) (K). (L) A hypothetical model for KC2 ferroptosis in HS mice. Summary data are presented as the mean ± SEM. Significance was calculated using a one-way ANOVA with Tukey's post hoc test.

Article Snippet: Genetic Modifications: Mice with specific knockout of Hmox1 in KCs were generated by crossing C57BL/6J- Clec4f em1(cre)Glass /J (033296, Jackson lab) with C57BL/6J- Hmox1 em1Cflox /Cya (S–CKO-02911, Cyagen Biosciences) mice, resulting in Clec4f-cre x Hmox1 flox/flox mice.

Techniques: Immunofluorescence, Staining, Western Blot, Expressing, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Luciferase, Transfection, Quantitative RT-PCR, Binding Assay