Fig 1: Exogenous Nrg4 limits septic liver injury after CLP (a) Survival curve of mice within 24 h post-CLP. n = 16 per group. b Murine sepsis scores at specified intervals (0, 3, 6, 12, and 24 h post-CLP). n = 6 per group. c, d Serum ALT and AST concentrations 24 h post-CLP. n = 6 per group. e Protein expression of HMGB1 in liver tissue 24 h post-CLP. n = 6 per group. f Histological changes in liver tissue observed via HE staining (the arrows indicate infiltration of inflammatory cells), scale bar: 100 μm. g Oil Red O staining of liver sections highlighting lipid droplet accumulation, scale bar: 100 μm. n = 6 per group. h Representative confocal microscopy images of liver sections stained for HMGB1 (red), albumin (green), and nuclei (blue); scale bar: 100 μm. Data are presented as mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant. CLP, cecal ligation and puncture
Fig 2: A schematic representation depicting the protective role of Nrg4, secreted by BAT, in septic liver injury During CLP- or LPS-induced sepsis, BAT is activated and releases Nrg4 into circulation, protecting the liver by inhibiting ferroptosis in septic liver injury. (Created using BioRender.com). BAT, brown adipose tissue; CLP, cecal ligation and puncture; LPS, lipopolysaccharide
Fig 3: Exogenous Nrg4 alleviates liver injury during sepsis by reducing inflammation and ferroptosis in the liver (a, b) Serum concentrations of TNF-α and IL-6. n = 5 per group. c qRT-PCR analysis quantifying Acox1, Cpt1a, Ehhadh, Fap27 and Ppargc1a mRNA levels. n = 5 per group. d Hydroxyl free radical scavenging capacity in mice liver tissue. n = 6 per group. e Fe2+ levels in mice liver tissue. n = 6 per group. f Prussian blue staining of liver slices highlights iron in liver tissue (the arrows indicate the stained hemosiderin in the tissue), scale bar: 100 μm. (g) GSH levels in mice liver tissue. n = 6 per group. h MDA levels in mice liver tissue. n = 6 per group. i Western blot analysis showing SLC7A11 and GPX4 expression in mice liver tissue. n = 6 per group. j Western blot analysis showing ACSL4, ALOX5, ALOX12, ALOX15, FPN1, NCOA4 and TFR2 expression in mice liver tissue. n = 6 per group. k qRT-PCR analysis quantifying Ncoa4, Trf, Tfr2, Fpn1, Hamp and Ltf mRNA levels. n = 5–6 per group. Data are presented as mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant
Fig 4: Nrg4 as a potential BAT adipokine for septic liver injury (a, c, e) qRT-PCR analysis quantifying Ucp1 and Nrg4 mRNA levels. n = 6 per group. b, d Serum concentrations of Nrg4. n = 6 per group. f Protein expression of UCP1 and Nrg4 in BAT. n = 6 per group. g Serum concentrations of Nrg4. n = 5–6. h Histological changes in BAT observed via HE staining, scale bar: 100 μm. i Representative confocal microscopy images of BAT sections stained for Nrg4 (red), PLIN (green), and nuclei (blue); scale bar: 100 μm. j Serum concentrations of Nrg4 at specified intervals (0, 6, 12, and 24 h post-rNrg4 administration). n = 5 per group. Data are presented as mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant. qRT-PCR, quantitative real-time polymerase chain reaction; HE, hematoxylin and eosin; BAT, brown adipose tissue; PLIN, perilipin
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