Fig 1: Administration of recombinant APOH protein protects septic mice. a Ligation of the caecum at designated positions to establish a severe CLP model. The caecum was ligated (indicated by the dotted green line) at a 60% distance between the distal pole and the base of the caecum (yellow dotted line). b The procedures for rAPOH treatment in CLP-induced severe sepsis. c Survival of mice with CLP after treatment with BSA and rAPOH (n = 8). d Lung, liver, and kidney tissues were stained with H&E in the BSA and rAPOH groups. e Semiquantitative scores of tissues were calculated in the BSA and rAPOH groups (n = 7). (f) Serum cytokine levels in the BSA and rAPOH groups at 24 h after severe CLP (n = 7). g Cytokine levels in the PLF in the BSA and rAPOH groups at 24 h after severe CLP (n = 7). The data are presented as the means ± standard deviations (S.D.). “*” indicates a difference between groups. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Apolipoprotein H, APOH; recombinant murine APOH, rAPOH; bovine serum albumin, BSA; caecal ligation and puncture, CLP; peritoneal lavage fluid, PLF; haematoxylin and eosin staining, H&E
Fig 2: APOH levels are significantly decreased in patients with sepsis. a LC–MS/MS was employed to identify potential biomarkers for sepsis in a pilot cohort, and ELISAs were used to confirm the expression of APOH in a validation cohort. A murine model of CLP-induced sepsis was employed to evaluate the therapeutic impact of APOH. b LC‒MS/MS was employed to identify the level of APOH in patients with sepsis and healthy controls in the pilot cohort. c LC‒MS/MS was employed to identify the level of APOH in survivors and non-survivors with sepsis in the pilot cohort. d Serum levels of APOH in 36 paediatric patients with sepsis and 69 healthy controls in the validation cohort. e The dynamics of APOH levels in the serum of paediatric patients with sepsis at 24, 48 and 72 h in the validation cohort. The data are presented as the means ± standard deviations (S.D.). “*” indicates a difference between groups. *p < 0.05, **p < 0.01, *** p < 0.001, ****p < 0.0001. Apolipoprotein H, APOH; liquid chromatography tandem mass spectrometry, LC‒MS/MS; enzyme-linked immunosorbent assay, ELISA.
Fig 3: APOH inhibits the TLR4/NF-κB pathway in macrophage cells treats with LPS. a The mRNA expression levels of TLR4, MyD88, TRAF6, JNK and P65 in PMs. b–e Western blotting analysis of the relative expression of the TLR4, MyD88, and TRAF6 proteins in PMs. f–g Western blot analysis of the expression of phosphorylated JNK in PMs. h–i Western blot analysis of the phosphorylated P65 in PMs. j The mRNA expression of TLR4, MyD88, TRAF6, JNK and P65 in RAW 264.7 cells. k–n Western blotting analysis of the relative expression of TLR4, MyD88, and TRAF6 proteins in RAW 264.7 cells. o–p Western blot analysis of the expression of phosphorylated JNK in RAW 264.7 cells. q–r Western blot analysis of the expression of phosphorylated p65 in RAW 264.7 cells. Primer sequences in mRNA were listed in Additional file 5: Table S2. The data are presented as the means ± standard deviations (S.D.). “*” indicates a difference between groups. *p < 0.05, **p < 0.01, *** p < 0.001, **** p < 0.0001. Apolipoprotein H, APOH; peritoneal macrophages, PMs; lipopolysaccharide, LPS; Toll-like receptor 4, TLR4; myeloid differentiation factor 88, MyD88; nuclear factor-κB, NF-κB; TNF receptor associated factor 6, TRAF6; Jun N-terminal kinase, JNK
Fig 4: The effect of APOH administration on macrophage polarization. a Flow cytometry was employed to determine the change in the M1 and M2 polarization in PMs. b–c The percentage of M1- and M2-polarized PMs was detected. d Flow cytometry was employed to determine the change in the M1 and M2 polarization in RAW 264.7 macrophages. e–f The percentage of M1- and M2-polarized RAW 264.7 cells were detected. g Western blot analysis of iNOS and Arg-1 in PMs. h–i Relative expression levels of iNOS and Arg-1 in PMs. j Western blot analysis of iNOS and Arg-1 in RAW 264.7 cells. k–l Relative expression levels of iNOS and Arg-1 in RAW 264.7 cells. The data are presented as the means ± standard deviations (S.D.). “*” indicates a difference between groups. *p < 0.05, **p < 0.01, *** p < 0.001, **** p < 0.0001. Apolipoprotein H, APOH; peritoneal macrophages, PMs; lipopolysaccharide, LPS; inducible nitric oxide synthase, iNOS; Arginase 1, Arg-1
Fig 5: Anti-APOH antibody worsens non-severe sepsis in mice. a Ligation of the caecum at designated positions to establish a non-severe CLP model. The caecum was ligated (indicated by the dotted green line) at a 40% distance between the distal pole and the base of the caecum (yellow dotted line). b The procedures for the administration of an anti-APOH antibody in mice with CLP-induced non-severe sepsis. c Survival of mice with CLP-induced non-severe sepsis after treatment with the anti-APOH antibody (n = 8). d Lung, liver, and kidney tissues were stained with H&E in the IgG control and anti-APOH groups. e Semiquantitative scores of tissues were calculated in the IgG control and anti-APOH groups (n = 7). f Serum cytokine levels in blood in the IgG control and anti-APOH groups at 24 h after non-severe CLP (n = 7). g Cytokine levels in the PLF in the IgG control and anti-APOH groups at 24 h after non-severe CLP (n = 7). The data are presented as the means ± standard deviations (S.D.). “*” indicates a difference between groups. *p < 0.05, **p < 0.01, *** p < 0.001, **** p < 0.0001. Apolipoprotein H, APOH; caecal ligation and puncture, CLP; peritoneal lavage fluid, PLF; haematoxylin and eosin staining, H&E
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