Fig 1.
Histological characterization of the rabbit PTE model.
Representative images of (A) H&E, (B) Masson and (C) Sirius Red staining of lung tissues before modeling and at 1, 7, and 14 days after modeling. Quantitative analysis of the collagen fiber area fraction from (D) Masson and (E) Sirius Red staining. For the quantitative analyses in (D) and (E), n = 6 biologically independent animals per group/time point. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Scale bars: 100 μm and 20 μm.
Fig 2.
Dynamic changes in serological indicators before and after PTE modeling.
(A) MPO-DNA. (B) vWF. (C) BNP. (D) D-dimer. (E) PAI-1. (F) t-PA. (G) VEGF. (H) HSP-47. n = 6 biologically independent animals per group/time point. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Fig 3.
DEGs and their GO and KEGG enrichment analysis in the PTE model group versus the control group at 1 (A), 3 (B), 7 (C), and 14 (D) days post-modeling.
Whole-blood RNA-seq was performed using n = 6 biologically independent animals per group/time point.
Fig 4.
Identification of common NETs/Ferroptosis and time point-specific upregulated DEGs following PTE.
(A) Venn diagram illustrating the common DEGs obtained from the intersection of upregulated DEGs across all post-PTE time points. (B, C) Venn diagrams showing the intersection of the common DEGs with gene sets for NETs (B) and ferroptosis (C) from the GeneCards database. (D-G) Venn diagrams identifying key time point-specific thrombus-associated DEGs at day 1 (D), day 3 (E), day 7 (F), and day 14 (G) post-embolization, revealed by intersecting the top 20 thrombus-associated DEGs with the time point-specific upregulated DEGs. RNA-seq input samples were derived from n = 6 biologically independent animals per group/time point.
Fig 5.
qRT-PCR validation of common NETs/ferroptosis-related and temporal-specific DEGs after PTE.
(A) Common NETs/ferroptosis-related DEGs at various time points post-PTE modeling. (B) Specific DEGs at day 1 post-PTE modeling. (C) Specific DEGs at day 3 post-PTE modeling. (D) Specific DEGs at day 7 post-PTE modeling. (E) Specific DEGs at day 14 post-PTE modeling. n = 6 biologically independent animals per group/time point. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Fig 6.
Changes and improvement rates of various indicators following rt-PA monotherapy and rt-PA + DNase I combination therapy on day 7 after PTE modeling.
(A) Collagen fiber area fraction. (B) PAT. (C) SpO2. (D) LVEF. (E) LVFS. (F) MPO-DNA. (G) vWF. (H) BNP. (I) PAI-1. (J) t-PA. (K) VEGF. n = 6 biologically independent animals per treatment group. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Fig 7.
Additional pharmacological validation of the NETs-TLR9-NF-kB/GPX4 axis in the rabbit PTE model.
Serum levels of (A) MPO-DNA, (B) TLR9, (C) phospho-p65, (D) total p65, (E) phospho-p65/total p65 ratio, and (F) GPX4 were measured by ELISA. Relative mRNA expression of (G) TLR9 and (H) GPX4 was measured by qRT-PCR. Data are presented as mean ± SD with all individual data points shown; n = 5 biologically independent animals per group. One-way ANOVA followed by Dunnett’s multiple-comparisons test was used, with the Model group as the reference. * P < 0.05; ** P < 0.01; *** P < 0.001 versus Model.