Fig 1: 13-Me-PLT decreases C5aR1-expressing neutrophils in the border zone of infarct area. (A) Experimental protocols. (B–C) Representative images and percentage of CD11b+ cells in total (n = 4, the same below). (D-E) Representative images and infiltrative percentage of neutrophils, macrophages and dendritic cells in gated CD11b+ cells. (F-G) Representative images of C5aR1+ in gated CD11b+Ly6G+ neutrophils, CD11b+Ly6C+Ly6G−F4/80+ macrophages and CD11b+Ly6C+Ly6G− CD11c+ dendritic cells among sham, I/R, and 13-Me-PLT groups. (H) Mean fluorescence intensity of C5aR1 in gated CD11b+Ly6G+ neutrophils, CD11b+Ly6C+Ly6G−F4/80+ macrophages and CD11b+Ly6C+Ly6G− CD11c+ dendritic cells among sham, I/R, and 13-Me-PLT groups. Data are presented as mean ± standard error of mean, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, vs. Sham; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. I/R; ns: no significance; Neu, neutrophils; Mac, macrophages; DC, dendritic cells.
Fig 2: 13-Me-PLT improves the myocardial injury and NETs formation mediated by C5aR1-overexpression. (A) Experimental protocols. (B–C) Representative images of echocardiography and statistical data on LVEF and LVFS (n = 7). (D) Representative images of heart sections with H&E staining (n = 4); Scale bar = 50 μm. (E) Plasma levels of C5a, C5b-9, CCL3, CXCL1, and NETs (n = 5–10 rats per group). (F) Transcript levels of C5ar1, Mpo and Ne genes in heart tissue (n = 6–8 rats per group). (G-H) Representative images and protein levels of C5aR1, MPO, NE and CitH3 (n = 6). (I) ROS levels in myocardial tissues (n = 5–6). (J-K) Images of immunofluorescent staining of DAPI, C5aR1 and NE at low magnification (scale bar = 50 μm) and enlarged images (scale bar = 10 μm (n = 4). Data are presented as mean ± standard error of mean, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. Control, #P < 0.05, ##P < 0.01, ###P < 0.001 vs. BM213.
Fig 3: C5a-C5aR1 axis inhibition exerts cardio-protective effects against myocardial I/R injury in rats. (A) Experimental protocols. (B–C) Representative images of echocardiography and statistical data on LVEF and LVFS (n = 7). (D) Representative images of cardiac sections with H&E staining (n = 4); Scale bar = 50 μm. (E-F) Representative images and quantitative analysis of IA/AAR ratio on myocardial sections of rats (n = 5–6). (G-H) Representative images and apoptotic cell counts. (n = 4). (I-J) Representative images and protein levels of BAX and BCL-2 (n = 6). Data are presented as mean ± standard error of mean, ∗∗∗P < 0.001 vs. Sham, #P < 0.05, ##P < 0.01, ###P < 0.001 vs. I/R. Red arrow: Ruptured myocardial fibers and neutrophil infiltration.
Fig 4: 13-Me-PLT inhibits C5aR1 and NET formation during myocardial I/R injury in rats. (A) Experimental protocols. (B) PCA among the Sham, I/R, and 13-Me-PLT groups. (C) Venn diagram showing the number of differentially expressed genes (DEGs), highlighting a total of 20 DEGs commonly identified in the I/R vs Sham group and 13-Me-PLT vs I/R group. (D) GSEA showing DEGs involved in NET formation in I/R vs Sham group and 13-Me-PLT vs I/R group. (E) Heatmap of DEGs associated with NET formation signaling pathway. (F) Transcript levels of C5ar1, Tlr7 and Ncf4 genes (n = 6). (G-H) Representative Western Blot and statistical data on C5aR1 protein levels in heart tissues (n = 6). (I) Molecular docking of 13-Me-PLT with the structure region of C5aR1 protein. (J) RMSD and Rg results on 13-Me-PLT and C5aR1 protein. (K) The response of 13-Me-PLT and C5aR1 in surface plasmon resonance (SPR) analysis. Data are presented as mean ± standard error of mean, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. Sham, #P < 0.05, ##P < 0.01, ###P < 0.001 vs. I/R.
Fig 5: C5a-C5aR1 axis inhibition decreases NETs formation in myocardial I/R injury of rat. (A) Plasma C5a, C5b-9, CCL3, CXCL1, and NETs levels (n = 5–8 rats per group). (B) Transcript levels of C5ar1, Mpo and Ne genes in myocardium. (n = 8). (C-D) Representative images and protein levels of C5aR1, MPO, NE and CitH3 in myocardium (n = 6). (E) ROS levels in myocardial tissues (n = 6). (F-G) Images of immunofluorescent staining of DAPI, C5aR1 and NE at low magnification (scale bar = 50 μm) and enlarged images (scale bar = 10 μm (n = 4). Data are presented as mean ± standard error of mean, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. Sham, #P < 0.05, ##P < 0.01, ###P < 0.001 vs. I/R.
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