Fig 2: The transcription factor SPI1 regulates S100A8 and S100A9 transcription. (A) Binding sites of SPI1 on the promoter of S100A8/A9; (B–C) Western blot and qRT-PCR detection of protein and mRNA expression of SPI1 in rat myocardial tissues, N = 6 rats/group, *p < 0.05 compared with the sham group; (D) ChIP assay determination of SPI1 enrichment in the promoter of S100A8/A9, * p < 0.05 compared with the anti-IgG group; (E) Dual-luciferase reporter gene assay to evaluate the binding relationship between SPI1 and S100A8/A9, * p < 0.05 compared with the si-NC group; (F–G) qRT-PCR and western blot assessment of the expression of related genes in primary rat cardiomyocytes, * p < 0.05 compared with the si-NC group. Cell experiments were repeated three times. SPI: salmonella pathogenicity island; qRT-PCR: quantitative real-time polymerase chain reaction; ChIP: chromatin immunoprecipitation; IgG: Immunoglobulin G; Si: silencing; NC: negative control.

Fig 3: Infarct wall thinning observed at myocardial infarction (MI) day 3 negatively correlated with neutrophils and positively correlated with macrophages. A, I: S100A8/A9 did not change neutrophil infiltration (Ly6b expression) in the left ventricle (LV) infarct in the S100A8/A9 group (n = 6) compared with saline group (n = 5), P = 0.09. Data analyzed using unpaired t test. A, II: neutrophils negatively correlated with the infarct wall thinning index at MI D3. B, I: S100A8/A9 increased macrophage infiltration (Mac-3 expression) in the LV infarct in the S100A8/A9 group (n = 6) compared with saline group (n = 5), P = 0.014. Data analyzed using unpaired t test. B, II: macrophages positively correlated with the infarct wall thinning index at MI D3. MI, myocardial infarction.

Fig 4: S100A8/A9 did not directly stimulate MMP-9 expression in vivo or neutrophil degranulation in vitro. A, I: S100A8/A9 did not increase MMP-9 expression in the left ventricle (LV) infarct at myocardial infarction (MI) D1. A, II: S100A8/A9 did not increase MMP-9 expression in the LV infarct at MI D3. Sample sizes were saline (n = 6) and S100A8/A9 (n = 5–7); data analyzed by unpaired t test. B: S100A8/A9 did not increase MMP-9 release from bone marrow-derived neutrophils stimulated with S100A8/A9 for 15 min in vitro. Samples were n = 3; data analyzed using paired t test.

Fig 5: AAV9-GFP expression in rats. (A) Distribution of GFP fluorescence in heart, kidney, and liver tissues after AAV9-GFP infection in rats (scale bar: 100 µm); (B) qRT-PCR examination of the expression of S100A8 and S100A9 in myocardial tissues after AAV-shS100A8 and AAV-shS100A9 infection in rats; (C) The expression of S100A8 and S100A9 in myocardial tissues measured by western blot after AAV-shS100A8 and AAV-shS100A9 infection in rats. *p < 0.05 compared with UCM + AAV-shNC group. N = 6 rats/group. AAV: adeno-associated virus; GFP: green fluorescent protein; qRT-PCR: quantitative real-time polymerase chain reaction; Sh: short hairpin.