Blockade of the Notch Signaling Pathway Promotes M2 Macrophage Polarization to Suppress Cardiac Fibrosis Remodeling in Mice With Myocardial Infarction

Figures

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  Figure 1. Blocking Notch signaling pathway improved myocardial injury in mice. (A–D): Left ventricular end-systolic diameter (LVES), left ventricular end-diastolic diameter (LVED), left ventricular ejection fraction (LVEF), and left ventricular fractional shortening (LVFS) were analyzed by micro-ultrasound machine, N = 23/per group; (E): Protein expression of RBP-J was detected, N = 6; (F): Infarction area was detected by TTC method. (G): TUNEL assay of the tissue apoptosis in the mouse heart. N = 3. Three independently repeated tests were performed, and the data were expressed as mean ±SD. One-way ANOVA was used for the analysis of variance, and Tukey's multiple comparisons test was used for the post-hoc test; ** P < 0.01 (compared with the sham group), ## P < 0.01, and # P < 0.05 (compared with the sh-NC group).

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  Figure 2. Blocking the Notch signaling pathway promoted the M2 polarization of cardiac macrophages and reduced the degree of fibrosis. (A): Identification of macrophage types in the myocardial infarction area of mice by immunofluorescence and flow cytometry, N = 6; (B): Observation of the degree of cardiac fibrosis in mice by MASSON staining, N = 8; (C): Detection of HYP content in the heart by ELISA kit, N = 6; (D): Detection of expression of TGF-β1, PDGF-B, COL1, and COL3 in the heart by qRT-PCR, N = 6; three independently repeated tests were performed, and the data were expressed as mean ± SD. One-way ANOVA was used for analysis of variance, and Tukey's multiple comparisons test was used for post-hoc test; ** P < 0.01, * P < 0.05 (compared with sham group), and ## P < 0.01, # P < 0.05 (compared with sh-NC group).

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  Figure 3. Blocking the Notch signaling reduced cardiac inflammation. (A): HE staining was conducted to observe the mouse heart, N = 8; (B): Immunohistochemistry staining was conducted to observe the macrophage and neutrophil infiltration in the mouse heart, N = 6; (C): Flow cytometry was performed to observe the macrophage and neutrophil infiltration in the mouse heart, N = 6; (D): ELISA was performed to detect the expression of inflammatory cytokines TNF-α, IL-1β, and IL-6 in the mouse heart, N = 6; three independently repeated tests were performed, and the data were expressed as mean ± SD. One-way ANOVA was used for analysis of variance, and Tukey's multiple comparisons test was used for post-hoc test; ** P < 0.01, * P < 0.05 (compared with sham group), and ## P < 0.01, # P < 0.05 (compared with sh-NC group).

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  Figure 4. Blocking the Notch signaling inhibits the activation of the NF-κB pathway. (A): p65 levels in mouse hearts were detected by immunohistochemistry staining; (B): Phosphorylation level of the NF-κB pathway was detected by Western blot; (C): mRNA expression of CYLD in mouse cardiac macrophages was detected by qRT-PCR; (D): Protein expression of CYLD in mouse cardiac macrophages was detected by Western blot; N = 6; three independently repeated tests were performed, and the data were expressed as mean ± SD. One-way ANOVA was used for the analysis of variance, and Tukey's multiple comparisons test was used for post hoc test; ** P < 0.01 (compared with the sham group), and ## P < 0.01 (compared with the sh-NC group).

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  Figure 5. Blocking of the Notch signaling pathway in vitro promoted M2 macrophage polarization and CYLD expression and inhibited fibrosis-related factors expression and cellular inflammatory levels. (A): Bone marrow-derived macrophages in mice were identified by flow cytometry; (B): Heart-derived fibroblasts were identified by immunohistochemistry. Macrophages with different treatments were isolated and divided into Mø-Sham, Mø-MI, Mø-sh-NC, and Mø-sh-RBP-J groups. Macrophages and fibroblasts stimulated with LPS + INF-γ for 24 h were co-cultured by Transwell to establish an in vitro inflammatory model; (C): Macrophage polarization was detected by flow cytometry; (D): p65 in macrophage nuclei of each group was detected by immunofluorescence; (E): Expression of fibrosis-related factors TGF-β1, PDGF-B, COL1, and COL3 in fibroblasts was detected by qRT-PCR; (F): Expression of inflammatory factors TNF-α, IL-1β, and IL-6 in macrophages was detected by ELISA; (G): The expression of CYLD in macrophages was detected by qRT-PCR. Three independently repeated tests were performed, and the data were expressed as mean ± SD. One-way ANOVA was used for analysis of variance, and Tukey's multiple…

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  Figure 6. Inhibition of CYLD expression reversed the results that blocking of the Notch signaling pathway inhibited fibrogenesis and the release of inflammatory factors. siRNA and siRNA Scramble of CYLD were transfected into bone marrow-derived macrophages Mø-sh-NC group and Mø-sh-RBP-J group and stimulated with LPS + INF-γ for 24 h. (A): mRNA expression of CYLD in macrophages of each group was detected by qRT-PCR; (B): p65 in macrophage nuclei was detected by immunofluorescence; (C): Expression of fibrosis-related factors TGF-β1, PDGF-B, COL1, and COL3 in fibroblasts was detected by qRT-PCR; (D): Expressions of inflammatory factors TNF-α, IL-1β, and IL-6 in macrophages were detected by ELISA; Three independently repeated tests were performed, and the data were expressed as mean ± SD. One-way ANOVA was used for the analysis of variance, and Tukey's multiple comparisons test was used for post- hoc test; ** P < 0.01 (compared with Mø-sh-NC + Scramble group), and ## P < 0.01 (compared with Mø-sh-RBP-J + Scramble group).

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  Figure 7. Mechanistic map of blocking the Notch pathway to improve cardiac fibrosis. Blocking of the Notch signaling pathway promoted M2 macrophage polarization and increased CYLD expression in macrophages, followed by inhibiting the activation of NF-κB pathway, inflammatory factors, and fibrosis-related factors, and ultimately suppressed cardiac fibrosis remodeling.