Icariin-mediated structural stabilization of TNF-α triggers a pro-regenerative signaling switch for endothelial recovery after myocardial infarction

Figures

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  FIGURE 1. Increased number of endothelial cells proliferation in post-infarction heart failure rats. (A) Schematic diagram of in vivo animal experiments. (B) Pathological changes in each group (n = 3). (C) Echocardiographic changes in each group (n = 12). (D) Changes in infarct size in each group in rats (n = 6). (E) Determination of CK, CK-MB and LDH in rat serum (n = 12). (F) Determination of ANP, BNP and Ang-2 in rat serum (n = 12). (G) Immunohistochemical changes in post-infarction heart failure rats (n = 3). (H) Immunofluorescence assay for detection of regenerating endothelial cell (n = 3). LADL, left anterior descending coronary artery ligation. BrdU marks regeneration cells and CD31 marks endothelial cells. Data are presented as the means ± SD. Compared with sham group, # p < 0.05, ## p < 0.01.

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  FIGURE 2. The TNF pathway plays an important role in heart failure. (A) Flowchart of the GEO database for online data retrieval. (B) Principal component analysis between groups. (C) Expression of differential genes between groups. (D) GO enrichment analysis of differential genes. (E) KEGG enrichment analysis of differential genes. (F) Enrichment of core genes of TNF signaling pathway. (G) mRNA expression of TNF, MYC and FOS. (H) TNF signaling pathway can regulate the cell cycle (n = 8).

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  FIGURE 3. In vitro experiments confirm TNF signaling pathway promotes endothelial cell proliferation. (A) Schematic diagram of in vitro cell experiments. (B) Cell survival was reduced with the addition of TNF inhibitors (n = 6). (C) Migratory capacity was reduced with the addition of TNF inhibitors (n = 6). (D) EdU + cell was reduced with the addition of TNF inhibitors (n = 3). (E) TNF pathway mRNA expression was reduced by the addition of TNF inhibitors (n = 3). (F) Protein expression of TNF pathway was reduced by the addition of TNF inhibitors (n = 3). Data are presented as the means ± SD. Compared with normal control group, # p < 0.05, ## p < 0.01. Compared with OGD/R group, * p < 0.05, ** p < 0.01.

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  FIGURE 4. In vivo experiments confirm TNF signaling pathway promotes endothelial cell proliferation. (A) Schematic diagram of animal experiments. (B) Changes in infarct size in each group (n = 3). (C) Pathological changes in each group. (D) Echocardiographic changes in each group (n = 8). (E) Determination of CK, CK-MB and LDH in rat serum (n = 8). (F) Determination of ANP, BNP and Ang-2 in rat serum (n = 8). (G) Immunohistochemical changes in post-infarction heart failure rats (n = 3). (H) Immunofluorescence assay for detection of regenerating endothelial cell (n = 3). BrdU marks regeneration cells and CD31 marks endothelial cells. (I) Western blot of TNF signaling pathway in each group (n = 3). LADL, left anterior descending coronary artery ligation. Data are presented as the means ± SD. Compared with sham group, # p < 0.05, ## p < 0.01. Compared with model group, * p < 0.05, ** p < 0.01.

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  FIGURE 5. Molecular docking screening of traditional chinese medicines potentially targeting TNF. (A) Flowchart of the GEO database for online data retrieval. (B) Binding Affinity Heatmap of TNF with small molecules (binding affinity < −5.0). (C) Visualization of molecular docking of TNF with small molecules. (D) Biolayer Interferometry of icariin and TNF-a.

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  FIGURE 6. ICA promotes structural stabilization of TNF. (A) Root-mean-square deviation of TNF and ICA. (B) Root-mean-square fluctuation of TNF and ICA. (C) Radius of gyration of TNF and ICA. (D) Typical diagram of TNF and ICA during molecular dynamics simulations. ICA, Icariin.

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  FIGURE 7. In vitro experiments confirm ICA promotes endothelial cell proliferation via TNF signaling pathway. (A) Schematic diagram of in vitro cell experiments of ICA. (B) Cell survival was induced with the addition of ICA (n = 6). (C) Migratory capacity was induced with the addition of ICA (n = 6). (D) EdU+ cells were induced with the addition of ICA (n = 3). (E) Western blot of TNF signaling pathway in each group (n = 3). Data are presented as the means ± SD. ICA, Icariin. Compared with NC group, # p < 0.05, ## p < 0.01. Compared with OGD/R group, * p < 0.05, ** p < 0.01. Compared with OGD/R + ICA group, ! p < 0.05, !! p < 0.01.