Proteomic results indicate inhibition of complement cascades is one of the mechanisms underlying the protective effects of Tongxinluo against myocardial infarction

FIGURE 1. The overview of the proteomic results of mice treated with NS or TXL. n = 6 mice per group for proteomic analysis. (A) The scheme of the proteomic analysis. (B) The PCA of proteomic results. (C) The heatmap of differentially expressed proteins (FC > 1.5 or <2/3, p < 0.05) between the AMI + NS and the AMI-TXL group.

FIGURE 2. Activation of complement cascades is significantly hampered by TXL treatment. (A) KEGG pathway enrichment analysis was corrected for multiple testing using the Benjamini–Hochberg method, and adjusted P-values are shown. (B) The expression of several key proteins associated with complement and coagulation cascades. C1s1 refers to the database-annotated protein entry identified in the proteomic analysis. The numbers in cells showed the averaged expression levels. (C,D) The volcano plots of differentially expressed proteins between the AMI + NS and the Sham groups (C) and between the AMI + TXL and the AMI + NS groups (D).

FIGURE 3. Representative predicted docking poses between of C1qa or the proteomics-identified C1s1 database entry with TXL-derived compounds. The left image shows the overall view, while the right image shows the partial view. The yellow sticks represent small molecules, the cyan cartoon represents the protein, the blue lines indicate hydrogen bonding interactions, and the gray dashed lines represent hydrophobic interactions.

FIGURE 4. Adding C1i did not further enhance the complement inhibition and cell-protective effects of TXL. n = 6 mice per group for WB and ELISA analyses; n = 4 mice per group for immunofluorescence. (A) The WB results of C1s and C1qa. (B) The quantitative analysis of C1s and C1qa. (C) The representative images of immunofluorescent staining of C1s and C1qa. Bar = 50 μm. (D) The quantitative analysis of immunofluorescent staining. (E) The serum C3a levels. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

FIGURE 5. C1-mediated complement cascades is one of the mechanisms underlying the protective effects of TXL against cell death. (A) The WB results of Bcl-2 and Bax, two essential survival-related proteins (n = 6). (B) The quantitative analysis of Bcl-2 and Bax. (C) The representative images of TUNEL staining (n = 4). Bar = 50 μm. (D) The quantitative analysis of TUNEL+ cells per HPF. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

FIGURE 6. C1-mediated complement cascades is one of the mechanisms underlying the cardioprotective effects of TXL. n = 6 mice per group for echocardiography, Sirius red staining, and WGA staining. (A) The representative images of echocardiography. (B–D) The quantitative analysis of LVEF at day 3 (B), 7 (C), and 21 days (D) after AMI. (E) The representative images of Sirius red staining. Bar = 1 mm. (F) The quantitative analysis of Sirius red staining. Fibrotic area is identified as red area. (G) The representative images of WGA staining. Bar = 50 μm. (H) The quantitative analysis of WGA staining. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.