Periplocymarin Alleviates Doxorubicin-Induced Heart Failure and Excessive Accumulation of Ceramides

Figure 1. Effects of periplocymarin on cardiac function and heart weight after 3-day doxorubicin (DOX) treatment in mice. The cardiac function of the mice was determined by the echocardiography in M-mode on the aspect of the short axis. (A) Representative images of echocardiography. (B–H) The ejection fraction (EF%) (B), fractional shortening (FS%) (C), left ventricular anterior diastolic wall thickness (LVAW; d) (D), left ventricular anterior systolic wall thickness (LVAW; s) (E), left ventricular posterior diastolic wall thickness (LVPW; d) (F), left ventricular posterior systolic wall thickness (LVPW; s) (G) and left ventricular mass (H) were calculated according to the M-mode echocardiography. Data are presented as mean ± SEM; n = 5–7, *p < 0.05, **p < 0.01, ***p < 0.001 vs. control; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. DOX. (I) The mice were euthanized and the representative images of the hearts were shown. Scale bar, 5 mm. (J) The ratios of heart weights to tibia length were calculated. Data are presented as mean ± SEM; n = 5–7. ***p < 0.001 vs. control, ##p < 0.01 vs. DOX. (K) The ratios of heart weights to body weights were calculated. Data are presented as mean ± SEM; n = 5-7.…

Figure 2. Periplocymarin improved cardiomyocyte apoptosis induced by DOX. (A) Representative micrographs of hematoxylin and eosin (H&E) staining. The left panel is 50X (Scale bar, 50 μm), the right panel is 200X (Scale bar, 50 μm). (B,C) Representative fluorescent images of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining of the hearts (B). Scale bar, 50 μm. Quantitative analysis of TUNEL staining in different groups (C). Data are presented as mean ± SEM; n = 4, *p < 0.05 vs. control, #p < 0.05 vs. DOX. (D) Western blot analysis of Bcl-2, cleaved caspase-3, and caspase-3 protein levels in heart tissues. (E) Quantitative analysis of Bcl-2, cleaved caspase-3, and caspase-3 expression in D. Data are presented as mean ± SEM; n = 3, *p < 0.05 vs. control, **p < 0.001 vs. control, #p < 0.05 vs. DOX. (F) Flow cytometric scatterplot of Annexin-V-FLTC staining of H9c2 cell apoptosis. (G) Flow cytometric histogram of Annexin-V-FLTC staining of H9c2 cell apoptosis. (H) Quantitative analysis of apoptotic H9c2 cells in different groups using bar graphs. Data are presented as mean ± SEM; n = 3. **p < 0.01, ***p < 0.001.

Figure 3. Periplocymarin regulated metabolic disorders induced by DOX. (A–D). The ceramides in mice serum were measured by liquid chromatography with tandem mass spectrometry (LC-MS/MS). The cluster heatmap of ceramides was presented (A). The score scatter plots of the quality control (QC) and the mice serum partial least squares discriminant analysis (PLS-DA) data were presented (B). Paired comparison of orthogonal partial least squares discriminate analysis (OPLS-DA) score scatter plots between the control group and DOX group (C). Paired comparisons of OPLS-DA score scatter plots between the DOX group and DOX + periplocymarin group (D). (E–H) Ceramides in H9c2 cells were measured by LC-MS/MS. The cluster heatmap of ceramides in H9c2 cell (E). The score scatter plots of the quality control (QC) and the H9c2 cells PLS-DA data were displayed (F). Paired comparisons of OPLS-DA score scatter plots between the control and DOX groups (G). Paired comparisons of OPLS-DA score scatter plots between the DOX and DOX + periplocymarin groups (H). The dots of different colors represented different groups, the green dots represented control samples, the purple dots represented the periplocymarin group,…

Figure 4. Periplocymarin regulated ceramides metabolism disorder induced by DOX. (A–D) Relative abundance of Cer16:0 (A), Cer18:0 (B), Cer24:1 (C), and Cer24:0 (D) ceramides in mice serum. (E–G) The ratios of Cer16:0 to Cer24:0 (E), Cer18:0 to Cer24:0 (F), and Cer24:1 to Cer24:0 (G) in mice serum. Data are presented as mean ± SEM; n = 6, **p < 0.01, ***p < 0.001 vs. control; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. DOX. (H–K) Relative abundance of Cer16:0 (H), Cer18:0 (I), Cer24:1 (J), and Cer24:0 (K) ceramides in H9c2 cells. (L–N) The ratios of Cer16:0 to Cer24:0 (L), Cer18:0 to Cer24:0 (M) and Cer24:1 to Cer24:0 (N) in H9c2 cells. Data are presented as mean ± SEM; n = 6, ***p < 0.001 vs. control; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. DOX.

Figure 5. Network pharmacology to explore the mechanism of periplocymarin in the treatment of heart failure. (A) Intersection targets of periplocymarin and heart failure. Periplocymarin:300 targets; heart failure:8,770 targets. (B) The network of heart failure-targets-periplocymarin. Green hexagon represent compound, red hexagon represents disease, squares represent targets. (C) Protein-protein interaction network of 246 intersecting targets. The darkness of colors and sizes of circles represent the degree of freedom. (D–G) Bubble plot of Gene Ontology (GO) enrichment analysis of shared targets by periplocymarin and heart failure. Bubble plot: letters on the left are GO names, numbers on the bottom are the proportions of genes, sizes of the circles indicate the numbers of enriched genes, and colors reflect p values. The redder the colors are, the more enriched the genes are, and the smaller the p values are. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses (D), biological process (BP) enrichment (E), cellular components (CC) enrichment (F), and molecular function (MF) enrichment (G) of the 246 intersecting genes. (H) Network of KEGG pathway and the 166 related targets.…

Figure 6. Periplocymarin regulated the expression of enzymes involved in ceramides metabolism. (A) CerS1-6 messenger RNA (mRNA) expression in the heart tissues was measured by real-time PCR. Data are presented as mean ± SEM; n = 5, *p < 0.05, **p < 0.01, ***p < 0.001 vs. control; #p < 0.05, ##p<0.01, ###p<0.001 vs. DOX. (B) CerS1-6 mRNA expression in the primary cardiomyocytes was measured by real-time PCR. Data are presented as mean ± SEM; n = 3, *p < 0.05, **p < 0.01, ***p < 0.001 vs. control; ##p < 0.01, ###p < 0.001 vs. DOX. (C) The expression of CerS1-6 in the RNA sequencing data (GSE157282).